Growth differentiation factor-15 encodes a novel microRNA 3189 that functions as a potent regulator of cell death.

According to the latest version of miRBase, approximately 30% of microRNAs (miRNAs) are unique to primates, but the physiological function of the vast majority remains unknown. In this study, we identified miR-3189 as a novel, p53-regulated, primate-specific miRNA embedded in the intron of the p53-target gene GDF15. Antagonizing miR-3189 increased proliferation and sensitized cells to DNA damage-induced apoptosis, suggesting a tumor suppressor function for endogenous miR-3189. Identification of genome-wide miR-3189 targets revealed that miR-3189 directly inhibits the expression of a large number of genes involved in cell cycle control and cell survival. In addition, miR-3189 downregulated the expression of multiple p53 inhibitors resulting in elevated p53 levels and upregulation of several p53 targets including p21 (CDKN1A), GADD45A and the miR-3189 host gene GDF15, suggesting miR-3189 auto-regulation. Surprisingly, miR-3189 overexpression in p53-/- cells upregulated a subset of p53-targets including GDF15, GADD45A, and NOXA, but not CDKN1A. Consistent with these results, overexpression of miR-3189 potently induced apoptosis and inhibited tumorigenicity in vivo in a p53-independent manner. Collectively, our study identified miR-3189 as a novel, primate-specific miRNA whose effects are mediated by both p53-dependent and p53-independent mechanisms. miR-3189 may, therefore, represent a novel tool that can be utilized therapeutically to induce a potent proapoptotic effect even in p53-deficient tumors.

A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis.

Most p53 mutations in human cancers are missense mutations resulting in a full-length mutant p53 protein. Besides losing tumor suppressor activity, some hotspot p53 mutants gain oncogenic functions. This effect is mediated in part, through gene expression changes due to inhibition of p63 and p73 by mutant p53 at their target gene promoters. Here, we report that the tumor suppressor microRNA let-7i is downregulated by mutant p53 in multiple cell lines expressing endogenous mutant p53. In breast cancer patients, significantly decreased let-7i levels were associated with missense mutations in p53. Chromatin immunoprecipitation and promoter luciferase assays established let-7i as a transcriptional target of mutant p53 through p63. Introduction of let-7i to mutant p53 cells significantly inhibited migration, invasion and metastasis by repressing a network of oncogenes including E2F5, LIN28B, MYC and NRAS. Our findings demonstrate that repression of let-7i expression by mutant p53 has a key role in enhancing migration, invasion and metastasis.

Inhibition controls for qualitative real-time PCR assays: are they necessary for all specimen matrices?

A retrospective analysis of 386,706 specimens representing a variety of matrix types used in qualitative real-time PCR assays determined the overall inhibition rate to be 0.87% when the inhibition control was added preextraction to 5,613 specimens and 0.01% when the inhibition control was added postextraction but preamplification in 381,093 specimens. Inhibition rates of ≤ 1% were found for all specimen matrix types except urine and formalin-fixed, paraffin-embedded tissue.

Real-time PCR in clinical microbiology: applications for routine laboratory testing.

Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.

Stability of premixed syringes of diamorphine and hyperbaric bupivacaine.

BACKGROUND: It is common clinical practice to add diamorphine to heavy bupivacaine when performing spinal anaesthesia for either obstetric or general surgical procedures. If pre-filled syringes were available potential problems arising due to the wrong mixture being administered could be reduced, whilst also providing greater assurances of sterility and accuracy of dosage. It is therefore necessary to establish whether diamorphine 100 microg/mL is stable in solution with 0.5% hyperbaric bupivacaine, to allow production of pre-filled syringes for use in spinal anaesthesia. METHOD: Diamorphine hydrochloride was dissolved in water for injection, and added to hyperbaric bupivacaine then stored in 5-mL plastic syringes. Eleven syringes were stored at 40 degrees C/75% relative humidity, 25 degrees C/60% relative humidity and 7 degrees C for 90 days. Samples were taken at five time points for measurement of diamorphine and bupivacaine concentrations using high performance liquid chromatography. RESULTS: Diamorphine concentrations fell over the study period. No significant changes were observed the bupivacaine content of the samples. There was 10% degradation of diamorphine after 4 days at 40 degrees C, after 7 days at 25 degrees C, and after 26 days at 7 degrees C. CONCLUSION: Diamorphine is stable in hyperbaric bupivacaine at 7 degrees C for long enough to allow preparation of pre-filled syringes in advance (by hospital pharmacy aseptic units) for use in spinal anaesthesia.

Premixed solutions of diamorphine in ropivacaine for epidural anaesthesia: a study on their long-term stability.

BACKGROUND: Local anaesthetics and opioid mixtures are commonly used to provide anaesthesia or analgesia during the perioperative period. In order to facilitate their preparation and storage it is necessary to establish the stability of such solutions. METHODS: In our study, diamorphine was added to ropivacaine 0.2% 200-ml polybags to give a concentration of 25 micro g ml(-1) and to ropivacaine 1% 50-ml syringes to give a concentration of 45 micro g ml(-1). The polybags and syringes were stored at 40 degrees C, 21 degrees C and 4 degrees C for up to 120 days. Samples were taken during this period for measurement of diamorphine and ropivacaine content and pH of the solutions. RESULTS: We found that the storage temperature and the initial concentration influenced the rate of degradation of diamorphine in both the polybags and the syringes. In the syringes, 10% degradation of diamorphine [T (0.9)] was: 6 days at 40 degrees C, 16 days at 21 degrees C and 30 days at 4 degrees C. In the polybags, diamorphine T (0.9) was 6 days at 40 degrees C, 28 days at 21 degrees C and 70 days at 4 degrees C. CONCLUSIONS: It is feasible to manufacture such solutions in pharmacy aseptic units and to store them for up to 1 month for routine use in epidural infusions.

Laboratory diagnosis of viral hepatitis.

Both serologic and molecular assays are useful in the diagnosis of viral hepatitis. They may detect early infections before other signs of disease appear, differentiate acute from chronic infections, and detect persistence of viremia or verify development of immunity. Molecular assays may also be used to monitor responses to antiviral therapy, and in the future, be a primary method to screen blood and organ donors (NAT). EIA serologies are used to diagnose acute HAV infections or establish immune status. Similar immunoassays are used to detect HBV infections, verify persistence of antigenemia and degree of infectivity, and indicate immunity (including the response to vaccination). HBV molecular assays can shorten the diagnostic window period, verify persistence of viremia, including monitoring response to antiviral therapy, and be useful in NAT screening of donors. Molecular assays play a major role in HCV diagnosis where serologic tests can document past or present infection but cannot differentiate one from the other. A variety of molecular tests can be used as sensitive (and early) detectors of viremia (and serve as confirmatory tests for positive serologies and as donor NAT methods), document its persistence as an indicator of chronic infection, and monitor responses to antiviral therapy. Both qualitative and quantitative molecular assays are available, and their efficient use requires familiarity with the sensitivity and dynamic ranges of each method.

Topical anaesthesia for phacoemulsification surgery.

AIM: To assess the discomfort experienced by patients undergoing phacoemulsification under topical anaesthesia. METHODS: Thirty patients had a collagen contact lens, soaked in 4% lignocaine, placed on the cornea 23-89 min preoperatively. Immediately preoperatively, additional ophthetic drops were inserted. The patients underwent phacoemulsification surgery with the insertion of a foldable intraocular lens. This group was compared with 30 age- and sex-matched controls who underwent phacoemulsification and the insertion of a non-foldable lens under peribulbar anaesthesia. (8 mL of an 80:20 mix of 0.5% bupivacaine, 4% lignocaine and hyaluronidase). The pain perceived by patients was assessed on a verbal analogue scale (0-10) at administration of anaesthesia, perioperatively and 3-6 h postoperatively. RESULTS: Topical anaesthesia was less painful than peribulbar anaesthetic at administration (difference in mean pain score 1.2, P < 0.05, Wilcoxon paired signed rank test). Patients experienced more pain during the operation under topical anaesthesia (difference in means 0.63, P < 0.05). There was no difference between the groups postoperatively. CONCLUSION: Topical anaesthesia does not carry the risk of injection into, or injury of, the many delicate structures of the orbit. Because it provides acceptable levels of anaesthesia for phacoemulsification, it could be used more frequently.

The use of micro-dose aprotinin with continuous infusion in coronary artery bypass surgery.

BACKGROUND: To evaluate the efficacy of aprotinin at a dose far less than standard. EXPERIMENTAL DESIGN: Retrospective, case-control study. SETTING: community-based, teaching hospital PATIENTS: one hundred one patients undergoing primary, non-emergent, coronary artery bypass during two, six-month periods were studied. INTERVENTIONS: during the first period aprotinin was not administered, and these patients served as controls (n = 52). During the second period all patients received aprotinin via a micro-dose regimen (n = 49). MEASURES: postoperative bleeding and blood product usage served as determinants of efficacy. RESULTS: A significant difference existed in postoperative bleeding with the mean thoracic drain outputs being reduced in the aprotinin group both at 6 hours (p = 0.0003) and in total (p = 0.0004). This was further supported by significantly higher hematocrits (p = 0.03) on the first postoperative day in patients receiving aprotinin. Likewise, there was a significant reduction in total blood product exposures (p = 0.04) and platelet usage (p = 0.02) in the aprotinin group with a tendency towards decreased red cell usage. Further, when all patients with a hematocrit < or =30% prior to bypass were excluded, the significant reduction in total blood product exposures persisted (p = 0.04), and there was a significant reduction in red cell usage (p = 0.04) with a trend towards decreased platelet usage (p = 0.06) in the aprotinin group. CONCLUSIONS: Micro-dose aprotinin significantly reduces postoperative bleeding and blood product usage in primary, non-emergent, CABG patients.

Clinical comparison of the Treponema pallidum CAPTIA syphilis-G enzyme immunoassay with the fluorescent treponemal antibody absorption immunoglobulin G assay for syphilis testing.

Recently, a treponema-specific immunoglobulin G (IgG) enzyme immunoassay (EIA), the CAPTIA Syphilis-G (Trinity Biotech, Jamestown, N.Y.), has become available as a diagnostic test for syphilis. A total of 89 stored sera previously tested by the fluorescent treponemal antibody absorption (FTA-ABS) IgG assay were evaluated by the CAPTIA EIA. The FTA-ABS IgG procedure was performed by technologists unblinded to results of rapid plasmid reagin (RPR) testing of the same specimens. Borderline CAPTIA-positive samples (antibody indices of >/=0.650 and 0.900, the sample was considered positive. Thirteen of 89 (15%) samples had discrepant results. Compared to the FTA-ABS assay, the CAPTIA EIA had a sensitivity and specificity and positive and negative predictive values of 70.7, 97.9, 96.7, and 79.7%, respectively. In another analysis, discrepancies between results were resolved by repeated FTA-ABS testing (technologists were blinded to previous RPR results) and patient chart reviews. Seven CAPTIA-negative samples which were previously interpreted (unblinded) as minimally reactive by the FTA method were subsequently interpreted (blinded) as nonreactive. One other discrepant sample (CAPTIA negative and FTA-ABS positive [at an intensity of 3+], unblinded) was FTA negative with repeated testing (blinded). For the five remaining discrepant samples, chart reviews indicated that one patient (CAPTIA negative and FTA-ABS positive [minimally reactive], blinded) had possible syphilis. These five samples were also evaluated and found to be negative by another treponema-specific test, the Treponema pallidum microhemagglutination assay. Therefore, after repeated testing and chart reviews, 2 of the 89 (2%) samples had discrepant results; the adjusted sensitivity, specificity, and positive and negative predictive values were 96.7, 98.3, 96.7, and 98.3%, respectively. This study demonstrates that the CAPTIA IgG EIA is a reliable method for syphilis testing and that personnel performing tests which require subjective interpretation, like the FTA-ABS test, may be biased by RPR test results.

Should angiographically disease-free saphenous vein grafts be replaced at the time of redo coronary artery bypass grafting?

BACKGROUND: Controversy exists regarding the management of angiographically disease-free saphenous vein grafts at the time of redo coronary artery bypass grafting (CABG). Some authorities favor replacement of these disease-free grafts, arguing that occlusion is likely in the near future. Others believe that these grafts are "biologically privileged" and should not be replaced. METHODS: One hundred thirty-two consecutive patients (113 men, 19 women, aged 46 to 88 years, mean 67 years) underwent redo revascularization with one or more angiographically disease-free saphenous vein grafts at the time of redo CABG. Thirty-six patients had the disease-free grafts replaced (R) and 96 did not (NR). The mean interval from the first CABG was 9.25 years. RESULTS: Surgical mortality was comparable in the NR and R groups (5 of 96 or 5.2% versus 3 of 36 or 8.3%, respectively; p < 0.5). Survival at 1 and 3 years was higher in the NR group than the R group (98% versus 80%, and 95% vs. 66% respectively; p < 0.0001). Late myocardial infarction was less common in the NR group than in the R group (12 of 91 or 12.9% versus 12 of 33 or 36.4%; p < 0.003). Recurrent angina was less common in the NR than in the R group (21 of 91 or 23.1% versus 15 of 33 or 45.5%; p < 0.015). Cardiac hospitalization was required less commonly in the NR than in the R group (11 of 91 or 12.1% versus 12 of 33 or 36.4%; p < 0.002). In nondiseased grafts undergoing angiographic evaluation late after redo CABG, rate of new stenosis was lower in NR grafts than in R grafts (2 of 12 or 16.7% versus 2 of 3 or 66.7%; p < 0.05). CONCLUSIONS: With a conservative approach that does not replace nondiseased saphenous vein grafts at redo CABG (1) there is no increase in operative mortality, (2) good late survival is obtained, (3) clinical ischemia related to the NR saphenous vein grafts is uncommon, and (4) NR grafts continue to be patent. We conclude that disease-free vein grafts may not require routine replacement at redo CABG. A randomized study is required for definitive resolution.

Seroconversion to human herpesvirus 6 following liver transplantation is a marker of cytomegalovirus disease.

Human herpesvirus 6 (HHV-6) infection is common after transplantation; HHV-6 is known to interact with other viruses and induce immunosuppression. Whether HHV-6 plays a role in the occurrence of cytomegalovirus (CMV) infection after transplantation was investigated. In a cohort of 247 liver transplant recipients, HHV-6 seroconversion was identified as a significant risk factor for development of symptomatic CMV infection (P < .001), including CMV organ involvement (P < .001), even in the presence of the other significant risk factors: D+/R- CMV serologic status (P < .001) or use of OKT3 after transplantation (P = .002). Subgroup analysis indicated that HHV-6 seroconversion was significantly associated with symptomatic CMV infection in the D+/R+ but not in the D+/R- CMV serologic group (P < .001 and P = .11, respectively). These results indicate that HHV-6 seroconversion is a marker for CMV disease after transplantation and suggest that additional studies using more sensitive diagnostic techniques are warranted to determine the relationship between HHV-6 and CMV infection after transplantation.

Corticosteroid usage and binding to arginine: determinants of corticosteroid hypersensitivity.

Hypersensitivity to topical corticosteroids is a common cause of allergic contact dermatitis. The development of contact allergy is dependent on individual susceptibility, exposure to the potential allergen and the ability to penetrate the epidermis and react with epidermal protein. We looked at corticosteroid binding to arginine and relative usage of corticosteroids to see if these variables explain the number of allergic reactions seen to these structurally similar chemicals. A linear relationship was found between a measure of corticosteroid binding to arginine, the log of relative corticosteroid usage and the log of the relative number of corticosteroid allergies. Using multiple regression this association was significant (P = 0.01). Statistically, these two variables accounted for 73% of the variation in the results. Our results showed that the number of corticosteroid allergic reactions was dependent on usage and the intrinsic ability of the corticosteroid to degrade and bind to arginine. While total corticosteroid usage is unlikely to change, the prescription of individual corticosteroids with a reduced potential to degrade and bind to protein, but with equal efficacy, might reduce the overall prevalence of corticosteroid hypersensitivity.

Comparison of shell vials and conventional tubes seeded with rhabdomyosarcoma and MRC-5 cells for the rapid detection of herpes simplex virus.

Shell vials (SV) and conventional tubes (CT) were seeded with rhabdomyosarcoma (RD) and MRC-5 cells and inoculated with clinical specimens, and the systems were evaluated for the rapid diagnosis of herpes simplex virus (HSV) infections by detection of cytopathic effects (CPE) (for CT, for 7 days) and by using fluoresceinated monoclonal antibodies (for SV, 16 h postinoculation). Of 245 genital specimens (16 from males and 229 from females) 56 (23%) seeded with MRC-5 cells (14 type 1 and 42 type 2) and 55 (22%) seeded with RD cells were detected in CT; however, CPE were recognized in only 26 (46%) of the total HSV-positive cultures 1 day postinoculation. Forty-eight (86% sensitivity, MRC-5) and 46 (84% sensitivity, RD) HSV strains were detected immunologically in SV 16 h postinoculation. Early CPE in CT or fluorescent foci in SV were easier to detect in MRC-5 than in RD cell cultures. MRC-5 and RD cells were equally sensitive to infection with HSV. CT cell cultures were more sensitive than SV but less rapid for the detection of HSV infection (P less than 0.01). We recommend using SV for the rapid diagnosis of HSV infections, but in addition, CT must be inoculated with MRC-5 or RD to ensure maximum detection of this virus.

A system for the quantitative determination of hydrocarbons in human breath.

A method for the analysis of hydrocarbons in exhaled human breath samples has been developed and its quantitative performance optimized and exhaustively validated. The method involves preconcentration on a solid absorbent at 0 degree C and desorption at 250 degrees C to a packed column gas chromatograph. Calibrations for ethane and pentane are reproducible and linear over the concentration ranges found in human breath samples. The technique is now available for study of conditions, such as cystic fibrosis, in which an oxidative stress component in tissue injury is suspected.

Detection of Chlamydia trachomatis in genital specimens by the Chlamydiazyme test.

Cotton swabs were used to collect two specimens each from 416 patients (206 males, 210 females) attending a sexually transmitted disease clinic. The first swab was transported in Specimen Storage Reagent and extracted in Specimen Dilution Buffer for enzyme immunoassay by Chlamydiazyme (Abbott Laboratories); the second swab was extracted into 2SP and inoculated into McCoy cell cultures. In the first phase of the study (215 patients: 111 males, 114 females) enzyme immunoassay results were positive (optical density greater than or equal to 0.1) in 30 of 35 instances in which Chlamydia trachomatis was isolated (sensitivity, 86%). Of 18 false-positive enzyme immunoassay results, 15 (83%) were cervical swabs (specificity, 90%). In a phase II study, using a modified Chlamydiazyme kit, 201 patients were tested (95 males, 106 females). Of 41 chlamydial isolates, 8 were not detected by the Chlamydiazyme test (sensitivity, 81%). Only three positive Chlamydiazyme test results could not be confirmed by culture (specificity, 98%). Overall, Chlamydiazyme assay provided a rapid (4 h), sensitive, and specific assay for the detection of chlamydial antigens.

alpha,alpha-Diphenylsuccinimide: evaluation of anticonvulsant and hydrophobic properties.

The anticonvulsant potencies (ED50) of alpha,alpha-diphenylsuccinimide, phenytoin, and phenobarbital were evaluated in mice by a standard maximal electroshock technique. Potencies were expressed in terms of intraperitoneal dosage and blood and brain concentrations. Overt neurotoxicity (TD50) was assessed by the rotorod method. These data were compared with relative hydrophobicities for the above compounds and three others [carbamazepine, cyheptamide, and (diphenylacetyl)urea] taken from the literature. An approximate parabolic dependence of anticonvulsant potency on hydrophobicity was observed regardless of the means of expressing potency (intraperitoneal dosage, blood concentration, or brain concentration); approximate optimal hydrophobicities were in the range of 2.18-2.23 (log P). Calculated therapeutic indices (TD50/ED50) also displayed a parabolic dependence on hydrophobicity, while toxic potency (TD50) displayed a linear dependence (within the limited range of log P values studied). Implications of the parabolic dependence of anticonvulsant potency and linear dependence of toxic potency on hydrophobicity are discussed with respect to the possible mechanisms involved.

The stability of trifluorothymidine: hydrolysis in buffered aqueous solutions.

The decomposition of trifluorothymidine in aqueous solution, and borate and phosphate buffers has been studied using an h.p.l.c. method. In aqueous solution accelerated studies demonstrate that a storage life, to 10% loss, of more than 30 years at 4 degrees C is predicted, but steam sterilization causes extensive breakdown. Considerable differences occur in the kinetics and routes of decomposition in borate and phosphate buffers in the pH range 4-8. Hydroxide ion attack producing 5-carboxy-2'-deoxyuridine is suppressed in borate buffers. Complex kinetics of decomposition in phosphate buffer are found and possible explanations suggested. Steam sterilization in buffered solution also leads to unacceptable breakdown.