A pilot study comparing hearing thresholds of soldiers at induction and after completion of one year in high altitude area.

BACKGROUND: Despite so much research in high altitude area, our existing knowledge is still lacking on otological effects of long-term stay in high altitude. This pilot study was conducted to compare the hearing thresholds of army soldiers at induction and after completion of one year in high altitude area (HAA). METHODS: Hearing thresholds of 433 soldiers posted in HAA were recorded using pure tone audiometry at the time of induction and second thresholds after one year of stay in high altitude for frequencies of 500Hz, 1KHzs, 2 KHzs and 4 KHzs. The two sets of hearing thresholds for air conduction were compared using paired "t" test for any statistical significance. RESULTS: The mean Pure Tone Audiometry (PTA) thresholds for 433 left ears worsened from 9.43dB to 9.65dB at 500 HZs ; 14.02dB to 14.32dB at 1 KHZs ; 15.04dB to 16.09dB at 2KHzs and 18.63dB to 22.59dB at 4 KHZs. Similarly for right ear, PTA thresholds worsened from 9.43dB to 9.69dB at 500HZs; 13.95dB to 14.34dB at 1 KHZs; 15.38dB to 17.26dB at 2 KHZs and from 18.59dB to 23.06dB at 4KHZs. These results are found to be statistically significant (p<0.05) for all frequencies. CONCLUSION: This pilot study shows deterioration of hearing thresholds in tested frequencies in both ears after a long stay (one year) in high altitude area. We recommend further structured research on otologic effect of long term stay in high altitude.

Twelve tips for establishing a new medical school.

Many new medical programs have been established during the last 20 years, and this trend seems set to continue as the health care needs of the world's populations become more complex and demand increases for more physicians to provide the necessary health care. In this paper, we address how best to establish a new medical school, based on our experiences in new ventures in several countries. Success requires a combination of boldness of vision, support from many stakeholder groups, adequate financial and human resources, educational expertise, confidence, patience, and persistence.

Concurrent infection of Bluetongue and Peste-des-petits-ruminants virus in small ruminants in Haryana State of India.

Bluetongue (BT) and peste-des-petits-ruminants (PPR) are major transboundary diseases of small ruminant, which are endemic in India. Testing of bluetongue virus (BTV) and peste-des-petits-ruminants virus (PPRV) from recent outbreaks (2015-2016) in different regions of Haryana State of India revealed that 27.5% of the samples showed the presence of dual infection of BTV and PPRV. Analysis of Seg-2 of BTV (the serotype-determining protein) showed the presence of BTV-12w in several isolates. However, analysis of N gene fragment amplicons showed that viruses belong to lineage IV were most closely related to a pathogenic strain of PPRV from Delhi. This is the first report of co-circulation of PPRV lineage IV and bluetongue virus serotype 12 in the state.

p28, a first in class peptide inhibitor of cop1 binding to p53.

BACKGROUND: A 28 amino-acid (aa) cell-penetrating peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen Pseudomonas aeruginosa, produces a post-translational increase in p53 in cancer cells by inhibiting its ubiquitination. METHODS: In silico computational simulations were used to predict motifs within the p53 DNA-binding domain (DBD) as potential sites for p28 binding. In vitro direct and competitive pull-down studies as well as western blot and RT-PCR analyses were used to validate predictions. RESULTS: The L1 loop (aa 112-124), a region within the S7-S8 loop (aa 214-236) and T140, P142, Q144, W146, R282 and L289 of the p53DBD were identified as potential sites for p28 binding. p28 decreased the level of the E3 ligase COP1 >80%, in p53wt and p53mut cells with no decrease in COP1 in p53dom/neg or p53null cells. Brief increases in the expression of the E3 ligases, TOPORS, Pirh2 and HDM2 (human double minute 2) in p53wt and p53mut cells were in response to sustained increases in p53. CONCLUSION: These data identify the specific motifs within the DBD of p53 that bind p28 and suggest that p28 inhibition of COP1 binding results in the sustained, post-translational increase in p53 levels and subsequent inhibition of cancer cell growth independent of an HDM2 pathway.

A first-in-class, first-in-human, phase I trial of p28, a non-HDM2-mediated peptide inhibitor of p53 ubiquitination in patients with advanced solid tumours.

BACKGROUND: This first-in-human, phase I clinical trial of p28 (NSC745104), a 28-amino-acid fragment of the cupredoxin azurin, investigated the safety, tolerability, pharmacokinetics and preliminary activity of p28 in patients with p53(+) metastatic solid tumours. METHODS: A total of 15 patients were administered p28 i.v. as a short infusion three times per week for 4 weeks followed by a 2-week rest under an accelerated titration 3+3 dose escalation design until either a grade 3-related adverse event occurred or the maximum tolerated dose (MTD) was reached. Single-dose and steady-state serum pharmacokinetics were characterised. Assessments included toxicity, best objective response by RECIST 1.1 Criteria, and overall survival. RESULTS: No patients exhibited any dose-limiting toxicities (DLTs), significant adverse events or exhibited an immune response (IgG) to the peptide. The No Observed Adverse Effect Level (NOAEL) and MTD were not reached. Seven patients demonstrated stable disease for 7-61 weeks, three a partial response for 44-125 weeks, and one a complete response for 139 weeks. Three patients are still alive at 158, 140, and 110 weeks post therapy completion. CONCLUSION: p28 was tolerated with no significant adverse events. An MTD was not reached. Evidence of anti-tumour activity indicates a highly favourable therapeutic index and demonstrates proof of concept for this new class of non-HDM2-mediated peptide inhibitors of p53 ubiquitination.

Are medical student results affected by allocation to different sites in a dispersed rural medical school?

INTRODUCTION: As medical education becomes more decentralised, and greater use is made of rural clinical schools and other dispersed sites, attention is being paid to the quality of the learning experiences across these sites. This article explores this issue by analysing the performance data of 4 cohorts of students in a dispersed clinical school model across 4 sites. The study is set in a newly established medical school in a regional area with a model of dispersed education, using data from the second to fifth cohorts to graduate from this school. METHODS: Summative assessment results of 4 graduating cohorts were examined over the final 2 years of the course. Two analyses were conducted: an analysis of variance of mean scores in both years across the 4 sites; and an analysis of the effect of moving to different clinical schools on the students' rank order of performance by use of the Kruskal-Wallis test. RESULTS: Analysis revealed no significant difference in the mean scores of the students studying at each site, and no significant differences overall in the median ranking across the years. Some small changes in the relative ranking of students were noticed, and workplace-based assessment scores in the final year were higher than the examination-based scores in the previous year. CONCLUSIONS: The choice of clinical school site for the final 2 years of an undergraduate rural medical school appears to have no effect on mean assessment scores and only a minor effect on the rank order of student scores. Workplace-based assessment produces higher scores but also has little effect on student rank order. Further studies are necessary to replicate these findings in other settings and demonstrate that student learning experiences in rural sites, while popular with students, translate into required learning outcomes, as measured by summative assessments.

Rural internships for final year students: clinical experience, education and workforce.

INTRODUCTION: The James Cook University School of Medicine is the only complete medical school in northern Australia, and it has a mission to prepare graduates to meet the unique needs of the region with a particular emphasis on rural, remote, Indigenous and tropical health. Eight-week 'rural internships' have been undertaken by all sixth-year medical students at James Cook University since 2005. Each student had previously completed at least 12 weeks of structured rural placements in years 2 and 4, as well as other core teaching in rural health including the year 2 subject, 'Rural, Remote, Indigenous and Tropical Health'. Students worked in rural hospitals across northern Australia developing and practising clinical skills under the supervision of senior staff. Students undertook full-time inpatient and outpatient responsibilities under supervision, being rostered for after-hours work with appropriate support. Assessment involved a learning portfolio, including multi-source feedback from peers, supervisors and patients, and a population health project and a telephone referral exercise. METHODS: This article describes the development, implementation and assessment of the first years of the program, from 2005 to 2007. Evaluation included student questionnaires, site visits and interviews, and follow-up teleconferences with preceptors. RESULTS: The rural internship provides senior medical students with valuable experience by active participation in the healthcare team. Students reported a rich and varied clinical experience. Students accept limited supervised responsibility and further their ability and confidence to undertake the role of the intern. Importantly, they proved not to be a burden to the system. This rotation therefore appears to meet educational needs without compromising the local workforce (and indeed may add to it). Students felt welcomed by their communities and enjoyed the social and cultural aspects of their attachment, as well as the clinical aspects and the opportunity to further their understanding of rural communities, rural health care and the healthcare team. Preparation of the students, the preceptors and the communities emerged as a key element of success. CONCLUSION: This model extends and enhances the traditional apprenticeship model by its rural focus and distributed nature, and involvement of the entire student cohort. In addition, the contribution to patient care by senior students and junior doctors enables a consultant-registrar-resident model, in which experienced rural doctors function as consultants providing advice, support and tuition rather than predominantly face-to-face patient care. This approach also provides a means to address an emerging paradox: rural preceptors and communities want to teach students, appreciating the long-term workforce implications, but are increasingly constrained by resources, particularly time. Similar innovative approaches should be explored in other settings.

Resveratrol inhibits rhabdomyosarcoma cell proliferation.

Rhabdomysarcoma is the most common soft tissue tumour in children under the age of 15. Although the introduction of multimodal treatment programmes, including chemotherapy, radiation therapy and excision have increased the overall survival, the chemotherapeutic agents currently used for the treatment of rhabdomyosarcoma exhibit considerable toxicity. The aim of this study was to investigate the effects and possible mechanism(s) of action of resveratrol on human embryonal rhabdomyosarcoma (RD) cells. Resveratrol is a natural polyphenolic compound produced in a number of edible plants and has received considerable attention as a potential chemopreventive and/or chemotherapeutic agent against various types of cancers. In the present study, resveratrol was shown to inhibit cell proliferation of RD cells in a dose-dependent manner with an IC50 of 48.1 micromol/l and induce an arrest in the S/G2 phase of the cell cycle. As evident from immunocytochemical data, resveratrol treatment increased the size of the RD cells. Furthermore, resveratrol treatment resulted in a significant downregulation of cyclin B expression as demonstrated by western blot analyses. In conclusion, the present study shows that resveratrol exerts a strong inhibition of rhabdomyosarcoma cell proliferation in part by arresting cells in S/G2 phase of the cell cycle. These findings warrant further investigation to establish potential use of resveratrol as a relatively non-toxic chemotherapeutic agent for the treatment of rhabdomyosarcoma.

Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity.

Azurin is a copper-containing protein involved in electron transfer during denitrification. We reported recently that purified azurin demonstrates cytotoxicity to macrophages by forming a complex with the tumour-suppressor protein p53, thereby stabilizing it and enhancing its function as an inducer of proapoptotic activity (Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T. K., and Chakrabarty, A. M. 2002, Infect Immun70: 7054-7062). It is, however, not known whether the oxidoreductase (redox) activity of azurin or the involvement of copper is important for its cytotoxicity. We have isolated apo-azurin devoid of copper and site-directed mutants that are redox negative because of either replacement of a cysteine residue (Cys-112) involved in co-ordination with copper or mutational replacement of two methionine residues (Met-44 and Met-64) that are present in the hydrophobic patch of azurin and allow interaction of azurin with its redox partner cytochrome c551. We demonstrate that, although the wild type (wt) and the Cys-112 Asp mutant azurin can form complexes with the tumour-suppressor protein p53 and generate high levels of reactive oxygen species (ROS), the redox-negative Met-44LysMet-64Glu mutant azurin is defective in complex formation with p53, generates low levels of ROS and lacks appreciable cytotoxicity towards macrophages. Thus, complex formation with p53 and ROS generation, rather than azurin redox activity, are important in the cytotoxic action of azurin towards macrophages.

The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53.

Two redox proteins, azurin and cytochrome c(551) elaborated by Pseudomonas aeruginosa, demonstrate significant cytotoxic activity towards macrophages. Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis. Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin. Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level. A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity. Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein.

In vitro transformation of human congenital naevus to malignant melanoma.

The incidence of melanoma is estimated to be growing at the second fastest rate among all cancers in the United States. The progression of the melanocyte to a malignant melanoma involves various sequential steps: development of benign naevocellular naevus, preneoplastic dysplastic naevus, primary melanoma, and metastatic melanoma. Despite these clearly defined stages, very little is known about the molecular events leading to melanoma progression. We established a human congenital naevus cell line (UISO-CMN-1). UISO-CMN-1 cells were confirmed to have melanocytic origin by S100 immunoreactivity and the presence of melanin granules and melanosomes. UISO-CMN-1 cells, even though they showed structural and numerical abnormalities in karyotype, were non-tumorigenic when transplanted into athymic mice. However, following frequent exposure to ultraviolet C radiation, UISO-CMN-1 cells acquired tumorigenic potential. Transformation of UISO-CMN-1 cells into tumorigenic cells was accompanied by induction of ganglioside-2 expression without any significant changes in cellular ganglioside-3. These transformed and non-transformed UISO-CMN-1 cell lines can serve as excellent research tools for studying the molecular changes associated with melanoma development and progression, and for identifying agents that might prevent development of malignant melanoma.

Betulinic acid reduces ultraviolet-C-induced DNA breakage in congenital melanocytic naeval cells: evidence for a potential role as a chemopreventive agent.

Melanoma transformation progresses in a multistep fashion from precursor lesions such as congenital naevi. Exposure to ultraviolet (UV) light promotes this process. Betulinic acid (BA) was identified by our group as a selective inhibitor of melanoma that functions by inducing apoptosis. The present study was designed to investigate the effect of BA and UV-C (254 nm) on cultured congenital melanocytic naevi (CMN) cells, using the single-cell gel electrophoresis (comet) assay to detect DNA damage. Exposure to UV light induced a 1.7-fold increase in CMN cells (P = 0.008) when compared with controls. When a p53 genetic suppressor element that encodes a dominant negative polypeptide (termed GSE56) was introduced into the CMN cells, the transfected cells were more sensitive to UV-induced DNA breakage. This suggests that p53 can protect against UV-induced DNA damage and subsequent melanoma transformation. Pretreatment with BA (3 microm) for 48 h resulted in a 25.5% reduction in UV-induced DNA breakage in the CMN cells (P = 0.023), but no changes were observed in the transfected cells. However, Western blot analysis revealed no changes in the p53 or p21 levels in BA-treated cells, suggesting that BA might mediate its action via a non-p53 pathway. These data indicate that BA may have an application as a chemopreventive agent in patients with congenital naevi.

Prognostic significance of periodic acid-Schiff-positive patterns in primary cutaneous melanoma.

The patterns of periodic acid-Schiff (PAS) staining of extracellular matrix in histological sections of certain melanomas may be predictive of outcome. Recent in vitro and molecular genetic data suggest that the appearance of these patterns in both uveal and cutaneous melanoma is a function of aggressive tumor cells. We studied 96 patients with primary cutaneous melanomas treated at the University of Illinois at Chicago who were monitored for disease-free survival. Survival probabilities were determined by Kaplan-Meier estimates, and prognostic factors were evaluated by multivariate analysis. By univariate analysis, there was a significant decrease in disease-free survival among patients whose tumors contained parallel with cross-linking or network patterns (PXNs; P = 0.0070). Stepwise regression with Cox models that included the combinations of the PAS-positive patterns, tumor thickness, female gender, ulceration, and age yielded a model with thickness and the PAS-positive parallel with cross-linking or networks. Despite the relatively small sample size in this study, the detection of the PAS-positive parallel with cross-linking or networking in cutaneous melanoma was associated with a decrease in disease-free outcome. Additional studies of the prognostic significance of these patterns is warranted on larger data sets.

A novel use for the comet assay: detection of topoisomerase II inhibitors.

BACKGROUND: The simple and quick comet assay can quantitatively detect DNA cleavage in cells. This study aimed to determine whether the comet assay could be used to detect topoisomerase (topo) II inhibitors. MATERIALS AND METHODS: HT-29 colon cancer cells were pre-incubated with aclarubicin, a topo II antagonist, then treated with topo II poisons: etoposide (VP-16), teniposide (VM-26), 4'-(acridinylamino) methansulfon-m-anisidide (m-AMSA) and adriamycin (doxorubicin). We also tested a topo I poison (camptothecin) and a microtubule depolymerization inhibitor (taxol). RESULTS: Aclarubicin significantly reduced DNA cleavage induced by topo II poisons, but not that induced by camptothecin. In HL-60/MX2 cells (containing no topo II beta and reduced topo II alpha), DNA breakage induced by topo II poisons was lower. Also, aclarubicin antagonized topo I-mediated camptothecin-induced DNA cleavage in these resistant cells. CONCLUSIONS: The comet assay can be used to detect topo II poisons in cultured cells. Also, aclarubicin has a dual topo I and topo II antagonism, with "preferential antagonism" of topo II when topo II beta catalytic activity is normally expressed.

Micropthalmia transcription factor: a new prognostic marker in intermediate-thickness cutaneous malignant melanoma.

Micropthalmia transcription factor (Mitf) is involved in melanocyte development and differentiation. The current study was undertaken to determine whether there is a relationship between Mitf expression and survival in patients with intermediate-thickness (1.0-4.0 mm) melanoma. Original paraffin blocks or slides of the primary tumor were accessible in 63 such patients. Mitf expression was evaluated by immunocytochemistry and analyzed visually. Slides were graded as follows according to the percentage of cells whose nuclei stained positive for Mitf: (a) 0, 0%; (b) +1, 1-25%; (c) +2, 26-50%; (d) +3, 51-75%; and (e) +4, > 75%. Median follow-up was 50 months. Mean thickness was 2.2 +/- 0.7 mm. Mean overall survival was 171.90 +/- 13.12 months. Mean disease-free survival was 168.53 +/- 13.96 months. Fifty-two melanomas (82.5%) stained positive for Mitf. By univariate analysis, mean overall survival and disease-free survival in patients whose melanomas did not express Mitf were 80.89 +/- 17.98 months (median, 51 months) and 71.36 +/- 19.87 months (median, 40 months), respectively. This compares with 187.90 +/- 13.41 months (median, not reached) and 186.78 +/- 13.84 months (median, not reached), respectively, for patients whose melanomas expressed Mitf (P = 0.0086 and P = 0.0054). These findings persisted in multivariate analysis. In addition, patients with > 50% Mitf expression had significantly fewer nodal metastases after node dissection than patients with < or = 50% Mitf expression (P = 0.04). Our data suggest that Mitf may be a new molecular prognostic marker in patients with intermediate-thickness melanoma.

Engineering and characterization of a single-chain antibody fragment (scFV1924) against the human sarcoma-associated antigen p102.

BACKGROUND: Monoclonal Antibody (MAb) 19-24 against the human sarcoma-associated antigen (SAA) p102 was previously characterized, and it demonstrated potential for sarcoma immunodiagnosis and immunotherapy. Due to the limitations of using intact murine MAbs in human clinical settings, we engineered a single-chain antibody fragment of MAb 19-24 (scFV1924). MATERIALS AND METHODS: Monoclonal Antibody (MAb) 19-24 against the human sarcoma-associated antigen (SAA) p102 was previously characterized, and it demonstrated potential for sarcoma immunodiagnosis and immunotherapy. Due to the limitations of using intact murine MAbs in human clinical settings, we engineered a single-chain antibody fragment of MAb 19-24 (scFV1924). RESULTS: The results from competition radioimmunoassay showed that binding of MAb 19-24 to SAA p102 on sarcoma cell membranes was inhibited by scFV1924. CONCLUSIONS: This indicates the potential of further application of the scFV1924 in human sarcoma immunotherapy.

Induction of differentiation by 1alpha-hydroxyvitamin D(5) in T47D human breast cancer cells and its interaction with vitamin D receptors.

The role of the active metabolite of vitamin D, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), in cell differentiation is well established. However, its use as a differentiating agent in a clinical setting is precluded due to its hypercalcaemic activity. Recently, we synthesised a relatively non-calcaemic analogue of vitamin D(5), 1alpha-hydroxyvitamin D(5) (1alpha(OH)D(5)), which inhibited the development of carcinogen-induced mammary lesions in culture and suppressed the incidence of chemically induced mammary carcinogmas in rats. In the present study, we determined the differentiating effects of 1alpha-(OH)D(5) in T47D human breast cancer cells and compared its effects with 1,25(OH)(2)D(3). Cells incubated with either 10 or 100 nM of the analogues inhibited cell proliferation in a dose-dependent manner, as measured by the dimethylthiazolyl-2,5-diphenyltetrazolium bromide (MTT) assay. Similar growth-inhibitory effects were also observed for MCF10(neo) cells. Both vitamin D analogues induced cell differentiation, as determined by induction of casein expression and lipid production. However, MCF10(neo) cells failed to respond to either vitamin D analogue and did not undergo cell differentiation. Since the cell differentiating effect of vitamin D is considered to be mediated via the vitamin D receptor (VDR), we examined the induction of VDR using reverse transcriptase-polymerase chain reaction (RT-PCR) in both cells. The results showed that, in T47D cells, both 1,25(OH)(2)D(3) and 1alpha(OH)D(5) induced VDR in a dose-dependent manner. Moreover, both analogues of vitamin D upregulated the expression of vitamin D response element-chloramphenicol acetyl transferase (VDRE-CAT). These results collectively indicate that 1alpha-(OH)D(5) may mediate its cell-differentiating action via VDR in a manner similar to that of 1,25(OH)(2)D(3).

Genistein induces apoptosis and topoisomerase II-mediated DNA breakage in colon cancer cells.

The present study was undertaken to determine if (a) genistein induces topo II-mediated DNA damage in HT-29 colon cancer cells; and (b) if this damage is required to induce apoptosis. DNA damage was evaluated using the comet assay. Apoptosis was determined by the ethidium bromide/acridine orange staining technique. DNA breakage was noted within 1 h of treatment. Apoptosis was only induced with high concentrations (>/=60 microM) of genistein. Marked inhibition of HT-29 cell growth was evident at concentrations ranging from 60 to 150 microM. This was associated with a cell cycle arrest at G(2)/M. Similar findings were obtained in SW-620 and SW-1116 colon cancer cell lines. Aclarubicin, a topo II antagonist, reduced genistein-induced DNA breaks but did not reduce apoptosis. These data suggest that, in colon cancer cells, topo II serves as the enzymatic target of genistein. Furthermore, topo II-mediated DNA cleavage is not required for the induction of apoptosis.