The effect of lipid extraction on carbon and nitrogen stable isotope ratios in oyster tissues: Implications for glycogen-rich species.

RATIONALE: Extraction of lipids from tissues prior to carbon stable isotope analysis (SIA) has become a common practice, despite a lack of species-specific data to indicate when lipid extraction is needed. Marine invertebrates, including bivalves, are known to store carbon as glycogen and less in the form of lipids than other species, potentially reducing the need for lipid extraction even when C:N values are above 3.5, a value that previous studies suggest indicates a need for lipid extraction of animal tissues. METHODS: We investigated the need for lipid extraction on individual tissues (adductor muscle, gut gland, gill) and whole tissue of a glycogen-storing species, the oyster Crassostrea virginica. Bulk and lipid-extracted samples were analyzed for their C and N stable isotope ratios by continuous flow isotope ratio mass spectrometry (IRMS). Samples were analyzed on a 20-20 isotope ratio mass spectrometer (PDZ Europa) after combustion in an elemental analyzer (PDZ Europa Automatic Analyzer-Gas Solid Liquid). RESULTS: Although the C:N values for most bulk (unextracted) tissue samples were greater than 3.5, the lipid-extracted δ13 C values did not differ from the bulk values. Lipid extraction, however, affected δ15 N values in all tissue types except adductor muscle, indicating that separate SIA may be required when tissues are lipid extracted. CONCLUSIONS: These data demonstrate that it is not necessary to lipid extract oyster tissues in all cases, and that C:N thresholds for lipid extraction in other species may not be reliable for organisms such as oysters that store glycogen. Our data indicate that minimizing unnecessary lipid extraction through preliminary testing will save researchers time and expense by avoiding superfluous sample handling, reducing concern over secondary effects on data quality, and reducing the costs of reagents and additional separate stable isotope analysis to ensure analytical accuracy. Copyright © 2016 John Wiley & Sons, Ltd.

Protein kinase inhibitors in the treatment of inflammatory and autoimmune diseases.

Protein kinases mediate protein phosphorylation, which is a fundamental component of cell signalling, with crucial roles in most signal transduction cascades: from controlling cell growth and proliferation to the initiation and regulation of immunological responses. Aberrant kinase activity is implicated in an increasing number of diseases, with more than 400 human diseases now linked either directly or indirectly to protein kinases. Protein kinases are therefore regarded as highly important drug targets, and are the subject of intensive research activity. The success of small molecule kinase inhibitors in the treatment of cancer, coupled with a greater understanding of inflammatory signalling cascades, has led to kinase inhibitors taking centre stage in the pursuit for new anti-inflammatory agents for the treatment of immune-mediated diseases. Herein we discuss the main classes of kinase inhibitors; namely Janus kinase (JAK), mitogen-activated protein kinase (MAPK) and spleen tyrosine kinase (Syk) inhibitors. We provide a mechanistic insight into how these inhibitors interfere with kinase signalling pathways and discuss the clinical successes and failures in the implementation of kinase-directed therapeutics in the context of inflammatory and autoimmune disorders.

Forced oscillations in the clinical setting in young children with neonatal lung disease.

The extent of respiratory dysfunction is not well characterised in children with neonatal chronic lung disease (nCLD) too young to perform spirometry. Forced oscillations are easily performed by healthy young children; however, they may be more difficult for those with nCLD. The present study aimed to describe the feasibility of using the forced oscillation technique in children with nCLD in a routine clinical setting and to investigate the influence of neonatal factors on subsequent lung function. Respiratory function tests were attempted in 64 patients with nCLD aged 3.2-6.6 yrs. Respiratory resistance and reactance at 6, 8 and 10 Hz were expressed as z-scores derived from a healthy reference population. The within-test variation and between-test repeatability were also assessed. Technically, satisfactory data were obtained from 77% of children. On grouped data, z-scores for all oscillatory indices were different from zero and related to hospital oxygen administration in the neonatal period. In conclusion, the forced oscillation technique was feasible in preschool children with neonatal chronic lung disease in the clinical outpatient setting. These children had lung function significantly worse than that predicted from healthy children. Respiratory function assessed using forced oscillations appeared to reflect the severity of lung disease during the neonatal period.

Sources of inoculum for Phytophthora ramorum in a redwood forest.

ABSTRACT Sources of inoculum were investigated for dominant hosts of Phytophthora ramorum in a redwood forest. Infected trunks, twigs, and/or leaves of bay laurel (Umbellularia californica), tanoak (Lithocarpus densiflorus), and redwood (Sequoia sempervirens) were tested in the laboratory for sporangia production. Sporangia occurred on all plant tissues with the highest percentage on bay laurel leaves and tanoak twigs. To further compare these two species, field measurements of inoculum production and infection were conducted during the rainy seasons of 2003-04 and 2004-05. Inoculum levels in throughfall rainwater and from individual infections were significantly higher for bay laurel as opposed to tanoak for both seasons. Both measurements of inoculum production from bay laurel were significantly greater during 2004-05 when rainfall extended longer into the spring, while inoculum quantities for tanoak were not significantly different between the 2 years. Tanoak twigs were more likely to be infected than bay laurel leaves in 2003-04, and equally likely to be infected in 2004-05. These results indicate that the majority of P. ramorum inoculum in redwood forest is produced from infections on bay laurel leaves. Years with extended rains pose an elevated risk for tanoak because inoculum levels are higher and infectious periods continue into late spring.

Diagnosis of equine infectious anaemia during the 2006 outbreak in Ireland.

In 2006 there was an outbreak of equine infectious anaemia (EIA) in Ireland. This paper describes the use of the diagnosis of clinical and subclinical cases of the disease. In acute cases the ELISAs and the immunoblot were more sensitive than the AGID. In one mare, fluctuating antibody levels were observed in all the serological assays before it seroconverted by AGID. Viral RNA and DNA were detected by RT-PCR and PCR in all the tissues from the infected animals examined postmortem. The PCR detected viral DNA in plasma regardless of the stage of the disease. In contrast, the RT-PCR detected RNA in only 52 per cent of the seropositive animals tested and appeared to be most sensitive for the detection of virus early in infection. Both PCR and RT-PCR demonstrated potential to detect acutely infected horses earlier than some of the official tests. The serological data suggest that the usual incubation/seroconversion period for this strain of the virus was approximately 37 days but may be more than 60 days in a few cases.

Denosumab: a case of MRONJ with resolution.

A patient was referred by a clinical oncologist regarding a non-healing area of bone in the lower jaw. The patient was taking denosumab (Prolia(®), Xgeva(®)) when they had a tooth extracted and the area failed to heal. Following suspension of the drug, the area healed with mucosal coverage. This new class of drugs are being increasingly used as an alternative antiresorptive drug to bisphosphonates and are licenced in the UK for prevention of osteoporotic fractures and prevention of skeletal-related events (SREs) in patients with metastatic cancer.

Mutation of the p53 gene in human soft tissue sarcomas: association with abnormalities of the RB1 gene.

Soft tissue sarcomas have been examined for alterations in the p53 gene. In six sarcomas, loss or rearrangement of both alleles of this gene was detected while in a further seven sarcomas, point mutation or absence of transcription of the p53 gene was observed. Abnormalities of the p53 gene were found in several classes of soft tissue sarcoma, including leiomyosarcomas, rhabdomyosarcomas and malignant fibrous histiocytomas. Our studies also show that abnormalities of the RB1 suppressor gene and of the p53 gene frequently occur together. These results are consistent with the idea that the p53 gene is a tumour suppressor gene and indicate that coincident inactivation of more than one tumour suppressor gene may, in some cases, be required for tumour development.

Detection of transforming genes by transfection of DNA from primary soft-tissue tumours.

A series of adult soft-tissue tumours were screened for the presence of activated oncogenes by transfecting tumour DNA into NIH3T3 mouse fibroblasts. In these studies an activated K-ras gene that contained a mutation at the second position of codon 12 (GGT----GAT) was found in a leiomyosarcoma. In addition, following transfection of DNA from a liposarcoma, we identified an activated gene that failed to hybridize to probes prepared from 10 known human oncogenes (K-ras, H-ras, N-ras, ret, met, trk, mas, dbl, raf and hst) that have previously been detected in DNA transfection experiments. BamHI-BamHI fragments of this activated gene of 3.5 and 8.5 kb were cloned from NIH3T3 secondary transfectants using a probe that detects the human alu family of highly repetitive DNA sequences. Repeat-free subclones of these BamHI fragments were used to map this gene to human chromosome 19 (p13.2-q13.3). Our studies also demonstrate that a subclone from one of the BamHI fragments detects a 3.0 kb transcript in primary and secondary transfectants.

Combination carboplatin and radiotherapy in the management of stage II testicular seminoma: comparison with radiotherapy treatment alone.

PURPOSE: To assess the results of treatment in 33 patients with stage IIA/B seminoma who were treated with carboplatin and radiotherapy (RT) between January 1989 and December 1996. PATIENTS AND METHODS: Thirty patients received single course single agent carboplatin (400 mg/m2 or area under curve (AUC 7), two patients received two courses carboplatin, and one patient received single course carboplatin and etoposide, all 4-6 weeks prior to infra-diaphragmatic RT. Results were retrospectively compared with those obtained for 80 patients treated from 1970 to 1998 with radiotherapy alone. RESULTS: There was minimal toxicity associated with the use of carboplatin prior to RT. With a median follow-up of 4 years (range 2-70 months) 2/33 patients treated with chemotherapy and RT have relapsed, 5-year relapse free survival (RFS) = 96.9% (95% confidence interval (CI) 72.9-99.4%), and one patient has died of progressive disease, 5-year overall survival (OS) = 96.7%. With a median follow-up of 11.2 years (range 6 months to 25.8 years) 15/80 patients treated with RT alone have relapsed, 5-year RFS = 80.7% (95% CI 70.1-87.9%), including 13/61 patients treated with infra-diaphragmatic RT, 5-year RFS = 77.9%, and 2/19 treated with additional supra-diaphragmatic RT, 5-year RFS = 89.5% (P = 0.277). Eleven out of 80 patients have died, 5-year OS = 94.7%. For stage IIA, 1/14 patients treated with chemotherapy and RT have relapsed, 5-year RFS = 92.3%, compared with 5/34 treated with infra-diaphragmatic RT alone 5-year, RFS = 84.9% (P = 0.527). For stage IIB, 1/19 patients relapsed (at 69 months) following chemotherapy and RT (5-year RFS = 100%), whereas 8/27 relapsed following infra-diaphragmatic RT alone, 5-year RFS = 69.4% (P = 0.0595). CONCLUSION: Infradiaphragmatic RT alone cures the majority of patients with stage II seminoma, but the relapse rate remains high particularly for patients with stage IIB disease. As compared with historical controls, carboplatin with RT appears to reduce the relapse rate in stage II seminoma with minimal additional toxicity and the results approach statistical significance for stage IIB patients. Confirmation would require a phase III randomized comparison.

Radiotoxicity of bismuth-213 bound to membranes of monolayer and spheroid cultures of tumor cells.

Monoclonal antibody 13A to murine CD44 was used to bind the alpha-particle emitter 213Bi to cell surfaces of cultured EMT-6 or Line 1 tumor cells. Data on kinetics and saturation of binding, cell shape and nuclear size were used to calculate the absorbed dose to the nuclei. Treatment of monolayer cells with [213Bi]MAb 13A produced a classical exponential survival curve with no apparent shoulder. Microdosimetry analyses indicated that 1.4-1.7 Gy produced a 37% surviving fraction (D0). Multicellular spheroids were shown to bind [213Bi]MAb 13A mainly on the outer cell layer. Relatively small amounts of activity added to the spheroids resulted in relatively large absorbed doses. The result was that 3-6-fold less added radioisotope was necessary to kill similar fractions of cells in spheroids than in monolayer cells. These data are consistent with the interpretation that the alpha particles from a single 213Bi atom bound to one cell can penetrate and kill adjacent cells. Flow cytometry was used to sort cells originating from the periphery or from the interior of spheroids. Cells from the outside of the [213Bi]MAb 13A exposed spheroids had a lower surviving fraction per administered activity than cells from the interior. Cells were killed efficiently in spheroids up to 20-30 cells in diameter. The data support the hypothesis that alpha-particle emitters should be very efficient at killing cells in micrometastases of solid tumors.

Strain in the anteromedial bundle of the anterior cruciate ligament under combination loading.

Strain within the anteromedial bundle (AMB) of the anterior cruciate ligament (ACL) was measured in 13 human knee specimens in order to determine the combination of external loads most likely to cause injury. Using a load application system that allowed 5 df with the flexion angle being fixed, pure loads of anterior/posterior force, medial/lateral force, varus/valgus torque, and internal/external axial torque were applied at three flexion angles: 0 degrees, 15 degrees, 30 degrees. Combined loads were applied in pairs at two flexion angles: 0 degrees and 30 degrees. Liquid mercury strain gauges were used to measure strain in the ACL. Anterior tibial force was the primary determinant of strain in the anteromedial bundle. This strain was significantly larger at 30 degrees flexion than at 0 degrees. The strain sensitivity of the AMB to medial force was approximately one-half that to pure anterior force. The effect of anterior and medial forces was additive when applied in combination. Neither pure axial torque nor pure varus/valgus torque was observed to strain significantly the AMB at any of the flexion angles investigated. However, valgus torque in combination with anterior force resulted in a significantly larger strain than pure anterior force. Internal axial torque in combination with anterior force also resulted in a larger strain than pure anterior force.

Direct measurement of strain in the posterolateral bundle of the anterior cruciate ligament.

The general objectives of the work reported in this article were to describe and validate a method for directly measuring strain in the posterolateral bundle (PLB) of the anterior cruciate ligament (ACL). The method is a procedure for gaining surgical access to the posterior fibers of the PLB through a portal incised in the joint capsule and then suturing a liquid metal strain gage (LMSG) on to these fibres. Because the incision possibly alters the load-displacement mechanics of the joint, validation included performing experiments to test the hypothesis that the incision did not significantly affect load-displacement relations. To illustrate the utility of the method, strains in both the anteromedial bundle (AMB) and PLB were measured and compared over the full range of flexion. Validation experiments revealed that the capsular incision as well as other incisions had no measurable effect on the load-displacement mechanics of the joint. Also, the PLB strain was significantly different from the AMB strain during passive flexion with a reciprocating function in load sharing evident between the two bundles.

Strain in the medial collateral ligament of the human knee under single and combined loads.

Strain within the medial collateral ligament (MCL) was measured in 13 human knee specimens to determine both the single and combined external loads most likely to cause injury. Using a load application system which allowed six degrees of freedom with flexion angle being fixed, both single loads of anterior/posterior force, medial/lateral force, varus/valgus torque, and internal/external axial torque and all pairs of these loads were applied at flexion angles of 0 degrees and 30 degrees. Liquid mercury strain gages were used to measure strain at four sites in the MCL. Two of the sites were the anterior fibers superior and inferior to the joint line and the other two were posterior of the two anterior sites. A factorial analysis revealed a significant interaction between the site experiencing the greatest strain and flexion angle. The posterior superior site experienced significantly greater strain at 0 degree flexion whereas strains was significantly greater at the anterior superior site at 30 degree flexion. Of the single moments, external axial was more damaging than valgus in that the strain developed at equivalent load was significantly greater. None of the moment-moment combinations was identified as being significantly more damaging. A similar result held for the force-moment combinations.

Ludwig's angina: an update.

Despite a reduction in preantibiotic mortality rates that exceeded 50%, Ludwig's angina remains a potentially lethal entity primarily because of rapidly progressive airway obstruction. Since the reports of several large series in the 1940's, there have been put sporadic case reports because of widespread use of antibiotics in orodental infection, improved dental care, as well as adherence to strict diagnostic criteria. Since this entity is now uncommon, unnecessary delay in diagnosis and management may occur and may result in serious complications. This presentation will consist of an historical review, discussion of pathogenesis followed by clinical presentation, bacteriology and treatment, as well as a detailed analysis of our most recent 20 cases. There were no complications and no deaths. The infection resolved with medical therapy in 11 patients, while 9 patients required surgical procedures. Penicillin, clindamycin or chloramphenicol were started initially in all cases. Four of these 9 patients developed a localized abscess, while on antibiotics, which required drainage. Tracheotomy or intubation was necessary in 7 patients. Early and aggressive use of appropriate antibiotics and protection of the airway are the mainstays of a successful treatment regimen. Judicious surgical intervention is indicated in those patients who develop localized abscesses while on antibiotics or are unresponsive to medical management.

Treatment of the clinically negative neck in floor of the mouth carcinoma.

A retrospective study of 61 consecutive patients with floor of mouth carcinoma and a clinically negative neck was done to determine the value of elective treatment to the neck. Subsequent histologically proven nodal disease was analyzed according to initial treatment modality (23 surgical, 38 irradiation), stage (29--T1,27--T2,5--T3), hemi-neck at risk (midline lesions placed both hemi-necks at risk), and primary control for a minimum of 2 years (50 patients, 78 hemi-necks at risk). Neck failures were classified by T stage, extent of neck treatment (upper neck or complete) and by primary control at the time of manifest neck metastasis. Of the hemi-necks at risk with the primary controlled, 10% (17% of patients) developed nodal disease without complete neck treatment; 86% (6/7) of these patients were salvaged. The occult positive node incidence, conservatively calculated by elimination of all patients treated with elective partial or total neck irradiation, was 10% for patients with T1 and T2 lesions (7% for hemi-necks at risk). Based on the clinical course of patients with primary control, only 1 patient (2%) might have benefited from initial complete elective neck treatment. These results suggest that elective neck treatment in early (T1 and T2, N0) floor of mouth carcinoma is of doubtful value. Supraomohyoid , rather than suprahyoid dissection, is recommended if surgical treatment of the neck is undertaken.

A clinically applicable technique to study cytoskeletal dynamics in normal and abnormal polymorphonuclear leukocytes isolated from small volume blood samples.

Shape change and motility of polymorphonuclear leukocytes (PMNs) are essential for host defense and require dynamic reorganizations of microfilamentous cytoskeleton by reversible polymerization of G-actin into filaments (F-actin). Although clinical disorders of actin polymerization are rare, recently described simple methodologies for assaying actin dynamics in PMNs make the technique readily applicable to clinical studies. To develop a clinically useful F-actin assay, the authors investigated the optimal preparation conditions for PMN isolation that resulted in the least in vitro cytoskeletal activation and evaluated the variability in actin dynamics in acutely and chronically infected patients. Basal and chemotactic factor-activated PMN F-actin content was measured by a previously described flow cytometric technique in fixed, permeabilized, NBDphallacidin-stained PMNs isolated by centrifugation in Percoll or Ficoll-Hypaque density gradients or by countercurrent elutriation. F-actin content is expressed as mean fluorescent channel or relative fluorescence intensity. Basal F-actin in PMNs prepared from countercurrent elutriation (mean fluorescent channel = 79.0 +/- 4.5, n = 6) or by Ficoll Hypaque (82.0 +/- 3.5, n = 4) was significantly higher than endotoxin free, Percoll purified PMNs, whether purified in bulk (56.1 +/- 7.9, n = 8) or by the small volume modification applicable to clinical studies (53.3 +/- 8.7, n = 15). Basal Ficoll Hypaque purified PMNs have evidence of shape change, whereas endotoxin free, Percoll purified PMNs are smooth and round and represent the most basal cell equivalent in F-actin content to a circulating PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Avascular necrosis in patients treated with BEP chemotherapy for testicular tumours.

Avascular necrosis (AVN) is known to occur after combination chemotherapy for lymphomas and leukaemias that includes high dose corticosteroids, but it has been reported rarely in patients with solid tumours. We describe five recent cases in young men with testicular tumours (three of which were of good prognosis), who had been treated with chemotherapy using dexamethasone as an antiemetic. Dexamethasone is a low cost and effective antiemetic, but it may be responsible for inducing AVN in patients receiving chemotherapy for solid tumours. A prospective survey of the frequency of AVN is justified to quantify the extent of the problem.

Facial keratoacanthoma.

The keratoacanthoma is a common tumor of hair follicle origin that appears predominantly on sun-exposed skin of elderly persons. Approximately 80% of the tumors arise on the face. It is characterized by the rapid growth of a painless, dome-shaped, 10 to 25 mm solitary lesion with a central keratinous plug that undergoes spontaneous involution over 6 to 12 months, leaving behind a puckered scar. Alarming growth and early histologic appearance (pseudocarcinomatous infiltration, cell atypicality, mitoses) are suggestive of squamous cell carcinoma, but biologic behavior and tumor architecture confirm its benign nature. However, evidence that all keratoacanthomas regress is lacking, malignant transformation has been reported, and less than excisional biopsy may render a difficult histologic diagnosis either inconclusive or erroneous, even in expert hands. Excisional biopsy is recommended as expedient and definitive management that offers optimal prognosis. Local flap or full-thickness graft closure affords cosmetic results that are superior to those provided by other treatment methods.

Reinnervation of skeletal muscle with a neuromuscular pedicle.

In the past decade the otolaryngologist has become interested in the problem of muscle reinnervation as it relates to laryngeal and facial paralysis. Although reinnervation by neuromuscular pedicle transfer has shown promising results in the laboratory and clinic, some investigators have had difficulty in achieving reliable results with this procedure. To further assess the technique's validity, we investigated the neuromuscular pedicle. This study utilized a strap muscle neuromuscular pedicle transfer to a contralateral strap muscle in the rabbit. The results were analyzed by the use of a number of independent measures, including electrical stimulation of the nerve, muscle contractibility, electromyography, enzyme histochemistry, reduced-silver staining for normal fibers, and the retrograde transport of the enzyme marker horseradish peroxidase. The physiologic and anatomic results demonstrated that morphologic and functional reinnervation of the experimentally isolated muscle by the transferred neuromuscular pedicle occurred. The most convincing data were produced by gross electrical stimulation, twitch and tetanic contraction, and horseradish peroxidase labeling. Electromyographic activity and other histologic findings supported the above conclusions.