Current methods for accurate quantification of nucleic acids typically begin with a template preparation step in which DNA and/or RNA are freed of bound proteins and are then purified. Isolation of RNA is particularly challenging because this molecule is sensitive to elevated temperatures and is degraded by RNases, which therefore have to be immediately inactivated upon cell lysis. Many protocols for nucleic acids purification, reverse transcription of RNA and/or amplification of DNA require repeated transfers from tube to tube and other manipulations during which materials may be lost.Xist, Sry and the two heat-inducible hsp70i (hsp70.1 and hsp70.3), as well as their RNA transcripts in single mouse embryos and in isolated blastomeres.This paper introduces a novel and highly reliable single-tube method for rapid cell lysis, followed by quantitative preparation and analysis of both RNA and/or DNA molecules in small samples. In contrast to previous approaches, this procedure allows all steps to be carried out by sequential dilution in a single tube, without chemical extraction or binding to a matrix. We demonstrate the utility of this method by quantification of four genes, This method virtually eliminates losses of nucleic acids and is sensitive and accurate down to single molecules. Real-time polymerase chain reaction (PCR) in combination with reverse transcription (RT) provides a powerful tool for accurate quantification of DNA and RNA copy numbers and has opened the way to the study of subtle modulations of gene expression in small numbers of cells, as well as small-scale genetic analyses aimed at establishing chromosome numbers, the presence of mutations, or allele dropout. The reliability of these measurements, however, depends on the accuracy of each step, including preparation and recovery of RNA and/or DNA, reverse transcription of RNA into cDNA, and quantifiable and specific amplification of all desired sequences. The importance of optimizing each of these steps is well recognized , as is tOur laboratory has already demonstrated that bound proteins prevent reliable PCR amplification of genomic DNA and that a thorough proteolytic digestion followed by heat inactivation solves this problem . For accThe chaotropic agent guanidine isothiocyanate (GITC) has long been the chemical of choice for nucleic acid preparation. It is particularly useful for RNA studies ,9, becauIn view of these limitations we devised an alternative strategy in which the sample is collected and denatured in a minimal volume of a GITC solution, briefly heated to allow dry storage, and, when needed, is directly analyzed in the same tube by performing quantitative RT-PCR in a volume large enough to lower GITC concentration to negligible levels. This new procedure, hereafter referred to as PurAmp (patent pending) and described here in full for the first time, is the only available method that allows a whole sample, such as a single embryo or cell, to be processed from lysis to RT-PCR in the same tube, under conditions that permit precise quantification of both RNA and genomic DNA copy numbers. This fully optimized method is sensitive enough to detect specific sequences within a single chromosome in one cell, yet robust enough to measure the presence of thousands of RNA molecules released from hundreds of cells.Xist, responsible for X-inactivation and dosage compensation in female cells [hsp70.1 and hsp70.3, jointly called hsp70i [Xist RNA is a noncoding transcript that exerts its particular function of gene-silencer by coating the inactive X-chromosome. Unlike hsp70i RNA and most other mRNAs, it is therefore localized in the cell nucleus and particularly challenging to extract. Besides their biological relevance, both Xist and the hsp70i genes offer the advantage of naturally-occurring unambiguous controls for the specificity of transcripts amplified with RT-PCR. Xist RNA is, in fact, virtually absent from male cleavage stage embryos [hsp70i RNA is predominantly synthesized in response to stress although minimal levels of hsp70i transcripts are normally present in embryonic cells. A careful quantitative analysis of hsp70i heat and culture stress-response in preimplantation embryos at different developmental stages, and its implications for development, will be presented elsewhere (C. Hartshorn, A. Anshelevich and L. J. Wangh, in preparation). In addition, we have been able to detect and quantify the genomic sequences of Xist, the hsp70i and the male sex-determining gene Sry [PurAmp has made it possible for us to conveniently investigate expression levels of two gene types essential for early mouse embryo development: le cells -13, and d hsp70i ,15. Xist embryos -19, whilgene Sry . BecauseThe PurAmp method presented in this study is performed in a single tube from cell lysis to cDNA or genomic DNA amplification, thus eliminating possible loss of template molecules due to procedures such as phase separation and recovery, repeated washing and re-suspension of nucleic acid pellets, elution from binding matrices and vessel-to-vessel transfer. This strategy offers an immediate improvement in the precision of gene expression analyses, at the same time shortening considerably the experimental protocol compared to traditional methods.Xist RNA content of a group of female mouse embryos at the blastocysts stage and quantified Xist and Sry DNA copy numbers in their male counterparts. Our previous analysis of these parameters in single female and male mouse embryos at different developmental stages provided us with an ample pool of data obtained with commercially available nucleic acids preparation methods [In order to validate our method, we initially measured the  methods -19, whicXist/Sry amplicons was detected using two molecular beacons conjugated to different fluorescent dyes. Both amplicons span intronless sequences of the genes [Sry and Xist signal. Three embryos were identified as male based on the presence of the Sry amplicon  as the Sry signal . This indicates the presence of the same number of copies of Sry and Xist templates, as expected for male blastomeres that contain one copy of the Sry gene on the Y-chromosome and one copy of the Xist gene on the X-chromosome. Neither gene is expressed in male blastocysts [Xist and Sry DNA. The three remaining embryos did not generate any Sry signal , the accumulation of Xist RNA per female blastocyst averages above 12,000 copies, considerably higher than our previous measurement of 6797 ± 2894 copies obtained using a multistep nucleic acids isolation procedure [Xist and Sry sequences is neither decreased nor increased by the presence of diluted denaturing solution (0.4 mM GITC, as in the PurAmp protocol) during real-time PCR. In fact, additional experiments revealed that Xist/Sry real-time PCR was unaffected by a GITC concentration as high as 20 mM (not shown). Taken together these results suggest that the higher levels of Xist cDNA measured using the PurAmp method are due to an improvement in RNA recovery at the initial step of cell lysis.Quantification of the real-time PCR data obtained from the three male embryos in Fig. astocyst . Both vaastocyst , a phenorocedure . The datXist and Sry genes and thousands of copies of Xist transcripts, with rather wide sample-to-sample fluctuations. In order to more carefully determine the quantitative capability of the new assay, we next analyzed embryos at earlier developmental stages containing lower and, in some cases, precisely known numbers of template copies. Figure Xist amplicons generated in the course of two separate experiments by, right-to-left, i) a 3-cell male embryo (yellow); ii) a 4-cell male embryo (green); iii) a single blastomere isolated from a 4-cell female embryo (light purple); iiii) a 4-cell female embryo (red); iiiii) a female blastocyst (blue). The gender of each embryo was confirmed by the detection of an Sry-specific fluorescent signal in male samples , denoting the presence of Xist transcripts , albeit at considerably lower levels than those measured in the female blastocyst . These measurements are consistent with other studies demonstrating that Xist transcripts are accumulated in the developing embryo beginning at the late 2-cell stage [Xist developmental profile [As shown above, blastocysts are comprised by many cells and contain hundreds of copies of the ther two , an obsele cells ,17. Convll stage  and withXist signal generated by a single blastomere isolated from a 4-cell female embryo arose 2.2 cycles later than the Xist signal of the whole 4-cell female embryo  RNAs.In order to more extensively test the validity of the PurAmp approach to template quantification in single cells, we measured transcript levels of the heat shock-inducible genes  protein ,15. For hsp70i expression. Like Sry, the hsp70i are naturally intronless genes and therefore once again our pair of PCR primers simultaneously amplified both genomic DNA and cDNA sequences. The data in Table hsp70i template numbers due to the presence of thousands of copies of hsp70i RNA, although these numbers were considerably lower when samples were prepared with a multistep/multitube phenol-chloroform extraction [hsp70i RNA levels. Based on these results, the duration of the recovery period was increased to at least two hours in all following experiments, eliminating the finding of "non responsive" embryos.A preliminary set of experiments was carried out on embryos at the blastocyst stage, when heat shock response is fully established , with thtraction ,18 rathehsp70i RNA were then quantified in whole 8-cell embryos that had or had not been exposed to a temperature increase, as summarized in Table hsp70i genomic DNA copies measured in the absence of RT was consistent with the presence of four copies of the genes per cell, one hsp70.1 and one hsp70.3 on each chromosome 17, and with the fact that some of the cells analyzed had already duplicated their DNA. Only a minimal amount of hsp70i RNA, calculated as the difference of template copy numbers obtained with and without reverse transcription (hsp70i cDNA + genomic DNA less hsp70i genomic DNA), was present in non-heated embryos, indicating that the embryos were not stressed by culture conditions [Copy numbers of nditions .hsp70i RNA increased sharply after a 30-minute heat treatment, followed by a recovery period of either 2 or 3 hours necessary for transcripts synthesis and accumulation. Some of the heat-shocked embryos were harvested intact, while others were dissociated in single blastomeres. The average numbers of hsp70i template copies per blastomere were calculated from the isolated cells  and compared to the average per blastomere values obtained from whole embryos. The results show that the amount of hsp70i RNA + genomic DNA per cell calculated by these two approaches is very similar. A post-heating recovery period of 3 hours rather than 2 hours increased the hsp70i RNA levels only slightly, indicating that the onset of transcription and the major build-up in RNA occur quickly. Single cells derived from non-heated embryos contained only trace amounts of hsp70i RNA, consistent with whole embryo measurements, and PCR efficiency was, again, unaffected by the PurAmp components (not shown).Expression of Xist/Sry PurAmp protocol and analyzed the efficacy with which the genomic DNA was degraded. Genome numbers in embryos at the morula stage were calculated by counting Xist and Sry copies in five male samples (as detailed for blastocysts, see above) and averaged at 21.3 ± 8.9, consistent with the fact that embryos at this stage are normally comprised of 16-to-32 cells. After treatment with DNase I, only 0.8 ± 1.5 genomes per embryo were still present in a group of 15 male samples, demonstrating that the enzyme had successfully degraded 96.3% of the DNA. Figure Xist (upper panel) and Sry (lower panel) DNA in a group of eight male and eight female single embryos. As expected, control male samples (blue lines) contained equal numbers of Xist and Sry copies, as shown by the equal CT values, corresponding to the genomic DNA copy number. Both amplicons were absent from DNase-treated embryos that could be identified as male because they were devoid of Xist RNA (yellow lines). In contrast, control female samples (red lines) contained Xist RNA and DNA but lacked Sry. DNase treatment caused a delay in the Xist signals arising from female embryos (green lines), consistent with the elimination of all 42 copies of genomic Xist DNA in each embryo plus some decrease in the number of Xist transcripts. The amount of RNA recovered in these samples averaged at 75% of control levels. The fact that some RNA is lost during the DNase step is not surprising as it is well known that some RNA hydrolysis is unavoidable (see Discussion), and is not linked to the single-tube procedure. We anticipate that further optimization of the available DNase protocols and reagents will minimize this problem. Thus, the data in Fig. The PurAmp method described above automatically results in the quantitative recovery of genomic DNA, which can then be used as a quality control and a convenient internal standard for the simultaneous recovery and measurement of mRNA -19, see , see 19,It is increasingly clear that individual cells in a population do not exhibit identical patterns of gene expression and, hence, that expression profiling is more informative if it is quantitative and carried out at the single cell level -27. ThisXist RNA, are freed and made available to reverse transcription as well as cytoplasmic mRNA molecules, a result unattainable by mild detergent treatment that leaves nuclei intact [First, cell lysis and protein denaturation occur very rapidly upon delivery of the sample to crystalline GITC, thus ensuring both protein removal from DNA and RNA and inactivation of cellular nuclease that would otherwise quickly degrade RNA . Transcri intact . Second,Xist RNA levels in developing mouse embryos highlight one of several merits of the PurAmp method over the traditional, multistep approach to nucleic acids purification [Xist RNA levels are higher with the single-tube protocol. The same culture conditions and procedures were used in the two groups of experiments, making it unlikely that differences in embryo quality were the cause of the increase in Xist RNA. We, therefore, conclude that the higher levels of Xist RNA observed using the new procedure reflect improved template preparation with efficient inactivation of RNases and reduced loss of RNA molecules. Xist RNA is known to trigger X-chromosome silencing through interactions with numerous proteins and possibly with the nuclear matrix scaffold [hsp70i templates in heat-shocked blastocysts supports the view that larger pools of RNA are detected in PurAmp-treated samples than when using phase separation-based nucleic acid extraction. The later method, in fact, presents several steps that require extreme care to avoid loss of material, including complete recovery of the upper phase, thorough precipitation of all nucleic acids molecules, and repeated re-suspension and washing of barely visible pellets. All these manipulations render the results obtained with this technique particularly operator-dependent, while, in contrast, PurAmp simply requires sequential addition of reagents into the same tube. Once the sample is delivered to the LysoDot in the reaction vessel  were obtained from Embryotech Laboratories, Inc. (Wilmington, MA), and were cultured as previously described  until thXist measurements) or pre-compaction 8-cell embryos (for hsp70i measurements) after drilling the zona using a ZILOS-tk™ zona infrared laser optical system (beam = 1480 nm) (Hamilton Thorne Biosciences, Inc., Beverly, MA), according to a procedure developed in our laboratory and described elsewhere [Single blastomeres were isolated from either 4-cell embryos  to the inside surface of the lids of PCR-grade reaction tubes (Applied Biosystems, Foster City, CA). Precise measurement of the droplets size was obtained following the method previously described by Wangh . The denImmediately before harvesting, individual embryos were placed in 3 ml of Dulbecco's PBS devoid of calcium and magnesium chloride . DulbeccIn order to perform reverse transcription, each sample was carefully re-solubilized in the lid by addition of 6 μl of Random Hexamers mixture (4.2 ng/μl) in DEPC-treated water . Tubes were closed, inverted, briefly centrifuged and incubated for 5 minutes at 65°C in order to allow primer/RNA hybridization. The remaining reagents needed for RT were then added to the tube in a volume of 4 μl, and the reaction was carried out according to the protocol suggested by the manufacturer. As previously described -19, all Xist and Sry genomic DNA + cDNA templates was thus performed in a final volume of 100 μl, as detailed elsewhere, in the presence of 4 units of Taq DNA polymerase (Promega, Madison, WI) [® 7700 Sequence Detector (Applied Biosystems, Foster City, CA) and fluorescence readings were taken at the annealing temperature.The full volume of each sample was then mixed with 89.5 μl of complete PCR amplification cocktail containing sequence-specific molecular beacons as detection probes . Multiplson, WI) ,19. Realhsp70i assay was carried out similarly to the one for the Xist/Sry multiplex, by sequential dilutions of denaturant, RT and PCR reagents. The procedure for collection and lysis of the samples was the same. Dry samples were re-solubilized with 6 μl of random decamer primers (8.3 μM) in nuclease-free water . After a 3 minute incubation at 75°C to optimize primer binding to RNA, all other reagents needed for RT were added to the sample and the reaction was carried out according to the manufacturer's instructions. As for the Xist/Sry assay, all RT mixture components were used at the suggested concentrations, but volumes were halved so that RT was performed in a final volume of 10 μl. An RNase H digestion step was included at the end of RT, as in the case of the Xist/Sry assay.Embryos were heat-shocked at 43°C for 30 minutes, followed by a recovery period of 30–40 minutes (blastocysts), or 2–3 hours (8-cell embryos, as indicated) at 37°C. The hsp70i primers were localized at positions 1245/1305 of the hsp70.1 GenBank sequence with accession number M35021  and were identical to sequences within the hsp70.3 gene, previously known as hsp70A1 (GenBank sequence with accession number M76613) [hsp70i was the only amplicon generated in this assay, it was not necessary to design a sequence-specific detection probe. In this case, SYBR® Green, a fluorescent dye that binds to double-stranded DNA, was used as fluorescent probe for real-time PCR. The specificity and purity of the amplicon was confirmed by both gel electrophoresis and analysis of the melt profile, as previously described [hsp70i PCR was the following: 50 mM Tris, pH 8.3, 3 mM MgCl2, 0.3 μM each primer, 0.25 mM each dNTP, 1:62,500 SYBR Green (from a "10,000X concentrate in DMSO" purchased from FMC BioProducts, Rockland, ME), and 4 units of Taq DNA polymerase (Promega, Madison, WI). The polymerase was incubated at a 1:1 (v/v) ratio with Platinum® Taq antibody (Invitrogen) for 5 minutes before addition to the reaction mixture (hotstart PCR). The cycling profile was: 95°C for 5 minutes; 10 cycles consisting of the following four steps: 95°C (20 sec), 64°C (30 sec), 72°C (30 sec), 84°C (15 sec); 35 cycles with the following four steps: 95°C (20 sec), 59°C (30 sec), 72°C (30 sec), 84°C (15 sec). Fluorescence readings were acquired at 84°C, in order to exclude fluorescent signals due to the possible formation of primer dimers late in the reaction.Real-time PCR was carried out in a final volume of 100 μl, by adding the PCR reagents to the sample after completion of RNase H digestion. The chosen  M76613) . Becauseescribed . The comhsp70i genomic DNA copy numbers in the absence of cDNA [A number of embryos were also processed as "No RT" controls, with the same protocol used for the other samples but without inclusion of reverse transcriptase in the RT mixture. These controls were used to quantify  of cDNA .hsp70i expression in blastocysts, some of the embryos were processed using a commercially available multistep/multitube kit, as previously described [For the preliminary studies on escribed . BrieflyT) at which each fluorescent signal was first detected above background. CT values were compared to standard scales obtained from analysis of male mouse genomes at known copy numbers, as detailed previously [T determinations. A lower CT value indicates an earlier detection of the fluorescent signal and therefore more templates present at the start of the reaction [reviewed in ref. [Sry (Y-chromosome), one copy of Xist (X-chromosome) and four copies of hsp70i (two of hsp70.1 and two of hsp70.3, both on chromosome 17).Calculation of template copy numbers was based on the "threshold cycle"  and 1 unit of DNase I (Ambion) in nuclease-free water. After an incubation of 20 minutes at room temperature, the reaction was terminated by adding 1 μl of a 10 mM EDTA solution, pH 8.0 (Invitrogen). The nuclease was inactivated by heating the samples at 65°C for 10 minutes, according to the protocol recommended by Invitrogen. One μl of a 25 ng/μl RT primer solution was then added to the sample, so that the final primer concentration was now the same as in the assay without DNase (see above). RT and PCR were then carried out as detailed for the "No DNase" assay.Embryos were grown to the morula stage and were individually harvested and lysed as detailed above. Controls were processed for multiplex detection of Xist/Sry measurements and drafted the manuscript. AA established heat shock conditions and performed the hsp70i quantification experiments. LJW coordinated the study and contributed to all aspects of its design.CH devised the finalized form of the PurAmp method, carried out the


Although modest improvements in the survival of patients with non-small cell lung cancer (NSCLC) can be achieved with cisplatin-based chemotherapy (CT), its value is disputed in the geriatric setting. In this study, we evaluate the feasibility of vinorelbine/cisplatin CT for elderly NSCLC patients.2, on day 1 and 8, and cisplatin on day 1, in 28 days-cycles. After stratification for age (up to 75 years), younger patients were sequentially allocated to moderate cisplatin doses (80 mg/m2 or 90 mg/m2), and older patients were allocated to lower cisplatin doses (60 mg/m2 or 70 mg/m2). We recruited patients aged over 70 years with newly diagnosed NSCLC, clinical stage III or IV, Karnofsky performance status ≥ 70%, normal serum creatinine, peripheral neuropathy ≤ grade 1, and no prior cancer therapy.In this pilot phase I/II trial, all patients received CT with vinorelbine 25 mg/mAnalysis was by intention to treat. Main toxicities (grade 3–4) was as follows: neutropenia, 20%; anemia, 11%; and thrombocytopenia, 2%; alopecia, 55%; fatigue, 11%; and peripheral neurotoxicity, 2%. No grade 3–4 emesis or renal toxicity occurred. Global median time to progression (TTP) and overall survival (OS) were 27.0 (95% CI: 10.1 to 43.7) weeks and 30.1 (95% CI: 24.4 to 35.8) weeks; 1- and 2-year survival rates were 36.3% and 13.2%, respectively. Overall response rate was 50.0% (95% CI: 35.4% to 64.5%), with 1 complete response; no difference on response rate was noticed according to cisplatin dose. Median overall survival was 30.1 weeks, with 1- and 2-year survival rates of 36.3% and 13.2%, respectively.Age does not preclude assessment on the role of cisplatin-vinorelbine CT for elderly NSCLC patients with good performance status and adequate bodily functions. Lung cancer is a disease with a great incidence in older people. In Brazil, its incidence rate in male sex was expected to reach 18.8:100.000 in 1999, and aged patients may have accounted for 57% of all lung cancer deaths. Unfortunately, up to two thirds will present at diagnosis with advanced disease, requiring chemotherapy (CT) [Vinorelbine, a semi synthetic vinca alkaloid, is a highly active drug for NSCLC and its association with cisplatin is worthwhile. European and Southwest Oncology Group trials demonstrated that vinorelbine/cisplatin (VP) offer therapeutic advantage over both drugs alone -4, previAlthough modest improvements in the survival of patients with non-small cell lung cancer (NSCLC) can be achieved with cisplatin-based CT , its val9/L and platelets ≥ 100 × 109/L), bilirubin under 1.25 times upper normal value (UNV), aspartate aminotransferase/alanine aminotransferase (AST/ALT) under 2 times UNV, and renal function (creatinine level under 120 μmol/L); no symptomatic brain metastasis; no prior cancer therapy; no indication for palliative radiation therapy; no previous or concomitant malignancy; and adequate social support. Exclusion criteria were symptomatic peripheral neuropathy and comorbidity regarded as an impediment for CT, such as renal disease, heart failure, coronary heart disease, uncontrolled infection, and cognitive impairment.This study enrolled patients older than 70 years with unresectable locally advanced or metastatic NSCLC. Informed consent was obtained from patients and their relatives, as approved by the Institutional Ethical Committee. Inclusion criteria: all patients had to have histologically confirmed NSCLC; Karnofsky performance status ≥ 70; measurable disease; adequate bone marrow reserve  and biochemistry; chest x-ray; bone scintigraphy scan; chest, abdominal and brain computed axial tomography scans; and electrocardiogram. Although pretreatment bone scan and brain CAT scan are recommended only when signs or symptoms of disease are present, they were added here due to Institutional Protocol Reviewers recommendation.2 on days 1 and 8, administered intravenously in bolus, followed by intravenous cisplatin over 1 hour. CT was administered every 4 weeks. Prophylactic anti-emetic drugs (intra venous dexamethasone 20 mg and ondansetron 16 mg) and fluid hydration  was used to minimize renal toxicity. Dexamethasone 4 mg PO BID plus metoclopramide 10 mg PO QID for 4 days was used to prevent delayed emesis. Patients were time-sequentially assigned to one of two groups, from lower to higher doses, according to age strata. Study doses were defined based on previous reports on renal tolerance of cisplatin in geriatric patients [2 and those aged 70 to 75 received cisplatin 80 or 90 mg/m2. Assignation to high dose groups (70 mg/m2 and 90 mg/m2) occurred after evaluation of toxicity at inferior doses, evaluated according to National Cancer Institute criteria. The protocol required a minimum of 6 and a maximum of 18 patients per group after the 1st cycle for safety analysis.Treatment consisted of vinorelbine 25 mg/mpatients . Age str9/L and platelets were <100 × 109/L, treatment was delayed by 1 week. Treament on days 8 had to be cancelled if neutrophil counts were <1.0 × 109/L and platelets were <100 × 109/L. If treatment could not be given after a 2-week interval because of haematological toxicity, it had to be discontinued and the patient withdrawn from the study. Concomitant use of hematopoietic growth factors were not allowed in the first treatment cycle but were administered subsequently on individual basis. Neurological toxicity above grade 2 resulted in suspension of treatment; ototoxicity grade 2 or 3 resulted in a 50% dose reduction of cisplatin.Chemotherapy doses were reduced for haematological, neurological, hepatic and renal toxicities. Toxicity was graded according to National Cancer Institute (NCI) common toxicity criteria guidelines. Changes in dosage were based on WBC results obtained on day 1 of treatment; if neutrophils were <1.5 × 10The following dose modifications of vinorelbine were set based on AST/ALT  and bilirubin values on day 1 or day 8 of treatment: if AST/ALT were between 5.1 and 20.0 × UNV or bilirubin was between 1.5 and 3.0 × UNV, dosing was cancelled and the patient was reassessed 1 week later. If AST/ALT were >20.0 × UNV or bilirubin was >3.0 × UNV, vinorelbine was discontinued. If serum creatinine was grade >1, the dose was delayed by 1 week and the test repeated. After a 2-week delay, the patient was taken off the study. WBC and biochemistry were also performed on day + 14 of treatment.We intended to administer a maximum of four CT cycles followed by radiation therapy in responding patients with stage III disease and six CT cycles in patients with wet IIIB or stage IV disease. Notwithstanding radical radiation therapy (RT) should deliver a total dose down to 66 Gy, covering tumor site and regional lymph nodes, and palliative therapy could use doses under to 45 Gy, the final choice of dose, fractioning, irradiated volume, and energies of radiation was at the radiation oncologist's discretion. Treatment interruption was allowed in case of disease progression, severe adverse events, or patient preference.Chest x-ray was performed before each cycle, and CAT scans every two cycle for response evaluation. Tumor response was recorded according to World Health Organization criteria and measured by the same observer (JRP). All responses had to be confirmed 3–4 weeks from initial evaluation. We reported here the best response designation recorded from the start of treatment until disease progression. Patients stopping treatment with an unconfirmed response, or only short stabilisation were considered as inevaluable, unless the response or stabilisation was further confirmed in the absence of any treatment. Patients were monitored for the first month off-study then followed up every 2–3 months.The dose intensity was calculated for both drugs by dividing the actual dose delivered by the length of therapy. Toxic death was defined as death occurring during the chemotherapeutic phase (including four weeks after its end) and due to drug toxicity. Early death was defined as death within four weeks after a chemotherapy cycle without severe toxicity and not related to the malignant disease. Response and survival were calculated by intention-to-treat. Progression was defined in relation to the best response obtained. The time to tumor progression lasted from the first day of treatment to the date of the first observation of progressive disease. Survival was defined as the time elapsed from the beginning of CT until death or last follow-up visit. Time-to-event analysis was performed using the Kaplan-Meier product-limit estimator. All analyses were carried out using a computer program (SPSS version 8.0, Chicago, USA).2 and eight received 70 mg/m2; in the former, fifteen patients received 80 mg/m2 and fourteen received 90 mg/m2. Patient characteristics are given in Table Forty-four patients were recruited from July 1996 to June 1998; twenty-nine aged 70–75 year and fifteen older than 75 years. Cisplatin doses were as follows: in the older cohort, seven patients received 60 mg/mTreatment results are shown in Table Toxicity data are presented in Table 2 on day 1 plus vinorelbine 25 mg/m2 on days 1 and 8, repeated each 28 days per in the maximum four cycles, was feasible for elderly NSCLC patients. Neurological and renal tolerance was particularly good. At the time we developed the protocol, no quality-of-life instrument had been validated for use in Brazil. Thus, a drawback in our study is the absence of quality-of-life analysis, which precludes evaluation of key dimensions in geriatric oncology.Treatment of elderly NSCLC patients with cisplatin-based regimens has been a less contentious matter nowadays but toxicity remains a major issue. In our pilot study, chemotherapy with cisplatin 70–80 mg/mCisplatin induces a sensory neuropathy due to axonal damage that is dependent on the total-dose and single-dose intensity , and it Cisplatin renal toxicity has been attributed to drug-protein interactions and the inactivation of specific brush border enzymes, resulting in damage of the loops of Henle, the distal tubules, and collecting ducts. Patients aged above 70 or even 80 are regarded as susceptible to cisplatin-induced renal damage as the younger counterparts ,16 and cThe observed response rate here was in the usually range of NSCLC phase II trials, but the absence of external review of radiological data and the widened confidence intervals expected because of small sample sizes in each group limit assertions that could otherwise be drawn. The 1-year survival rate (36%) was good but essentially equivalent to the reported elsewhere  for vinoVinorelbine is a cytotoxic agent that clearly has expanded the therapeutic options for elderly NSCLC patients ,21. The Although sequential administration of drugs is an attractive option for aged or frail patients, a setting where minimal treatment-related toxicity should be pursued, research on the role of non-platinum combinations for elderly NSCLC patients aroused attention. The Southern Italy Cooperative Oncology Group (SICOG) evaluated whether the association of vinorelbine and gemcitabine would be better than vinorelbine alone. To date, final results of this trial have not to come. Despite an article focusing on the interim analysis of 120 patients (60 at each group) claimed a survival advantage for the combined arm , furtherThe question whether or not cisplatin-containing regimens should be used to treat aged patients remains an important, still open, issue. As observed for paclitaxel-carboplatin , gemcitaThis study was supported by Institutional funds only. Authors did hot received reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this paper in the past five years.NSCLC: non-small cell lung cancerCT: chemotherapyNCI: National Cancer InstituteELVIS: Elderly Lung Cancer Vinorelbine Italian StudySICOG: Southern Italy Cooperative Oncology GroupGFR: glomerular filtration rateJRP was responsible for study conception, protocol conduction, and results interpretation. SJM carried out the data analysis and results discussion. SMN and FKI were responsible for patient care and data gatheringThe pre-publication history for this paper can be accessed here:


Medical ethics has existed since the time of Hippocrates. However, formal training in bioethics did not become established until a few decades ago. Bioethics has gained a strong foothold in health sciences in the developed world, especially in Europe and North America. The situation is quite different in many developing countries. In most African countries, bioethics – as established and practiced today in the west- is either non-existent or is rudimentary.Though bioethics has come of age in the developed and some developing countries, it is still largely "foreign" to most African countries. In some parts of Africa, some bioethics conferences have been held in the past decade to create research ethics awareness and ensure conformity to international guidelines for research with human participants. This idea has arisen in recognition of the genuine need to develop capacity for reviewing the ethics of research in Africa. It is also a condition required by external sponsors of collaborative research in Africa. The awareness and interest that these conferences have aroused need to be further strengthened and extended beyond research ethics to clinical practice. By and large, bioethics education in schools that train doctors and other health care providers is the hook that anchors both research ethics and clinical ethics.This communication reviews the current situation of bioethics in Africa as it applies to research ethics workshops and proposes that in spite of the present efforts to integrate ethics into biomedical research in Africa, much still needs to be done to accomplish this. A more comprehensive approach to bioethics with an all-inclusive benefit is to incorporate formal ethics education into health training institutions in Africa. Medical Ethics, which became the template on which the code of ethics of the American Medical Association was based in 1847 [Before the modern discipline of bioethics evolved, ethics had been on the centre stage of medical practice for more than two millennia, since the time of Hippocrates. In 1803, Thomas Percival published his book on  in 1847 . Of the The scope of bioethics has continued to expand in response to changes in societal dynamics, medical technology and health care practices. Perhaps more because of its antecedents than for want of content, most of the discourse and writings in the early days of bioethics centred on issues of patient-physician relationship, respect for person, best interest of the patient, and justice in health care delivery. Today, traditional bioethics discussions and literature are changing as new ethical concerns evolve around dilemmas posed by new technology on the subjects of end of life issues, organ donation, human reproduction and human genomics. Moreover, the bioethics agenda has expanded to include the subjects of resource allocation, organizational ethics and public health ethics, among others.Bioethics in its present form is rooted in and largely dominated by western culture. The tempo and content of bioethics discourse are largely influenced by the technological creations of the developed world. However, ethics is not exclusively the domain of the west. Core ethical values are essentially the same for all human communities leaving aside each community's customs, culture and preferences . AccordiIn the bioethics literature, bioethical discourse and arguments have been most prominent and intense concerning research involving human participants. One major achievement in this regard is the creation of an oversight body that sees to the proper design and conduct of research that conforms to generally acceptable and established ethical guidelines. There resides in this body the duty of ensuring that research sponsors and investigators abide by established conventions for carrying out clinical research. They also perform the role of assuring the safety of research participants and ensuring that participants (and/or society) benefit from the outcome of research. The relevance of this body to modern day health care and research is partly exemplified in the absence of any major scandals in the form and magnitude of those already recorded in history. The establishment of research ethics boards has not solved all the ethical problems of biomedical research though. There is still a lot to do to re-structure, re-empower and re-position the board to match the complexities of the present-day technology-driven medical research and practice.Various bodies within and outside Africa have pioneered the movement towards ensuring that medical research in Africa conforms to international ethical guidelines. This is the aspect of bioethics that is most visible in Africa and has been anchored partly by the Pan African Bioethics Initiative (PABIN), a pan-African organization that was established in 2001 to foster the development of bioethics in Africa with a particular focus on research ethics (5). This idea has arisen in recognition of the genuine need to develop capacity for reviewing the ethics of research in Africa. It is also a condition required by external sponsors of collaborative research in Africa. Ethics workshops and conferences have been held in different parts of Africa, including Tanzania , Zambia While the present efforts and achievements are commendable, much still needs to be done for the effects to filter through to the grassroots, which is the main arena of research activities and where the burdens of research are most felt. I say this for the following reasons. First, the present efforts are still limited in extent and effect. Hitherto, most of the conferences have taken place in two or three geographical zones of the continent and have been limited to a few days of activities. Of course, the interests and motives of the sponsoring agencies together with the presence on the ground of those who are available to organize the conference locally determine where, when, for how long and the number of participants in the workshop. There is thus restriction on the number of researchers who can attend the conferences from all over Africa and on the amount of knowledge that can be imbibed in those few days. The consequence is that the same few people attend the conferences most of the time. These attendees from a few centres might not be able to change unethical research practices in their countries. Attempts by these few to train their colleagues locally are often constrained by lack of funds.Second, absence of national directories of research activities in most African countries makes the magnitude of biomedical and social sciences research in Africa to be underestimated. For example in Nigeria, five categories of research and researchers are easily identifiable. Individual or institution-supported research is done by students, clinicians (including resident doctors) and other scientists. This category constitutes a significant proportion of research in tertiary academic and health institutions. Industry-sponsored research is undertaken by researchers for pharmaceutical companies to promote new or old drugs. Such research protocol may be indigenously developed or be a part of multicentre trials. In most instances, these companies do not go through the institutions where the researchers are based, but deal directly with individual researchers, who may or may not subject the research protocol to an ethics board review. Collaborative research with colleagues from the developed countries is often externally funded. It includes hospital and community-based trials and mostly involves experimenting with drugs or vaccines. Of particular ethical concern in collaborative research is the fact that external sponsors may differ in their motives for conducting research and there may be limited applicability of research benefits to the country or local community . MoreoveThird, a majority of Africa's research participants are highly vulnerable given their low level of formal education and the political, social and economic milieu in which they live. The fourth reason is that Africa is a pluralistic society with diverse peoples and cultures. While general guidelines may apply in most cases, in some the peculiarities of each ethnic and cultural group will significantly affect what research is done and how it is done in those communities. Lastly, not every research centre has established an ethics review process. Where already established, most of the ethics review committees are grossly underfunded and unequipped for their duties.This is the branch of bioethics that addresses ethical conflicts that arise in daily clinical practice in health care institutions, through the establishment of hospital ethics committees and ethics consultation services. Fletcher and Siegler define ethics consultation "as a service provided by an individual consultant, team or committee to address the ethical issues involved in a specific clinical case. Its central purpose is to improve the process and outcomes of patents' care by helping to identify, analyze, and resolve ethical problems" . An ethiDo we need the services of hospital ethics committees or consultants in African hospitals or at the bedside? That may not be the point presently. However, clinical ethics is neither about committees and consultations, nor about technology and end-of-life issues alone. Common sense and intuition are insufficient to address all ethical issues that arise in patients' care. The well-intentioned clinical decisions and judgments of yesterday may turn out to be unsound in the searchlight of today's ethical scrutiny. Although core moral virtues have generally guided medical practice in Africa as elsewhere, there is the increasing need to apply both cognitive and behavioural ethical values to everyday decision making at the bedside by the physician as well as other health-care professionals . PerhapsIt is not here suggested that next on the agenda of health care in Africa is to devote attention and resources to training bioethics consultants for the bedside. Most people in Africa still do not have access to qualified health personnel and reasonable health care. The point is that deliberate efforts should be made to train present and future health care providers to be aware of the core moral virtues required of them in their duties to patients; and be sensitive to the ethical values of their patients, their families and the society.In the developed world education in ethics is no longer a "hidden curriculum"  that is It is now time for Africa to join the rest of the world by introducing ethics education into the curricula of all medical schools where it is not presently taught. This is where the future of bioethics and health care delivery and research in Africa lies. Apart from some countries in the southern and eastern part of Africa and a handful of universities in other parts of Africa, there is no formal ethics education in most of Africa's medical schools. Ethics education to medical students is a necessary and required commitment to accomplishing an all-round training of the doctor whose decisions are both technical and moral. Much attention has wholly focused on the technical aspect of medical education, leaving the student to develop his or her moral attitudes passively through observation and intuition. Formal ethics teaching aims at equipping students with a common framework on which to reconcile patients' medical needs with their values, perceptions, situations and beliefs ; and mayPertinent to any discussion about teaching bioethics in Africa is the issue of shortage of trained bioethicists to fill the vacancies that would be created in academic institutions in many African countries. Apart from South Africa and a few others, most countries in Africa lack the requisite bioethics manpower that would be needed in the medical schools. Even in institutions where bioethics is already part of the medical curriculum, it is unlikely that there are enough bioethics teachers. It is in this regard that the efforts of international agencies that fund training of developing world bioethicists are noteworthy. Those Africans who have undergone bioethics training in the developed world and have become pioneers in their institutions have an awesome responsibility of establishing credible training agenda for their countries. They are also well positioned to directly seek funding for such home-based programmes from international sponsors. At the beginning, such scholars may encounter some crisis of identity and acceptance within the established academic system. However, such difficulties would fizzle out as they persist in highlighting and proffering solutions to the myriad of contemporary ethical problems within the system and the society. The initial difficulty of publishing their works in established western bioethics literature can be overcome by patronizing local or regional journals that target the primary audience for which their work is meant and open access journals that reach far and wide. Moreover, most bioethics literature consists of commentaries, observations, personal opinions and philosophical reasonings. As African boethicists embark on qualitative research to highlight ethical issues in Africa and provide quantitative data to fill the gap and provide information which are frequently lacking in most western bioethics journals, access to western dominated journals would be enhanced.Bioethics, Chadwick and Schuklenk question the altruism behind training developing world bioethicists in the west and warn against bioethics colonialism. They opine that graduates of such programmes are subjected to western ethical views and ideologies and that the developing world is not funded to develop bioethics capacity based on its own thinking [In an editorial published in a recent issue of thinking . While IIn the light of the foregoing, it is imperative that African bioethics must evolve which should take cognizance of its unique needs and circumstances and which, though amenable to improvement as a result of continuing interactions with other cultures and values, yet is not overshadowed by those influences. The need for the individual clinician/researcher to be committed to upholding high ethical standards and principles that respect the social, cultural, economic, educational and religious values of the people can not be over-emphasized. More efforts are required towards increasing continent-wide awareness about ethical issues in biomedical practice and research through ethics conferences, workshops, national bioethics conferences, the public media and Non-Governmental Organisations (NGOs). Countries where bioethics presence is fairly strong should assist neighboring countries to establish a presence, especially in organizing ethics review committees at research centres and institutions. Within countries, the possibility of joint or regional ethics review committees should be explored.Continuing and expanded support from the international bioethics community is required now more than before, to develop capacity for training of academic faculty, clinicians, researchers, government health ministry officials, NGOs, and the media in bioethics. The initiatives of the National Institute of Health of the United States to provide training grants for bioethics programmes within and outside Africa and the support of other institutions and bodies like the Wellcome Trust, African Malaria Network Trust (AMANET) and European Forum for Good Clinical Practice (EFGCP) among others, to the course of bioethics in Africa are noteworthy. Their support for bioethics capacity building programmes should not be limited to one or two sub-regions but to the whole continent. Further sponsorship should be provided for academic institutions within Africa to establish more short- and long-term training programmes at sub-regional levels. More importantly, these institutions should support the movement towards formal incorporation of bioethics into the curricula of medical schools and other health training institutions in Africa. The present and future needs for this in Africa are most apparent now. According to an African adage, the best time to plant a tree is twenty years ago, the next best time is now.The author was a Fogarty Fellow (2003/2004) at the Joint Centre for Bioethics, University of Toronto 88, College Street, Toronto, Ontario, Canada M5G 1L4.The pre-publication history for this paper can be accessed here:


Lack of antihypertensive use among hypertensive individuals is a major public health problem. It remains unclear as to how much of this lack of treatment is because of failure to diagnose hypertension or failure to initiate drug treatment for those with a diagnosis of hypertension. The primary aim of this study was to determine the proportion of those untreated individuals who would be recommended to start drug therapy for control of blood pressure among those aware or unaware of their diagnosis of hypertension.The Canadian Heart Health Surveys (1986 – 1992), a national, cross-sectional descriptive survey (n = 23 129), was used to determine the proportion of individuals who were untreated, yet satisfied the 2004 Canadian hypertension guidelines for initiating drug therapy. Patients were divided into subgroups of those aware and unaware of having a diagnosis of hypertension according to self reported awareness from the survey.Of those with untreated hypertension (= 140/90 mmHg), only 37% were aware of their diagnosis. 74% of untreated individuals aware of their diagnosis of hypertension would require drug therapy, compared to 57% of those who were unaware. Of those >65 years of age, 52% of aware individuals needed drug therapy whereas only 34% of unaware elderly would need drug treatment.In both unaware and aware subgroups, the majority of patients with untreated hypertension would benefit from antihypertensive drug therapy according to the 2004 Canadian Hypertension recommendations. The proportion of untreated patients that still need drug therapy was higher among those who were aware compared to those who were unaware. This finding suggests that the major gap in hypertension control may be in initiating drug therapy rather than in diagnosing hypertension. Further studies are needed to confirm these results to ultimately help strategize public health efforts in controlling hypertension. Antihypertensive drug therapy can reduce cardiovascular morbidity and mortality by 25–30% -3 in thoFrom the 2004 Canadian Hypertension Guidelines, pharmacotherapy is recommended if the diastolic blood pressure is > 100 mmHg; the systolic pressure is > 160 mmHg; or the diastolic blood pressure is >90 mmHg in the presence of cardiovascular heart disease or risk factors. To determine the proportions of individuals who satisfied the guidelines for initiation of drug treatment for hypertension, we used the Canadian Heart Health Surveys (1986 – 1992). This national, cross-sectional descriptive survey (n = 23 129) contains data on individual blood pressure measurements using standardized technique as well as information on cardiovascular risk profile, antihypertensive medication use and self reported awareness of hypertension. Further details of this survey have been published elsewhere [Of those with untreated hypertension (= 140/90 mmHg), only 37% (n = 897 786) were aware of their diagnosis. Among those aware and unaware of their diagnosis of hypertension, 35% vs. 36% were female; 14% vs. 12% had any cardiovascular disease and 31% vs. 75% were under the age of 65 years respectively. The proportions of those who would be recommended drug therapy among those who are aware and unaware are presented in Table In both unaware and aware subgroups, the majority of patients with untreated hypertension would benefit from antihypertensive drug therapy according to the 2004 Canadian Hypertension recommendations. This analysis extends the findings from other studies ,7,8 by dThere are several limitations with this study. First, the Canadian Heart Health Survey data are 12 years old and there is some evidence to suggest that prescription rates of antihypertensive drugs have improved since then . HoweverThis study found a considerable number of both unaware and aware patients with untreated hypertension require drug therapy. However, the majority of those who needed drug therapy were actually aware that they had a diagnosis of hypertension compared to those who were not aware. This finding suggests that the major gap in hypertension control may be in initiating drug therapy rather than in diagnosing hypertension. Further studies are needed to confirm these results to ultimately help strategize public health efforts in controlling hypertension.The author(s) declare that they have no competing interests.NK, DW and NC contributed to the design of the project. NK contributed to data analysis. NK, DW and NC contributed to writing the manuscript and for substantive, intellectual editing contributions.The pre-publication history for this paper can be accessed here:


The process of identifying differentially expressed genes in miroarray studies with small sample sizes can be improved substantially by extracting information from a large number of datasets accumulated in public databases. t-test analysis with five samples in each group or to that of a regularized t-test analysis with three samples in each group. Our results are further improved by weighting the results of our approach with the regularized t-test results in a hybrid method.We demonstrate that the process of identifying differentially expressed genes in microarray studies with small sample sizes can be substantially improved by extracting information from a large number of datasets accumulated in public databases. The improvement comes from more reliable estimates of gene-specific variances based on other datasets. For a two-group comparison with two arrays in each group, for example, the result of our method was comparable to that of a  Microarray experiments are often used to identify potentially relevant genes in biological processes. By determining which genes are differentially expressed between different states, for example, hypotheses can be developed as to the role of those genes in the underlying biological mechanism -4. HowevOverall, the need for a large sample size is acute for expression profiling studies. The number of arrays needed in a study depends on many factors, including the study design, the magnitude of biological variation in the samples, technical variability introduced in the experiment, and the desired level of sensitivity and specificity for differential expression. Several studies have examined this issue. A model with additive and multiplicative noise was used to derive the number of samples necessary for detecting fold changes of given magnitude when false-positive and false-negative rates are specified . The dift-test, although other similar statistics such as the signal-to-noise ratios [p-values for multiple testing [In the context of finding differentially expressed genes, the null hypothesis for each gene is that it is not differentially expressed between two groups, usually against the two-sided alternative hypothesis that the gene is up- or downregulated. The most commonly used statistical test in this setting has been the two-sample e ratios  have oft testing  and the  testing . Ideally testing , but in  testing . The mett-test or variations thereof is crowded by a large fraction of genes for which large t-statistics are due to underestimation of variance by chance.A fundamental difficulty in drawing reliable conclusions from a small number of samples lies in accurate estimation of the gene-specific variances, or the variance of a difference in mean expression levels per gene, with which to determine the statistical significance of observed changes in expression. Because variances based on a very small number of samples tend to fluctuate wildly as a result of randomness in sampling from a population, our ability to assess differential expression is drastically impacted. A naive application of standard methods used for larger sample sizes can result in a large number of false positives for differential expression. For example, with a small sample size, the list of significant genes identified by the t-test. A Bayesian framework for combining the variance estimate with a background variance associated with neighboring genes was developed in [Many methods have been devised to address this problem. A popular approach has been some type of regularization of the loped in ; a metholoped in . In the loped in ; empiricloped in -17. Nonploped in , analysiloped in , and Bayloped in ,21 are aloped in .Whereas all the available methods attempt to improve the identification of differentially expressed genes essentially by gathering information across similar genes, we suggest another solution. We propose estimating the natural variance of individual genes using a large number of experiments performed previously. This provides a different and potentially more stable and accurate estimate of the variance for each gene than by simply looking at the variance of a small number of expression levels, especially in studies with very small sample sizes. Using these variances as the basis for determining differential expression offers an alternative method that can reduce the false-positive rate significantly. As the most effective method, we propose a hybrid method in which we combine the variance estimate from the current dataset with the estimate from previous experiments. This approach can also be incorporated in other settings, especially in a Bayesian framework with prior distribution for variance derived from the database. It can be applied more generally to other testing procedures such as ANOVA that benefit from more accurate estimation of gene-specific variances, and can be easily extended to the estimation of the covariance matrix in multivariate analysis.More reliable calculations of such variances based on many chips is becoming increasingly possible through large public databases of previous experiments. Public databases such as the Gene Expression Omnibus (GEO)  contain t-test, the new GEO-adjusted method, the regularized t-test, and a hybrid method combining the GEO method and the regularized t-test. The primary difference between these methods lies in the denominator of each method's t-statistic. The GEO-adjusted method replaces the sample variance estimate in the denominator with the gene-specific variance calculated from the GEO database . Hence, the genes are sorted using the modified t-statistic:We compared the performance of four methods in accurately assessing differential expression of genes: the standard μi1, μi 2are the means for the groups 1 and 2, respectively, for the ith gene, n1 and n2 are the sample sizes in the groups 1 and 2, and σ2 GEO,1 is the gene-specific variance from the GEO database.where t-test, ranking genes based on the modified t-statistic:The regularized method we used added a small constant to the denominator of the σ0 is the fifth percentile of all variances . The reFinally, we introduce a hybrid algorithm that combines the GEO method with others through a voting mechanism. This provides a portable solution that can be combined with a variety of other testing procedures and could potentially improve the performance of any other algorithms designed to determine differential expression in experiments with small sample sizes.t-test analysis for the full dataset as the master list; with a sufficiently large dataset, this master list is a close approximation to the true list. Thus, we used the large dataset to compute a true t-statistic for each gene and then treated random small subsamples of the arrays from the dataset as simulated observed datasets from which we could compute estimated ranks for small subsamples. Because exploring all realizations of possible subsets for a large full dataset would be prohibitively time-consuming (for example, more than 108 combinations of 3v3 subsets for our first dataset), we sampled repeatedly for subsets until we obtained convergent results. We then compared these lists of differentially expressed genes with the master list for overlaps or correlations in the orderings. Once the size of the subsets approaches the size of the full dataset, there can be a substantial overestimation in the overlap of genes, owing to the fact that the master list is generated using the dataset from which the subsamples are derived. However, this effect appears to be negligible in our simulations because of the large size of our full dataset and the small subsample sizes that are of our interest.To compare the effectiveness of the methods, we determined lists of differentially expressed genes in order of significance by applying each procedure to a large number of subsets of arrays of a given size. These genes were then compared with the 'master' list of differentially expressed genes to assess the accuracy of the method. Because we generally do not know the correct ordering of genes with differential expression, we substituted the list obtained by the The dataset primarily used for testing was a prostate cancer dataset  that hadt-test was compared to the GEO-adjusted method. The behavior of the correlation coefficient was tracked as the number of genes being analyzed was increased and the averaged values over many simulations are shown in Figure t-test as the sample size increases, and that the results of GEO tend to correlate better with the master list than the results of 2v2, 3v3, 4v4, or 5v5 t-test.The first measure that was used to assess the effectiveness of the GEO-adjusted method was the correlation between the rank of the top genes returned by various methods and the true rank of those genes. This method was also used in . In thist-test at determining differentially expressed genes in small sample sizes. Compared to a simple t-test, the GEO method performs substantially better, returning results from a 2v2 test that are comparable to the results returned by a 5v5 experiment using t-test. Using GEO variances on a 2v2 test returns 231% more of the top 50 genes than the unadjusted t-test. While we are not suggesting that a simple t-test is a recommended method of assessing differential expression in such small sample sizes, it illustrates the potential of this method. Using gene-specific variances developed from GEO databases is clearly more accurate than the variances that an uninformed t-test derives from small samples. We do not plot the error bars for each measurement in the figures owing to space constraints, but we have verified in the important cases that the separation between the curves is significant.To further assess the reliability of the results, tests were conducted to determine the number of top 50 genes from the master list that were accurately returned using various methods. In Figure t-test. By smoothing out the variance estimates, the regularized t-test returns a more accurate assessment of differentially expressed genes than the standard t-test. Thus the gains from the GEO method over the regularized t-test were less substantial than over the standard t-test but still significant, especially for shorter gene lists. Improving our ability to reliably detect the differentially expressed genes with a short list is generally more valuable than doing so with a longer list simply because these genes at the top are the ones that an investigator examines most closely. As shown in Figure t-test in those three areas (50, 100 and 150 genes) was more than 30%. The performance of GEO on a 2v2 analysis seems roughly comparable to the performance of the regularized t-test on a 3v3 sample analysis.The GEO-adjusted method also compared favorably to a regularized t-test and the GEO method both use different, yet effective, techniques to smooth out variance, they can both contribute to the differential expression analysis. By using a voting method that weights and averages the results returned from the regularized t-test and the GEO method, the performance improves further . The results of a 2v2 chip analysis using our voting method nearly match the performance of a 4v4 regularized t-test analysis, which is quite promising /((x + y)/2); and z-score based on local variance correction  across signal intensity, as implemented in SNOMAD [t-test. The method based on the z-scores performs slightly better than either of the standard methods, but GEO still returns 57% more of the top 50 genes. In the 1v1 case, the voting method proves useful, improving the results of both methods. By combining the z-score method and GEO's rankings, the results are superior to a 3v3 regularized t-test analysis. The voting method captures 83% more of the top 50 genes than the best of the standard methods. These results reflect the success of the voting method in combining GEO's rankings with a variety of other methods to significantly improve the overall performance.Tests were also performed on other sample sizes, namely 1v1 and 3v3. Although we view the first case especially as an inadequate design and do not recommend it, we have found that investigators are sometimes forced to perform analysis on such a small number of samples. We are therefore interested in improving the effectiveness of such exploratory analysis, the results of which must be verified using other techniques such as quantitative reverse transcription PCR (QRT-PCR). In our 1v1 analysis, we compared the GEO method to three methods of ordering genes on the basis of differential expression: fold ratio, n SNOMAD . Basic ft-test and GEO method were also compared on 3v3 comparisons. Whereas GEO still returns more reliable estimates than the regularized t-test, the improvement is smaller than in the case of the smaller sample size comparisons. In the 3v3 case, the GEO-method results are comparable to those of a 4v4 regularized t-test, returning 17% more of the top 50 genes than the 3v3 regularized t-test. However, we do find that the voting method again improves the results, returning very similar numbers of correct genes as a 5v5 regularized t-test . Because two samples capture a much higher percentage of the data in 11 chips than in 50 chips, we expect the usual tests on subsamples to naturally provide results more similar to the master list. Therefore, we expect to see less of an improvement from GEO than in our cancer dataset. As before, we see the GEO results consistently providing better results in the smaller sets of genes than the standard e Figure  and 40-1e Figure . While tt Figure . In the For this method to be successful, a significant number of previously run chips must be available. As public databases grow in size and number, this limitation will gradually diminish, but not all chip types currently have enough available chips to use this method. Whereas the most popular chip types (such as Affymetrix HG-U95A) have hundreds of previously run chips available, it is more difficult to find databases of the less popular ones. In an attempt to test for the number of chips sufficient to utilize this method, variance analysis was performed. In Figure t-test. If our gene-specific variances were based on even more reliable estimates (such as samples from the same tissue or same disease), the performance of the GEO method would probably be increased. As public databases grow in size and organization, this should become increasingly possible.When trying to estimate the gene-specific variances for a particular experiment, the best approximation would come from a database of similar experiments. Because gene expression profiles have the potential to vary widely in cell and tissue type, examining many other chips of the same tissue type should provide the best indication of the baseline variance. For example, it would make most sense to draw on a large database of cancer chips to derive relevant information for a cancer dataset. Unfortunately, because of the dearth of large datasets that match each other in tissue type, chip type, and post-processing, it is difficult at this time to test this theory. Because our public databases do not yet contain enough chips sorted by tissue type to perform this procedure, we are forced to mix all the chips of any given type together. Yet, even with only a database of totally unrelated chips, we still saw a significant increase in performance, even over already improved methods such as the regularized We have shown here how to derive more stable variances based on chips of the same type. The problem is much more complicated when multiple chip types are involved. In fact, we have observed that even different generations within the same platform do not give concordant results. When the same tissue samples were hybridized on both HG-U95A and HG-U133A chips, the dominant feature in the data was the chip type rather than the sample characteristic, and the lists of differentially expressed genes differed substantially between the two cases. Standardizing across these two types can be done for a portion of the genes but it is an involved process (Hwang KB, Kong SW, Greenberg SA, P.J.P., unpublished work). Efficiently combining data from single-channel and double-channel arrays is even more difficult. A more comprehensive database with a larger number of arrays spanning a greater variety of experiments would alleviate the problem to some extent, but methodologies for integrating data from multiple platforms will be essential, not only for better estimation procedures for differential expression but for other purposes as well.Public databases are an important resource for investigators to consider. With the stores of chips accessible online, valuable information concerning genes can be compiled and used to supplement new studies and avoid duplication of effort. This methodology would be improved by modification to the databases. Mainly, it is very important to start gathering the raw files instead of processed files. With Affymetrix chips, for example, .cel files should be stored, so that they can be processed using the latest methodology and maintain their usefulness. Already, many of the chips in the GEO database are less useful because they only report values processed through MAS 4.0, an outdated methodology, and comparing these with values generated through MAS 5.0 introduces another source of variation. In addition, the chips should be categorized and sorted according to tissue type, to further facilitate grouping and analysis. These modifications would improve the ability to use previously run chips, thus countering the high costs associated with microarray experiments and enabling the sharing of information to accelerate progress. Thinking about how to take advantage of these databases could provide further improvements to methodologies and enable more tools to be used to study gene functions.This work proposes that value lies in pooling information from previous studies. Specifically, gene-specific information can be collected from public databases housing many chips, supplementing new studies and ensuring more reliable results. We show that compiling information from databases provides us with a different and potentially more accurate estimate of gene-specific variance, improving our differential expression analysis in small samples. In addition, because this improvement seems largely independent of the method of analysis, we are able to combine it with regularization in a voting method, leading to superior results. There were particularly strong improvements in the identification of the smallest groups of most differentially expressed genes, which would probably be deemed most important by an investigator as they are easiest to validate. Overall, the scale of the improvement is significant, as it allows investigators to halve their costs in some cases and still retain similar accuracy. The same approach can also be formulated in other settings, especially in the Bayesian framework in which priors for the gene variances may be estimated from previous datasets. Furthermore, as public databases are steadily growing in size, we expect refinement of this method to deliver greater success in the future. Regardless of what method an investigator might use, public databases are clearly a useful source of information and should prove useful in supplementing microarray studies.Because the very nature of public datasets implies that many of the chips have been generated and processed in different manners, standardization of the data is paramount. To maintain comparability, the chips were filtered to remove any chips processed with an algorithm other than MAS 5.0. After removing other unusable chips  471 HG-U95A chips remained.Normalization of all of these chips is crucial, in order to guarantee that scales are similar. In an effort to preserve the general characteristics of each chip, conforming their scales while allowing for some chip-by-chip variability, experiments with multiple methods of normalization were carried out. The two major types included normalizing the trimmed mean and trimmed variance of each chip and using percentage ranks instead of numerical expression levels. In the first case, all of the data points were adjusted to align the mean and variance of the middle 90% of values. In the percentage ranks method, the values were assigned percentiles, removing most normalization effects. In addition, a scale was generated that related the percentile with the average rank change of that percentile. The average rank change for a gene in the middle of the scale was significantly larger than the average rank change at either extreme. This scale was used to adjust the variances on the basis of the rank. Because the results from both normalization methods were fairly similar, only the results of the trimmed mean, trimmed variance experiments are reported here.After all of the chips were normalized, the gene-specific variance was calculated. These variances were calculated in two separate ways, using a global variance and a pooled variance:xij is the expression level of array i in experiment set j;  the mean in the experimental set j;  is the mean in all arrays; D and Dj contain the indices for the experimental sets and the arrays in the jth set, respectively. The global variance tends to reflect the degree that a gene may vary between different tissue types and diseases while the pooled variance reflects the degree that a gene tends to vary within each experiment. The global variance proved slightly more effective in the cancer dataset, while the pooled variance was more effective in the muscular dystrophy dataset. This seemed to be correlated to the composition of our GEO background datasets. Our set of 471 GEO chips contained 210 cancer chips but only 42 muscle chips. Because a large proportion of the total chips were cancer chips, a global variance may have more accurately represented the information in the whole dataset. However, because so many of the GEO chips were non-muscle, readjusting them into a pooled variance format may have provided a better gene-specific assessment of general expression. We further filtered the variance calculations to eliminate artifacts created by improperly processed chips along with biases from experimentation . Thus, the highest and lowest 10% of values for each gene across the full set of GEO arrays was trimmed off for the variance calculation. The 10% parameter was chosen experimentally, by tracking how stable variance calculations were as various percentages were trimmed.where, for each gene, N(0,1) asymptotically. Because the GEO data are an aggregate of many experiments under different conditions often processed differently, we cannot assume the same underlying distribution in general and hence we do not know if the estimators necessarily approach the true variance. However, these estimators appear to be reasonably good approximations to the 'true' variance as demonstrated by the numerical results, and they certainly perform better than estimates based only on the current data.The statistical properties of these variance estimators are difficult to show rigorously. If the samples from the GEO datasets can be assumed to come from the same population as those in the current study, the estimators should be unbiased and the proposed test statistic should behave as t-test analysis. Then, the various methods were compared with each other through a process of subsampling to determine how accurately the results reflect the master list. After two samples from each group were randomly selected, all the genes were filtered out that did not have an expression level above 100 in any of the samples. The goal was to lower false positives among the non-GEO methods, as their results could easily be influenced by small expression levels that by chance ended up with virtually no variance and thus were assigned large t-statistics. After processing in this way, the top genes derived using the t-test, the regularized t-test, and the GEO-adjusted method were compared with the master list to determine their effectiveness. This subsampling procedure was repeated 500 times for each experiment, and the results were averaged. These methods are outlined in the Results section.A master list of the most differentially expressed genes in the dataset was determined by t-test and the regularized t-test, the greatest success was found by averaging the results of the regularized t-test method and the GEO method. By averaging the ranks that we receive using GEO and using the regularized t-test set, our results are improved concerning our most important genes. This is not seen when the results of the simple t-test and GEO are combined, because the lists produced by the simple t-test are simply too inaccurate. However, as GEO and regularized t-test produce lists that are similar in quality, yet different in nature, a boost can be obtained by averaging the lists. Because using just the GEO variances ignores some of our experimental data and using just our experimental variances ignores global data, it seems that an averaging or voting procedure is a superior way to optimize results. In particular, the system that we used averaged 75% of the value of the lower rank (nearer the top of the list) with 25% of the value of the higher rank. A final score was obtained by combining the results from each method in this way, and the genes were re-ranked on the basis of this score. By using the 75%/25% ratio, genes that have a particularly high ranking on one of the methods are given slightly more importance than genes that have average rankings in both methods. Empirical testing of a number of combinations showed that the 75%/25% combination returned superior results, although all combinations experimented with returned results that were better than either method alone.Although the GEO-adjusted method was superior to both the


It provides an excellent elementary introduction to this topic.Following an illustrative historical introduction, the author briefly reviews vascular physiology. This is followed by the basics of fluid mechanics as an introduction to the hemodynamics of large arteries. A dedicated chapter illuminating the dynamic consequences of vascular branching follows this chapter. This is the field to which Professor Li has made his substantial contributions.The following chapters cover the venous system and microcirculation. Finally the book reviews measuring techniques used to study hemodynamic behavior.The author suggests this volume to be "a companion" to his own treatise "The Arterial Circulation" . For thoWhile the new book adds useful information on the venous system and on microcirculation, a topic that has been neglected in classical treatments of hemodynamics, this new volume is substantially less comprehensive in most topics covered in both. Also its index is regretfully, substantially less detailed. Little new has been published in this field since the first book was published in 2000, with the notable exception of Zamir's book "The Physics of Pulsatile Flow" [In brief, this book, which is more affordable than its predecessor, should be regarded as a good introduction to the topic, to be used primarily by bioengineering students, rather than an updated authorative text by its erudite author.


A learning module based around the case of a man with ezema who presents with a rash around his ear and eye. Test your knowledge with our online quiz A 33-y-old man presented to his primary care practitioner with a rash beginning around the left ear and spreading into the periorbital region. He was known to have eczema (atopic dermatitis), and the diagnosis of secondary bacterial infection and periorbital cellulitis was made.Because of a history of penicillin allergy, the patient was started on erythromycin. This treatment had no effect, and the rash extended. On day 4, he presented to the hospital accident and emergency department. He was systemically unwell, complaining of drenching sweats and rigors. On examination he was alert, he was pyrexial at 39.2 °C, his pulse was 100 bpm, and his blood pressure was 128/70 mm Hg. There was a widespread erythematous rash covering his face, chest, and arms, which was described as wet with a yellowish exudate. The rash extended around both eyes, which could not be opened .The patient had a history of atopic eczema (since childhood), rhinitis, and asthma. He was taking inhaled corticosteroids for asthma and had last received a course of systemic steroids one year previously. There were no risk factors for HIV infection.In many patients with a rash, the morphology of the rash will lead to a provisional clinical diagnosis, and it may be sufficiently characteristic for no confirmatory tests to be needed. An example is the typical rash of shingles. There are two broad reasons for doing investigations in a patient presenting with fever and a rash. Firstly, specific tests—such as blood tests, cultures, or a skin biopsy—may be required to help establish the diagnosis. In this 33-y-old patient, the rash was thought likely to be a skin infection, and so the specific tests that were indicated included viral and bacterial cultures. A skin biopsy would have been considered if the diagnosis remained unclear and the patient was not responding to empirical antimicrobial therapy. Secondly, investigations are used to evaluate how ill the patient is and whether there is any organ involvement beyond the skin. In this case, we were most concerned about severe sepsis with a risk of organ failure.9/l ; and differential white blood cell count, 90% neutrophils with a left shift. The patient was hyponatraemic , but renal and liver function tests were normal. C-reactive protein was 140 mg/l . Chest X ray was normal. Blood cultures and skin swabs were taken along with samples for viral culture and polymerase chain reaction (PCR).Investigations showed the following: haemoglobin, 128 g/L ; white blood cell count, 9.9 × 10A provisional diagnosis of bacterially infected eczema was made, and, in view of his history of penicillin allergy, the patient was started on intravenous vancomycin. He was admitted and referred for dermatology, infectious diseases, and ophthalmology reviews.On closer inspection of the rash, it was apparent that there were multiple small vesicles 1–2 mm in diameter and shallow ulcers over the face and periorbital region . There wIn view of the characteristic picture and underlying eczema, the diagnosis was revised to one of severe eczema herpeticum. The patient denied any previous history of oral cold sores or genital ulceration. However, the severe degree of skin inflammation, high fever, raised C-reactive protein, and left-shifted neutrophils suggested co-existent bacterial sepsis.High-dose intravenous aciclovir (10 mg/kg every 8 h) was started. Aciclovir inhibits thymidine kinase, which is an essential enzyme for several herpes viruses. Aciclovir has excellent activity against herpes simplex virus (HSV), and, although less potent, it is also effective against varicella zoster virus [Staphylococcus aureus [This man had severe skin involvement with a high likelihood of complicating bacterial sepsis, which is most commonly due to s aureus . ErythroS. aureus, which was resistant to penicillin, erythromycin, and clindamycin but sensitive to all other agents. Skin and eye swabs also grew S. aureus. HSV DNA was identified by PCR on fluid taken from the face. Viral cultures were positive for HSV-1. Serology taken on admission was positive for HSV-1 IgG.Two separate sets of blood cultures subsequently grew The patient rapidly defervesced, and the vesicles desquamated on day 6 . He was S. aureus. As mentioned above, the eyes were proactively managed to avoid corneal involvement. The S. aureus bacteraemia could have progressed to severe sepsis or led to metastatic spread of infection. Echocardiogram was normal with no evidence of endocarditis, and intravenous vancomycin was continued for a period of 14 d to minimize the risk of metastatic spread. Close attention to infection control procedures is essential to prevent hospital-acquired infection of the damaged skin or intravenous line sites.In the acute stage of his presentation, our two main concerns for this patient were the extent of involvement of the eyes beneath the eyelids and the positive blood cultures for Kaposi's varicelliform eruption describes the widespread dermal infection caused predominantly by HSV-1 in patients with a variety of skin conditions . It is mPrompt use of antiviral therapy is highly effective in controlling HSV replication. Aciclovir is the antiviral agent of choice and is usually given for 7 d. Patients with moderate to severe disease require admission for intravenous aciclovir. Oral aciclovir is effective for mild cases, which can be managed on an outpatient basis . BacteriA proportion of patients may experience recurrent attacks of eczema herpeticum . RecurreA recent retrospective analysis of 100 cases of eczema herpeticum in patients with atopic ezcema highlighted some predisposing factors. The analysis found that patients with eczema herpeticum had developed atopic eczema earlier and had higher total serum IgE levels than control patients with atopic eczema alone . In addiThe rapidly progressive, widespread, crusted papules, vesicles, and erosions in a patient with atopic eczema are characteristic, and an experienced physician who has seen eczema herpeticum before will make a rapid clinical diagnosis. However, some patients present with atypical or purely crusted lesions and no obvious vesicles, making a clinical diagnosis more difficult. Thus, a high index of clinical suspicion is required, and both viral and bacterial cultures are essential when a patient with atopic eczema presents with a spreading vesicular or crusting rash . Rapid cA similar and potentially fatal disease may follow smallpox vaccination ,10. UntiEczema herpeticum is an uncommon but potentially fatal disorder that can present to primary care practitioners or to the emergency department.Diagnostic delay is common and may increase morbidity.First-line therapy is with acyclovir; intravenous high-dose therapy is required in severe cases.Patients should be referred to a dermatologist for specialist care and follow-up to optimise eczema management, so that recurrence is less likely.Intensive nursing care is indicated to minimise scarring and promote rapid healing.If the periorbital region is involved, refer urgently to an ophthalmologist.