A simplified method for combined immunohistochemistry and in-situ hybridization in fresh-frozen,cryocut mouse brain sections

A method is described to perform combined immunohistochemistry and in situ hybridization in mouse brain sections. The protocol is specific to sections mounted on glass slides. In contrast to earlier methods that require either paraffin embedding or perfusion of the brain with paraformaldehyde, this protocol can be carried out on fresh-frozen, cryostat cut post-fixed sections. This simple and concise protocol increases the applicability of the technique as the RNAse-free immunodetection of antigen is useful by itself for immunologically identifying specific cells of interest and then examining gene expression in those cells using techniques such as real-time PCR and microarray analysis. The use of fresh-frozen, cryocut sections enables reliable detection of easily perturbable post-translational modifications such as phosphorylation and improves the quality of results obtained in subsequent in situ hybridization by reducing the background signal and interference from lower cell layers. Inducible transgenic mice that express either a dominant negative mutant form of the cAMP response element binding protein (mCREB) or CREB, in discrete brain regions, were used in this study. The combined immunohistochemistry and in situ hybridization protocol was used to examine colocalization of enkephalin or dynorphin mRNA, both downstream targets of CREB-mediated gene expression, in cells expressing transgenic mCREB or CREB.     

Randomized trial evaluating a new 0.5% fluorouracil formulation demonstrates efficacy after 1-, 2-, or 4-week treatment in patients with actinic keratosis

The efficacy and safety of a new 0.5% fluorouracil topical cream were compared with vehicle control for the treatment of actinic keratosis (AK). Active treatment applied once daily for 1, 2, or 4 weeks was more effective than vehicle control in achieving reduction from baseline in lesion counts and lesion clearance. Active treatment also resulted in significantly better global assessments of overall improvement. Treatment was effective regardless of the number of baseline lesions. Although longer treatment duration correlated with greater efficacy, treatment for 1, 2, or 4 weeks was effective. This new microsphere-based fluorouracil formulation was generally well tolerated; adverse events were primarily limited to facial irritation that resolved quickly after treatment. This new treatment provides a safe alternative to the topical fluorouracil formulations currently available for the 1-, 2-, or 4-week treatment of AK.     

Is unplanned return to the operating room a useful quality indicator in general surgery?

HYPOTHESIS: To test our hypothesis that unplanned return to the operating room (OR) is a useful quality indicator, we examined how often and for what reasons patients go back to the OR in a broad-based general surgery practice. DESIGN AND SETTING: Prospective cohort study at a rural tertiary care center. PATIENTS: Consecutive series of 3044 patients undergoing general surgery procedures in the OR between September 1, 1998, and March 31, 2000. Information about all postoperative adverse events occurring before discharge or within 30 days (whichever was longer) was collected prospectively. Unplanned return to the OR was defined as any secondary procedure required for a complication resulting directly or indirectly from the index operation. MAIN OUTCOME MEASURES: Unplanned return to the OR, mortality, and hospital charges. RESULTS: Overall, 107 (3.5%) had an unplanned return to the OR. A relatively small number of inpatient procedures accounted for a disproportionate share of unplanned reoperations, including colon resection (18% of total reoperations), renal transplant (9%), gastric bypass (6%), and pancreatic resection (6%). As expected, hospital charges were markedly higher for patients with unplanned returns to the OR. Reoperation was also associated with higher mortality rates; statistically significant increases were noted for pancreatic resection (33% vs 3.7%; P =.04), esophagogastrectomy (100% vs 4.2%; P =.002), and laparoscopic Nissen fundoplication (50% vs 0%; P =.01). Overall, 91 reoperations (85%) were for complications occurring at the original surgical site, including those related to an anastomosis (n = 16), surgical wound (n = 21), infection (n = 16), bleeding (n = 12), and other (n = 26). CONCLUSIONS: Unplanned returns to the OR occur across a broad spectrum of general surgical procedures and carry significant implications. Because they most often reflect problems related to the procedure itself, reoperation rates may be useful for monitoring quality across hospitals and for identifying opportunities for quality improvement locally.     

The potential effect of the UK 1990 height centile charts on community growth surveillance

The UK 1990 height charts are derived from an up to date dataset and introduce a change in the centile lines, particularly the addition of the 0.4th centile. This study examined the likely impact of these changes. Height data from London school children (1990-1993) were examined using Tanner and Whitehouse (TW) and UK 1990 charts. Numbers of children with height below TW 3rd centile were compared with numbers below the UK 1990 3rd and 0.4th centiles. The TW charts identified only 1% of children below the TW 3rd centile, while the UK 1990 charts identified 3% below the 3rd and 0.4% below the 0.4th centiles. If the 3rd centile remains as the referral 'cut off' for short stature, the introduction of the UK 1990 charts would increase current workload two- to three-fold, while a change to the 0.4th centile would reduce it by 50%. A significant number of children with abnormalities may be excluded from further assessment as a result of this latter change. In this small scale community study it is not possible to assess the consequences of this change. The heights at diagnosis of children with growth hormone (GH) deficiency (peak GH < 20 mU/l during a standard provocation test) were therefore compared to the 0.4th centile (UK 1990 charts). Sixty eight children with heights < 2nd centile (UK 1990 charts) currently receiving GH replacement (17 female, 51 male, aged 9.7, SD 3.5, years) were assessed, and of these, 28 (41%) had heights at diagnosis between 0.4th and 2nd centile, with a mean height standard deviation score of -2.32 (SD 0.21). This suggests that if the 0.4th centile were to be used as the sole criterion for referral for slow growth, a significant proportion of children with abnormality would not be referred for further assessment. The UK 1990 2nd centile should replace the TW 3rd centile. Children below this should undergo an intermediary medical assessment to confirm height measurement, to exclude from referral children with mild familial short stature and to identify concerns regarding the child.     

Aetiology of chronic suppurative lung disease

Forty one (1%) of 4000 children referred for respiratory disease had chronic suppurative lung disease not due to cystic fibrosis. Further investigations showed congenital malformations in six (15%), primary ciliary dyskinesia syndrome in seven (17%), 11 had immunological abnormalities (27%), and two bronchiectasis due to aspiration (5%). Therefore the underlying cause for the disease was found in 63%. Identification of predisposing causes may facilitate prevention of further bronchial damage.     

Trichloroethylene induced vitamin B(12) and folate deficiency leads to increased formic acid excretion in the rat

Exposure of rats to trichloroethylene induces a sustained excretion of large amounts of formic acid in urine. Both of the major metabolites, trichloroethanol and trichloroacetic acid, were found to induce this response, but not the minor metabolite S-(1, 2-dichlorovinyl) cysteine. Other polychlorinated solvents, including carbon tetrachloride and chloroform, also increased urinary formate excretion. Addition of folic acid either to diet or drinking water modulated the response indicating that these rats were folate deficient. Two markers of vitamin B(12) deficiency, methylmalonic acid and 5-methyltetrahydrofolate, were also markedly increased in urine and plasma respectively. The increase in 5-methyltetrahydrofolate is consistent with a folate deficiency caused by an inhibition of the vitamin B(12) dependent methionine salvage pathway. Since both vitamin B(12) and chemicals containing polychlorinated carbon atoms readily form free radicals, it is suggested that trichloroacetic acid and trichloroethanol interact with vitamin B(12) through a free radical mechanism inducing a B(12) deficiency and, as a consequence, a folate deficiency. As a result of the folate deficiency, excess formic acid, which is normally utilised through this pathway, is excreted in urine.     

A sequence-specific polymerase chain reaction assay for mitochondrial DNA polymorphisms in human platelets and white cells

BACKGROUND: Because mitochondria are abundant in white cells and are also present in platelets, polymorphic sequences in mitochondrial DNA (mtDNA) represent a unique target for polymerase chain reaction (PCR)- based detection of donor material. STUDY DESIGN AND METHODS: A PCR assay was developed that uses sequence-specific primers (SSP) focused on two continent-specific mtDNA polymorphisms. Results were validated by the use of informative restriction endonucleases. Three commercially available methods to extract mtDNA from white cell-reduced human platelets was compared. In preparation for in vivo studies, in vitro mixing studies designed to mimic transfusion were conducted to investigate the performance of the SSP-PCR assay. RESULTS: The gene sequences of two representative examples of amplicons obtained with the new SSP-PCR matched the sequence expected from the published genetic code. Fifteen individuals were classified as either positive (n = 6) or negative (n = 9) for the Asian polymorphism by the use of published primers known to flank the polymorphic site followed by digestion with appropriate restriction enzymes. Results with SSP-PCR were nearly perfectly concordant with those of restriction enzyme analysis. Although the use of three DNA extraction methods allowed the preparation of mtDNA that was suitable for PCR, large and consistent differences (ranging from 10- to 1000-fold) in endpoint sensitivity were found. In vitro mixing studies reproducibly documented that the SSP-PCR assay could detect as little as 1 percent of donor platelets mixed with recipient blood. CONCLUSION: PCR-SSP can be reliably used to identify human mtDNA polymorphisms. By optimization of the method of mtDNA extraction, the sensitivity of PCR-SSP assay was greatly increased. This assay should prove useful in investigations of allogeneic platelet transfusions without cell labeling. It may also be applied to studies of the donor cell microchimerism that follows transfusion or transplantation.     

Computer graphics for digitally formatted images

The increasing use of digitally formatted imaging systems requires high-quality interactive gray-scale computer raster graphics systems for the management, display, and analog film recording of digital image and alphanumeric information. These systems are a combination of computer hardware and software and implement a set of graphics protocols. This paper describes a set of interactive graphics protocols that has been developed for clinical use.     

多波长数据线性双组合同步荧光法测定苯酚与间苯二酚

提出了多波长数据线性双组合法计算解析苯酚和间苯二酚混合物的同步荧光光谱,考察了△λ=λ_em-λ_ex,pH,光路中光照时间对二种酚的荧光强度影响,建立了同时测定混合物中苯酚和间苯二酚的新方法。用于复方雷琐辛涂剂中苯酚与间苯二酚的同时测定,平均回收率分别为:苯酚98.5%±1.4%;间苯二酚98.8%±1.3%。     

金刚大化学成分的研究

从百部科黄精叶钩吻属植物金刚大(Croomia japonica Miq)的根部分得五个成分,分别为粉蕊黄杨胺A(pachysamine A,Ⅰ)、金刚大啶(croomionidine,Ⅱ)、金刚大碱(croomme,Ⅲ)、脱氢金刚大碱(didehydrocroomine,Ⅳ)和β-谷甾醇。应用波谱(IP,MS,¹HNMR和¹³CNMR等)分析、理化数据测定和化学转化,确定丁它们的结构,其中金刚大啶为新甾体生物碱。