Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation.

Eukaryotic cells rely on a surveillance mechanism known as the spindle checkpoint to ensure accurate chromosome segregation. The spindle checkpoint prevents sister chromatids from separating until all kinetochores achieve bipolar attachments to the mitotic spindle. Checkpoint proteins tightly inhibit the anaphase-promoting complex (APC), a ubiquitin ligase required for chromosome segregation and progression to anaphase. Unattached kinetochores promote the binding of checkpoint proteins Mad2 and BubR1 to the APC-activator Cdc20, rendering it unable to activate APC. Once all kinetochores are properly attached, however, cells inactivate the checkpoint within minutes, allowing for the rapid and synchronous segregation of chromosomes. How cells switch from strong APC inhibition before kinetochore attachment to rapid APC activation once attachment is complete remains a mystery. Here we show that checkpoint inactivation is an energy-consuming process involving APC-dependent multi-ubiquitination. Multi-ubiquitination by APC leads to the dissociation of Mad2 and BubR1 from Cdc20, a process that is reversed by a Cdc20-directed de-ubiquitinating enzyme. The mutual regulation between checkpoint proteins and APC leaves the cell poised for rapid checkpoint inactivation and ensures that chromosome segregation promptly follows the completion of kinetochore attachment. In addition, our results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes.

Comparative evaluation of chlorhexidine varnish and fluoride varnish on plaque Streptococcus mutans count--an in vivo study.

AIM: The aim of the study was to assess and compare the effect of chlorhexidine varnish and fluoride varnish application on Streptococcus mutans counts in plaque of occlusal pits and fissures of permanent mandibular first molars. MATERIALS AND METHODS: The study was an in vivo comparative study, conducted among 50 schoolchildren aged 7-8 years under a field setting. The 50 subjects were randomly allocated into two groups. Baseline plaque samples were collected from all the subjects followed by the application of two varnishes, Cervitec and Duraphat. The varnish was applied to pit and fissures of occlusal surface of mandibular first molar. The varnish application was carried out on the first day, fifth day and tenth day after baseline plaque sampling. Subsequent plaque samples were collected at the end of 1 month and at the end of 3 months after the varnish application. RESULTS: The Cervitec varnish has shown a statistically significant reduction at the end of 1 month and at the end of 3 months (P < 0.05). Duraphat varnish did not show a statistically significant difference in reducing the plaque S. mutans count at the end of 1 month and third month (P > 0.05). CONCLUSION: Cervitec varnish was found to be effective in reducing S. mutans count for a 3-month period, when compared to Duraphat varnish.

Association of differential expression of immunoregulatory molecules and presence of targetable mutations may inform rational design of clinical trials.

BACKGROUND: Immune checkpoint inhibitors (ICIs) and genomic biomarker-driven targeted therapies have revolutionized the modern oncologic treatment arsenal. The next step has been to combine targeted agents and ICIs. In doing so, some combination regimens may be more logical than others. PATIENTS AND METHODS: Whole-exome and whole-transcriptome sequencing were performed on 2739 unselected later-stage clinical cases from 24 solid tumor subtypes in the NantHealth database, and data were also curated from 5746 similarly sequenced patients across 28 solid tumor subtypes in The Cancer Genome Atlas (TCGA). Significant differential expression of 10 immunoregulatory molecules [IRMs (genes)] was analyzed for association with mutant versus wild-type genes. RESULTS: Twenty-three significant associations between currently actionable variants and RNA-expressed checkpoint genes were identified in the TCGA cases; 10 were validated in the external cohort of 2739 clinical cases from NantHealth (P values were adjusted using Benjamini-Hochberg multiple hypothesis correction to reduce false-discovery rate). Within the same 5746 TCGA profiles, 2740 TCGA patients were identified as having one or more potentially oncogenic single-nucleotide variant (SNV) mutation within an established 50-gene hotspot panel. Of the 50 genes, SNVs within 15 were found to be significantly associated with differential expression of at least one IRM after adjusting for tissue enrichment; six were confirmed significant associations in an independent set of 2739 clinical cases from NantHealth. CONCLUSIONS: Logically combining ICIs with targeted therapies may offer unique treatment strategies for patients with cancer. The presence of specific mutations impacts the expression of IRMs, an observation of potential importance for selecting combinations of gene- and immune-targeted therapeutics.

Orthodontic uprighting of impacted mandibular permanent second molar: a case report.

The mandibular second molars can become impacted beneath the crown of the first molars due to various causes and fail to erupt normally. Presented herewith is a case report of orthodontic uprighting of a mesioangular impacted mandibular right permanent second molar. Though various treatment options were available, an uprighting push spring appliance was used as it is easy to fabricate and produces distal tipping and uprighting of the impacted tooth without the necessity of surgical assistance, bone removal, or splinting. The uprighting of the mandibular second molar was achieved within two months.

Strain and damage-sensing performance of biocompatible smart CNT/UHMWPE nanocomposites.

Herein, we report strain- and damage-sensing performance of biocompatible smart CNT/UHMWPE nanocomposites for the first time. CNT/UHMWPE nanocomposites are fabricated by solution mixing followed by compression molding. The surface morphology, microstructural properties, thermal decomposition and stability, glass transition temperature and thermal conductivity of the nanocomposites are characterized. The degree of crystallinity of CNT/UHMWPE nanocomposites is found to have a maximum value of 52% at 0.1 wt% CNT loading. The degree of crystallinity influences the mechanical properties of the CNT/UHMWPE nanocomposites. The electrical percolation threshold is achieved at 0.05 wt% of CNT and it follows a two dimensional conductive network according to percolation theory. The piezoresistive response of CNT/UHMWPE nanocomposites is demonstrated with a gauge factor of ~2.0 in linear elastic regime and that in the range of 3.8-96.0 in inelastic regimes for 0.05 wt% of CNT loading. A simple theoretical model is also developed to predict the resistivity evolution in both elastic and inelastic regimes. High sensitivity of CNT/UHMWPE nanocomposites coupled with linear piezoresistive response up to 100% strain demonstrates their potential for application in artificial implants as a self-sensing material.

A two centre observational study of simultaneous pulse oximetry and arterial oxygen saturation recordings in intensive care unit patients.

The influence of variables that might affect the accuracy of pulse oximetry (SpO2) recordings in critically ill patients is not well established. We sought to describe the relationship between paired SpO2/SaO2 (oxygen saturation via arterial blood gas analysis) in adult intensive care unit (ICU) patients and to describe the diagnostic performance of SpO2 in detecting low SaO2 and PaO2. A paired SpO2/SaO2 measurement was obtained from 404 adults in ICU. Measurements were used to calculate bias, precision, and limits of agreement. Associations between bias and variables including vasopressor and inotrope use, capillary refill time, hand temperature, pulse pressure, body temperature, oximeter model, and skin colour were estimated. There was no overall statistically significant bias in paired SpO2/SaO2 measurements; observed limits of agreement were +/-4.4%. However, body temperature, oximeter model, and skin colour, were statistically significantly associated with the degree of bias. SpO2 <89% had a sensitivity of 3/7 (42.9%; 95% confidence intervals, CI, 9.9% to 81.6%) and a specificity of 344/384 (89.6%; 95% CI 86.1% to 92.5%) for detecting SaO2 <89%. The absence of statistically significant bias in paired SpO2/SaO2 in adult ICU patients provides support for the use of pulse oximetry to titrate oxygen therapy. However, SpO2 recordings alone should be used cautiously when SaO2 recordings of 4.4% higher or lower than the observed SpO2 would be of concern. A range of variables relevant to the critically ill had little or no effect on bias.

Mutations in Drosophila enabled and rescue by human vasodilator-stimulated phosphoprotein (VASP) indicate important functional roles for Ena/VASP homology domain 1 (EVH1) and EVH2 domains.

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

The contralateral slip. An avoidable complication and indication for prophylactic pinning in slipped upper femoral epiphysis.

Between July 1994 and June 2004, 60 patients with 76 slipped upper femoral epiphyses were managed within the adult trauma service of three hospitals. Treatment was by a single cannulated screw. Of these cases, 53 were unilateral, in 17 of which uncomplicated prophylactic fixation of the contralateral hip was performed. Of the other 36 cases, nine presented with a subsequent slip despite ongoing out-patient care. The subsequent slip was unpredictable in timing and unrelated to the age at the initial slip. It was more often unstable and in one case avascular necrosis developed. The overall rate of avascular necrosis, although in accordance with the literature, was 60% in acute unstable slips with a slip angle greater than 40 degrees. In our experience, prophylactic fixation was safer than continued observation of the contralateral hip.

Autonomic symptom burden is associated with MS-related fatigue and quality of life.

BACKGROUND: Nonspecific symptoms such as fatigue and dizziness are common in multiple sclerosis (MS), even in patients with normal exams. Little is known about the relationship of autonomic dysfunction with these symptoms and quality of life. OBJECTIVE: Assess the association of autonomic symptom burden with fatigue, clinical status and quality of life. METHODS: Subjects completed an autonomic symptom (COMPASS-31), quality of life (MSQOL-54) and fatigue (FSS) questionnaire at their routine MS clinic follow-up. Demographic and clinical data were collected from the medical record. Pearson correlations were assessed between autonomic symptoms and fatigue, quality of life, disability and disease duration. RESULTS: One-hundred subjects completed the study (mean age 48 years; 78% female; 84% relapsing-remitting), mean disease duration was 14.7 years and mean EDSS 2.5. MSQOL-54 composite scores were 58 physical and 65 mental. COMPASS-31 correlated with MSQOL-54 (Physical R= -0.60; Mental -0.54; p<0.001) and FSS (R=0.51; p<0.001). There was no relationship between COMPASS-31 and EDSS (R=0, p=0.97) or disease duration (R= -0.02, p=0.84). CONCLUSIONS: Autonomic symptom burden is correlated with decreased quality of life and increased fatigue. Autonomic symptoms are present early in the disease and at low disability and may reflect aspects of disease burden that are not well-captured by current disability measures.

Nitric oxide inducing factor as a measure of antigen and mitogen-specific T cell responses in chickens.

We describe here an assay to measure responses of T cells to in vitro stimulation with antigens and a T cell mitogen (ConA). Spleen cells from chickens immunized with live viruses and an inactivated antigen produced macrophage activating factors (MAF) in response to in vitro stimulation with homologous antigens. The production of MAF, quantitated by the induction of NO in a retrovirus transformed macrophage cell line, HD11 (Beug et al., 1979, Cell 18, 375) was antigen-specific and correlated well with T cell proliferation. Further studies showed that production of MAF was abrogated by cyclosporin A, anti-CD4 and anti-CD8 monoclonal antibodies. These data suggested that production of MAF required T cell activation and can be used as measure of antigen and mitogen-specific T cell responses in chickens.

Flow cytometric analysis of the neutralizing immune response against infectious bursal disease virus using reticuloendotheliosis virus-transformed lymphoblastoid cell lines.

Infectious bursal disease virus (IBDV) is a lymphotropic virus with cytocidal effect on B lymphocytes of the bursa of Fabricius. We investigated the susceptibility of clonal populations of reticuloendotheliosis virus-transformed chicken B lymphocytes of both spleen and bursal origin to IBDV infection. The infected cells were metabolically-labelled and the viral polypeptides were analyzed by immunoprecipitation using monoclonal antibodies (MAbs). Virus adsorption and the effects of neutralizing convalescent antisera and MAbs on virus attachment were studied using flow cytometry. The results of the study indicate firstly that the transformed B cells support virus replication and provide an efficient system for studying IBDV-lymphocyte interactions. Secondly, results obtained also showed that the most potent neutralizing antibodies may not be those involved in preventing the receptor-mediated viral attachment but rather those involved in the inhibition of downstream events such as virus penetration or uncoating.

Analysis of inter-serotypic structural relationships of infectious bursal disease virus using detergent solubilization and radioimmunoprecipitation with monoclonal antibodies.

Monoclonal antibodies (MAbs) neutralizing only the infectious bursal disease virus strains (IBDV) belonging to serotype 1 also immunoprecipitated the heterologous major antigenic proteins of serotype 2 IBDV. Detergent-solubilization followed by radioimmunoprecipitation assays (RIPA) using the MAbs revealed structural similarities between the conformation-dependent antigenic determinants of IBDV of the two existing serotypes. The presence of non-ionic detergent Triton X-100 determined the binding of altered proteins by MAbs in RIPA.

Radioimmunoprecipitation of avian reovirus polypeptides using virus-specific IgM and IgG murine monoclonal and chicken polyclonal antibodies.

A simple and improved procedure for radioimmunoprecipitation (RIPA) for the identification of major immunogenic proteins of avian reovirus using murine monoclonal and chicken polyclonal antibodies is described. Bacterial proteins (Staphylococcus aureus-Protein A or Streptococcus species-Protein G) commonly used in RIPA procedures lack reactivities with low avidity monoclonals belonging to immunoglobulin (Ig)G or IgM subtypes, as well as avian Ig. Hence we used an indirect approach utilizing species-specific anti-IgM, IgG or chicken Ig antibodies followed by the precipitation of the immune complexes using Protein A. This improved the sensitivity of the RIPA enabling the antigenic analysis of the major antigenic proteins of avian reovirus. The results indicated that the virus is highly immunogenic in the natural host, chicken than in mice. Furthermore, there exists a direct correlation between a strong neutralizing antibody response and an increased precipitation of the sigma (sigma) proteins of the virus. The results also demonstrate a strong association between the conformational viral epitopes of the three classes of proteins, large (lambda), medium (mu) and small (sigma).

Cyclic alternating combination chemotherapy for small cell lung cancer.

Sixty-two patients with small cell carcinoma of lung received cyclic alternating non-cross-resistant combination chemotherapy. Radiation to the chest was given to all the patients. Patients were given a course of VP16, adriamycin and vincristine (VAV) followed by radiation (3,000 rads) to the chest and then a second course of VAV. Three weeks later, a course of cytoxan, CCNU, and methotrexate (CCM) was given (6 weeks). Subsequently, the treatment was cycled between two courses of VAV (6 weeks) and one course of CCM (6 weeks). Overall objective response rate of 73%, with 45% complete response, was noted. Overall median survival was 50 weeks, with 83 weeks for complete responders. Median survival for patients with regional disease was 58 weeks compared to 40 weeks for extensive disease. All the patients headed for complete response did so prior to receiving CCM. These results were not superior to conventional combination chemotherapy regimens.

Functional expression and peroxisomal targeting of rat urate oxidase in monkey kidney cells.

Humans and hominoid primates lack the enzyme urate oxidase, which catalyzes the oxidation of uric acid to allantoin. In rats and most other mammals, urate oxidase is present as a crystalloid core within the peroxisomes of liver parenchymal cells. To determine whether functionally active recombinantly expressed urate oxidase can be targeted to the peroxisome as well as display the crystalloid core-like structure, we expressed rat urate oxidase cDNA in African green monkey kidney cells (CV-1 cells) under the control of a cytomegalovirus promoter. Cell lines stably expressing urate oxidase were isolated. Northern blot analysis revealed a 1.3-kb transcript and immunoblot analysis confirmed the presence of urate oxidase in the stably transfected cells. The recombinant urate oxidase expressed in CV-1 cells was functionally active. Immunofluorescence microscopy revealed that the expressed protein was visualized as discrete granules in the cytoplasm. Electron microscopy and immunocytochemical localization studies showed that the recombinantly expressed protein formed distinct crystalloid core structures with bundles of tubules within single membrane limited cytoplasmic organelles. On cross section, the recombinant urate oxidase tubular structures are arranged as circles of 10 surrounding a slightly larger circle. This arrangement is reminiscent of urate oxidase-containing cores in rat liver peroxisomes. Immunocytochemical studies confirmed that the recombinantly expressed urate oxidase is correctly targeted to the catalase-containing peroxisomes in these CV-1 cells.

Pain and loss of function in head and neck cancer survivors.

Head and neck cancers are relatively uncommon malignancies and the characteristics of pain and functional impairments in survivors are not well studied. To characterize the incidence, location, severity, types and causes of pain; associated functional impairments, and pain management methods, the medical charts of 40 consecutive outpatients with biopsy-proven head and neck cancers were reviewed. Pain was severe in 52% (N = 21), and was located near sites of tumor origin. Pain was caused by tumor recurrence in 35% (N = 14), treatment sequelae in 30% (N = 12), multiple etiologies in 25% (N = 10), and unrelated causes in 10% (N = 4). Pains were mixed nociceptive and neuropathic pain in 37.5% (N = 15), nociceptive pain in 32.5% (N = 13), myofascial in 13.0% (N = 6), neuropathic in 7.5% (N = 3); and other mixed types in 7.5% (N = 3). Despite the high prevalence of dysphagia (82%), 60% used orally administered opioid-nonopioid analgesics. Physical disfigurement (87.5%; N = 35), dysphagia (62.5%, N = 25), and jaw dysfunction (40.0%; N = 16) were the most frequent physical impairments. Multiple regression analysis showed that the presence of skull base or mandibular bone involvement had significant influence on the severity of pain (P = 0.03, adjusted R2 0.25) We conclude that pain in head and neck cancer can be chronic, severe, and persistent despite completion of oncologic treatment.

Medical decision-making in a patient with a history of cancer and chronic non-malignant pain.

The selection of cancer pain treatment modalities depends on careful assessment to establish the pathophysiology of the pain complaint. Treatment may consist of a single modality--e.g., pharmacotherapy--or multiple modalities--e.g., pharmacotherapy, anesthetic intervention, and radiotherapy for bone pain. Cancer patients may present with pain and multiple concomitant medical problems related to their primary neoplastic disease, complications of cancer treatment, or unrelated conditions including preexisting pain of nonmalignant origin. We present the case of a patient with new onset of pain superimposed on chronic nonmalignant pain. This case emphasizes the need for careful assessment and the close cooperation required between the pain consultant and the referring oncology staff to make optimal treatment decisions in the context of a complex medical illness.

Subarachnoid neurolytic block under general anesthesia in a 3-year-old with neuroblastoma.

CASE REPORT: A 3-year-old boy with neuroblastoma complained of severe pain in the left lower extremity. Pharmacologic management had previously been attempted, but severe pain continued, and further upward titration was complicated by sedative effects. METHODS AND RESULTS: Because the focus of treatment had become the controlling of pain, a lumbosacral subarachnoid neurolytic block was performed under general anesthesia. One-time neurolysis was more acceptable to the family than a procedure like epidural analgesia, that requires greater management. Contrast medium was used to monitor the spread of the neurolytic. An epidural catheter was inserted during the neurolytic block procedure for possible future use. The short-term results were good--pain reports and opioid doses decreased greatly, although with increased incontinence. The boy had new abdominal distention and pain 5 days after neurolysis. Opioid doses and sedatives were increased. He died 3 days later.

Dose response of ethanol ingestion on antioxidant defense system in rat brain subcellular fractions.

This study investigated the response of the antioxidant defense system in brain subcellular fractions after oral graded doses of ethanol to rat. Four groups of male Fischer-344 rats were orally administered saline, ethanol 2 g, 4 g, and 6 g/kg, respectively, and sacrificed 1 hour post treatment. Brain cytosol, synaptosomes, microsomes and mitochondria were separated by density gradient differential centrifugation and assayed for antioxidant system. A significant and dose-dependent-decrease in superoxide dismutase (SOD) activity was observed in all brain subcellular fractions. Catalase (CAT) activity was significantly decreased in brain mitochondria (67% and 80% of control) at higher doses of ethanol; whereas, CAT activity was significantly increased in cytosol, synaptosomes and microsomes. Glutathione peroxidase (GSH-Px) activity was significantly increased in all brain subcellular fractions except in cytosol at higher dose of ethanol. Malondialdehyde (MDA) content was significantly increased in all brain subcellular fractions showing dose response of ethanol-induced oxidative stress. The increase in MDA levels in the brain synaptosomes and microsomes were higher at 6 g dose of ethanol (155% and 163% of control) when compared to mitochondria and cytosol. Glutathione (GSH) levels were significantly increased in brain cytosol and microsomes at higher dose of ethanol (164% and 159% of control); whereas, the GSH concentration was significantly decreased in brain synaptosomes and mitochondria. The antioxidant enzyme (AOE) activity ratios (GSH-Px/SOD and GSH-Px + CAT/SOD) were dose dependently increased in all brain subcellular fractions, particularly in synaptosomes. The GSH/GSSG ratio was dose dependently increased in brain microsomes. The perturbations in the antioxidant defense system and enhanced lipid peroxidation following graded doses of ethanol ingestion indicate a dose-dependent-oxidative 2133stress response in brain subcellular compartments of rats.

Autoantibodies to IA-2 and GAD65 in patients with type 2 diabetes mellitus of varied duration: prevalence and correlation with clinical features.

OBJECTIVE: To determine the prevalence of autoantibodies to IA-2 (IA-2Ab) and glutamic acid decarboxylase (GADAb) in type 2 diabetes, their relationship to disease duration, and their importance in management decisions. METHODS: We undertook a study of 101 patients with type 2 diabetes (defined as nonketotic hyperglycemia at diagnosis) of varied duration (median, 4 years). Results were compared with those from 36 patients with type 1 diabetes also of varied duration (median, 2 years). IA-2Ab and GADAb were measured by radioligand-binding assays with use of in vitro-synthesized, 35S-labeled antigens. RESULTS: Of the 101 patients with type 2 diabetes, 20 (20%) were positive for GADAb; only 4 of these 20 were positive for IA-2Ab. In comparison, 75% of patients with type 1 diabetes were positive for GADAb, IA-2Ab, or both (P<0.0001). The coincidence of IA-2Ab positivity in GADAb-positive patients with type 2 diabetes was significantly lower than in patients with type 1 diabetes (20% versus 73%, respectively; P = 0.002). All four IA-2Ab- and GADAb-positive patients with type 2 diabetes required insulin and were younger than those positive for GADAb alone (P = 0.018). GADAb positivity in patients with type 2 diabetes was highly associated with insulin requirement (P = 0.004), with an odds ratio of 5.8 in predicting insulin dependence. Among patients with type 2 diabetes receiving insulin therapy, disease duration was significantly shorter (P = 0.025) and body mass index was significantly lower (P<0.001) in GADAb-positive versus GADAb-negative patients. In contrast to type 1 diabetes, in which GADAb values were negatively correlated with disease duration (r = -0.34; P = 0.044), no significant correlation with disease duration was observed in type 2 diabetes (r = -0.166; P = 0.48). CONCLUSION: Irrespective of duration of disease, measurement of IA-2Ab and GADAb can help to identify those patients with type 2 diabetes most likely to require insulin therapy.