It already exists within the US Food and Drug Administration, argues a former clinical reviewer of psychotropic drugs at the FDA It already exists within the US Food and Drug Administration Over the past several years, there has been growing concern about selective publication of clinical trial results ,2. The dThe two most frequently suggested remedies for the selective reporting of clinical trials results have been to register all clinical trials and to make their results publicly available. Registries have been called for at least as far back as 1974; hundreds have in fact already been established . Thus, oIncreasing the pressure on pharmaceutical companies to include more trials in registries, the International Committee of Medical Journal Editors has announced that, as a condition of considering a trial for publication, member journals will require its registration in a public trials registry . FurtherIn this essay, I argue that a highly valuable but underused registry and results database for US trials already exists within the Department of Health and Human Services, specifically within the Food and Drug Administration (FDA).Before a pharmaceutical company can conduct a US trial that it intends to use in support of a new drug application (NDA), it must first register that trial with the FDA. Because the NDA forms the basis for marketing approval, it seems likely that the percentage of industry-sponsored trials that are registered with the FDA is very high. This registration takes the form of an investigational new drug (IND) application . The INDThe FDA then begins the NDA review process, during which a physician, a statistician, and a pharmacologist, among others, generate lengthy review documents . These rA Cochrane systematic review of antidepressants for generalized anxiety disorder  lists onp = 0.039) result on a secondary observed-cases analysis, but a nonsignificant (p = 0.38) result on the primary efficacy analysis defined a priori. The third study was clearly negative, with p-values of 0.33 and 0.57 on the primary and secondary analyses, respectively.Turning to the controlled-release formulation of paroxetine (Paxil CR) for panic disorder, a review article states in its abstract that the drug “demonstrated efficacy in three well designed studies in patients with panic disorder with or without agoraphobia” . In readhttp://www.accessdata.fda.gov/scripts/cder/drugsatfda/index.cfm). These NDA review documents are much more detailed than the resulting package insert and often more detailed than corresponding journal publications.This process occurs entirely outside of the public domain. However, in the interest of making the FDA more “transparent,” and in accordance with the Electronic Freedom of Information Act , the FDAFor example, while the clinical trials section of the package insert is typically a few paragraphs long, the efficacy portion of the clinical review usually runs tens of pages. Because the FDA is made aware of all studies that the sponsor plans to use in support of the NDA before they are conducted, and thus before there can be any selection based on outcome, these reviews cover not only studies that are positive , but also studies whose outcome was negative or indeterminate. The sidebar gives an example of how NDA review documents at the FDA give valuable information about paroxetine for anxiety disorders.In the examples discussed in the sidebar, our having access to the FDA review documents allows us to become aware of, and see beyond, apparent publication bias. It is in the best interest of the public health for the FDA to make as many reviews available as possible. According to the FDA Web site, “As FDA continues to be one of the world's leading agencies in its emphasis on openness and transparency, it is aware that making even more information available to the public will further the Agency's mission to protect and promote public health and improve its credibility. For example, FDA has aggressively implemented the Electronic Freedom of Information Act…” .Unfortunately, the availability of review documents on Drugs@FDA is sporadic. To take additional examples from psychiatry, NDA reviews have been posted on Drugs@FDA for some approved drug–indication combinations, such as fluoxetine for pediatric depression, and aripiprazole and quetiapine for schizophrenia. However, NDA reviews for many other drug–indication combinations have not been posted: the Prozac Weekly formulation of fluoxetine, clozapine for suicidal behavior in patients with schizophrenia or schizoaffective disorder, and quetiapine for mania, among others. A review on paroxetine for pediatric depression, the subject of Elliot Spitzer's suit against GlaxoSmithKline, is not posted. This is probably because this drug–indication combination was not approved; in fact, it is possible that GlaxoSmithKline did not file an NDA to be reviewed. However, I do not understand why, in cases where NDAs were both submitted and approved, such as the ones listed above, some reviews are posted while others are not.I therefore suggest that we increase access to the clinical trials registry and results database that already exist within the FDA. The agency could expand its implementation of the Electronic Freedom of Information Act and make all NDA reviews, at least for approved NDAs, available in the public domain. The act is written into the FDA portion of the Code of Federal Regulations as follows: “The Food and Drug Administration will make the fullest possible disclosure of records to the public, consistent with the rights of individuals to privacy, the property rights of persons in trade secrets and confidential commercial or financial information” .There would surely be obstacles. The pharmaceutical industry would vigorously invoke Exemption 4 of the Freedom of Information Act, the exemption for trade secrets and confidential business information . HoweverIt should be clarified that this resource does not compete with proposals by the AMA and other groups for clinical trial registries—rather, it complements them. The AMA has proposed the creation of a registry that is comprehensive in scope. The FDA's registry and results database are restricted to those trials aimed at supporting US marketing approval or a change in labeling in the US. While data from many studies conducted abroad are submitted to the FDA for this purpose, this is not the case for drugs for which the sponsor has elected not to seek approval for marketing in the US. Nor does the FDA review data from most trials funded by other US government agencies, such as the National Institutes of Health, or by foundations. And drug companies fund investigator-initiated trials that are often not registered with the FDA.To make the FDA review data more accessible and user-friendly, simple formatting changes would be needed. For those (few) reviews that are currently posted on Drugs@FDA, one can determine the indication being evaluated only after opening the document and paging through it.  DespitDespite the limitations of the FDA's database, making it public is a strategy that could be implemented both rapidly and easily by building upon existing infrastructure. While we await the creation of a clinical trials registry and results database that is truly comprehensive, we already have at our disposal one that could serve as a trove of in-depth and unbiased information on many, if not most, drugs currently marketed in the US.


The developments in echocardiography or ultrasound cardiography (UCG) have improved our clinical capabilities. However, advanced hardware and software capabilities have resulted in UCG facilities of dubious clinical benefits. Is tissue Doppler echocardiography (TDE) is one such example?TDE has been touted as advancement in the field of echocardiography. The striking play of colors, impressive waveforms and the seemingly accurate velocity values could be deceptive. TDE is a clear case of inappropriate use of technology.To understand this, a comparison between flow Doppler and tissue Doppler is made. To make clinically meaningful velocity measurements with Doppler, we need prior knowledge of the line of motion. This is possible in blood flow but impossible in the complex myocardial motion. The qualitative comparison makes it evident that Doppler is best suited for flow studies.As of now TDE is going backwards using an indirect method when direct methods are better. The work on TDE at present is only debatable 'research and publication' material and do not translate into tangible clinical benefits. There are several advances like curved M-mode, strain rate imaging and tissue tracking in TDE. However these have been disappointing. This is due to the basic flaw in the application of the principles of Doppler. Doppler is best suited for flow studies and applying it to tissue motion is illogical. All data obtained by TDE is scientifically incorrect. This makes all the published papers on the subject flawed. Making diagnostic decisions based on this faulty application of technology would be unacceptable to the scientific cardiologist. Echocardiography or Ultrasound Cardiography (UCG) is a key investigation in cardiology. Its non-invasive nature makes it a widely acceptable and safe form of investigation. In many cases it has surpassed cardiac catheterisation in diagnostic yield and has become the investigation of choice.The rapid development of UCG has made available to the clinician newer insights into the anatomy and physiology of the heart. With the availability of digital technology it is possible to manipulate raw data in different ways. This has also spawned UCG facilities of dubious clinical benefits. Tissue doppler echocardiography (TDE) is one such example.When we consider UCG we are interested in 2 distinct aspects of the heart:1. Structure 2. Blood flow. Both these ultimately indicate cardiac function. B-mode/M-mode imaging or simply Ultrasound Imaging (USI) studies structure. Blood flow is studied by Doppler studies (DS). In each stream every development should incrementally improve our understanding of the heart, its structure and function. Or the development should improve the ease of operation or permit better documentation.In USI we had the following developments and each development added to the diagnostic yield. M-mode – temporal resolution; 2D – spatial resolution; Harmonic imaging – improvement in image quality; B-color – enhanced tissue perception; Ultrasonic tissue characterization and acoustic quantification – quantitative analysis of tissue; Omni-plane M-mode – temporal resolution at different segments and 3D imaging – another dimension of imaging.In DS too we had many developments, each improving our perception. Continuous wave (CW) – detected and measured high velocity flows; Pulse wave (PW) – located the abnormal flow; Color flow mapping (CFM) – made it possible to 'image' blood flows and power amplitude Doppler – to study vascular flow. Contrast, trans-oesophageal and intravascular UCG are the clinical extensions and exploitation of these developmentsWhat we see in TDE is a retrograde development. In the context of UCG, DS is an indirect method to study blood flow since USI in its present state cannot image fast moving blood cells. Spontaneous echo contrasts could mark the beginning of USI of blood cells. By using TDE we are using an indirect method where direct USI methods are better. Besides, TDE is an inappropriate application of the Doppler methodology.TDE is a by-product of color flow mapping (CFM) technology. In CFM, tissue signals are suppressed and flow signals are analyzed. The reverse is true in TDE. Doppler velocities associated with tissue motion are slower than blood flow. Flow signals are eliminated on the basis of signal amplitude. The amplitude of tissue motion is about 40 db greater than the corresponding flow amplitude. Blood flow imaging applies a high pass filter to exclude the strong but low frequency tissue signals and other 'noise' before the signal is input into an autocorrelator that estimates velocity. ErroneouThe first report on the use of TDE appeared in 1989. However all data using this modality is flawed. You cannot even think of designing a study to prove the point because the mensuration technique itself is wrong. So we have to use the scientific methods of comparison and reductio ad absurdum to verify it. Any new modality of diagnosis or treatment should be compared with the existing systems. In clinical trials we usually employ quantitative comparison with statistical methods. In this case, since the data acquisition is flawed we have to use a qualitative comparison. This is accepted practice in clinical medicine – It is like comparing a weak limb with the strong one in neurology. One also needs to understand the Doppler methodology to test the hypothesis.The article explains the flaws in tissue Doppler echocardiography. As the concept itself is flawed, To estimate clinically significant peak Doppler velocity the following steps are required:prior knowledge of the line of motion of the object. Only if you know this line of motion, you can apply the interrogating signal along the path of motion. This is because of the directional bias of Doppler.1. You must have 2. Next you will know the direction of motion – whether the object is moving towards the interrogating signal or away.3. Only after the first two steps you can measure the clinically significant peak velocity.Based on the above discussion it is clear that the following information can be derived from Doppler:1. Is there motion? Does the object move? This is a random application of Doppler. This is one aspect of tissue Doppler. But this information is redundant as we can already see the movement better by ultrasound imaging.2. What is the direction of motion? Is it moving towards or away from the interrogating signal? This is also redundant information in tissue Doppler for the reason given above.significant velocity we must know the clear-cut line of motion. Application of Doppler along this line enables us to measure the meaningful peak velocity.3. To measure the peak anticipated flow. That is also why we do not measure the mitral flow velocity from the parasternal views. Next we come to learn whether the blood is flowing towards or away from the transducer. After all these steps it is possible to measure velocity. In tissue Doppler we can measure several velocities in several directions. But we can never know which is the clinically significant peak velocity.Thus the primary step is to know the line of motion. This is possible in flow Doppler but impossible in tissue Doppler. For example we know for sure that the blood moves from left atrium to left ventricle through the mitral valve. At the mitral valve this is a unique free linear motion towards the apex. This produces a clinically significant velocity. That is why we place the sample volume at the mitral valve from apical views so as to align the beam in the line of TDE is a clear case of misuse of technology. To understand this, a comparison between flow Doppler and tissue Doppler is in order. CFM allows us to 'see' what we cannot see with ultrasonic 'eyes' hence its value is great. In color TDE we see more or less what we already see by USI hence its value is marginal. In CFM the anatomic landmarks are intact as the color is superimposed on the B-mode image. In color TDE the B-mode is eliminated and the entire 'picture' is Doppler information. It is difficult to determine the different anatomic regions on TDE. For example it would be very difficult to delineate the blood-endocardial boundary. In fact, the word 'tissue Doppler' is a misnomer and this is one of the reasons for the prevailing confusion. It gives the impression that only myocardial tissues are studied. The appropriate term would have been low velocity Doppler. Any movement in the low velocity range will be detected by 'tissue Doppler'. Myocardial tissue movement is just one among them. The discriminatory power of this modality is unsatisfactory.Myocardial motion is very complex and not amenable to Doppler studies. In the cardiac motion there are translational, rotational and deformational movements. Besides many tissues near the heart move – due to transmitted cardiac motion, vessel pulsation, respiratory motion and involuntary muscle movements and these interact with cardiac motion further and cause false Doppler shifts. VelocityBlood flow is simple and suitable for Doppler study. In flow Doppler at a particular point the linear projectile motion of free moving blood cells are studied. At the current interrogation points there is a unique unidirectional flow in one part of the cardiac cycle. For example in mitral valve Doppler interrogation, the unique directional signal is obtained only in diastole. If there is a signal in the in systole, it becomes abnormal and this information has great value. In TDE the to- and- fro motion (systole and diastole) of a tethered interconnected syncytium of myocytes is imaged. Such information is useless. This can be even otherwise seen and analyzed by USI.Doppler in its present form, is best suited to study flow.In flow Doppler there are definite 'points of interrogation', which are the normal and abnormal orifices. In TDE there are no such definite points. While in flow Doppler higher velocities are studied, TDE studies lower velocities i.e. the study of hypo functioning myocardial segments. Lower velocities are difficult to appreciate. The higher velocities are easier to appreciate due to aliasing and variance. There are no such indicators for low velocity. Thus hypo function is difficult to analyze. The derivations from flow Doppler are based on accepted hydrodynamic formulae (like Bernoulli's equation) and allows us to get orifice size, amount of flow and pressure gradients, which are clinically of great importance. The derivations from TDE are useless. Thus It is important to note that in all the above papers the data acquired is flawed due to the basic problem in technology application. Hence the correlations obtained in these papers are spurious or non-sense correlations. One paper presents a misuse of statistics when tissue Doppler velocities have been correlated with myocardial interstitial fibrosis and myocyte interstitial beta-adrenergic receptor density.[TDE has shown some promise in the Wolff-Parkinson-White syndrome. But even density. By analoThe prime use of TDE is to study regional wall motion abnormalities. It is claimed that it is possible to quantify wall motion at rest and during stress. However with the above-mentioned limitations it is unlikely to prove more effective than USI. The measurements are ultra sensitive and represent gross movements rather than myocyte contractions. With the advent of omni-plane M-mode it is possible to get temporal resolution in inaccessible planes and study wall motion abnormalities in a better way.The Doppler methods use mathematical formulae to indirectly study motion that cannot be directly measured. Examples are movements of stellar bodies and in the present context movements of blood cells. We use the indirect method based on Doppler principle because we cannot 'image' blood cells by USI. When the imaging of blood cells become possible, Doppler studies would probably be relegated to the background. Color TDE would have been valuable if direct USI was not available.Tissue Doppler has been a disappointing modality in clinical ultrasound cardiology. This is due to the basic flaw in the application of the principles of Doppler. Doppler is best suited for flow studies and applying it to tissue motion is not acceptable. In flow Doppler we suppress the tissue 'noise' and display flow. In TDE ipotential areas for investigation and research.However the author is of the view that TDE could probably have some role in diagnostic UCG. What is required is that Doppler should be used for the purpose it was intended i.e. to study blood flow. Thus we will have 2 types of Doppler studies: High velocity flow Doppler (HVFD) studies and Low velocity flow Doppler (LVFD) studies. Conventional Doppler would be HVFD. TDE would be LVFD and we would be imaging and analyzing very low velocity flows. The possible uses of color LVFD studies would be the study of low flow thrombogenic states, flow in the atria, the blood-endocardial/endothelial interface, low velocity turbulences and blood flow in organs. Pulse wave spectral LVFD could be useful for the detection of low velocity turbulences as a cause for murmurs (not detectable with HVFD). These are It is time to look at TDE on more realistic terms. As a new modality of imaging it appears exciting. But its real clinical utility is doubtful. TDE does not give any additional information over the conventional modalities. In fact due to the above-mentioned deficiencies it could give misleading information. Making diagnostic and therapeutic decisions based on this faulty application of technology would be unacceptable to the scientific cardiologist.CFM Color Flow MappingCW Continuous WaveDS Doppler StudiesHVFD High Velocity Flow DopplerLVFD Low Velocity Flow DopplerPW Pulse WaveTDE Tissue Doppler EchocardiographyUCG Ultrasound CardiographyUSI Ultrasound ImagingNone declared.


Molecular detection of herpesviruses DNA is considered as the reference standard assay for diagnosis of central nervous system infections. In this study nested PCR and real time PCR techniques for detection of Herpes simplex virus type 1 (HSV-1), Cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in cerebrospinal fluid of HIV patients were compared.Forty-six, 85 and 145 samples previously resulted positive for HSV-1, CMV and EBV by nested PCR and 150 randomly chosen negative samples among 1181 collected in the period 1996–2003 were retrospectively reassessed in duplicate by real time PCR and nested PCR.Samples giving positive results for CMV, HSV-1 and EBV with nested PCR were positive also with real time PCR. One of the negative samples resulted positive for HSV and one for EBV. Real time PCR showed comparable sensitivity and specificity vs nested PCR.Real time PCR proved to be a suitable method for diagnosis of herpesvirus infections in CNS, showing comparable sensitivity and being less time consuming than nested PCR. Opportunistic infections as well as tumors and vascular and metabolic disorders are common in HIV-infected patients -3. GenerAlthough the rate of CNS complications is relatively low, if compared with the high prevalence of herpesviruses in the population, these viruses represent the most important pathogens associated with viral encephalitis and meningitis ,8, beingOne-step or nested polymerase chain reaction (PCR) has rapidly replaced immunological assays based on virus specific Ig antibodies in CSF for laboratory diagnosis of Herpesvirus infections, even if serological methods are considered an additional tool for defining clinical diagnosis.Although nested PCR is considered the method of choice in terms of specificity ,13,14, sIn the last years, introduction of real time PCR has markedly increased the ease and the speed in the virology laboratory due to the relevant technology that permits rapid temperature cycling within a close system. Considering the importance of relationship between viral load in CSF and severity and outcome of disease, an additional advantage of Real Time PCR is the capability to perform simultaneous qualitative and quantitative analysis.Here is reported our experience gained in the diagnosis of herpesvirus infections of the CNS in HIV patients by means of nested PCR and Real Time PCR, which has been recently applied in our laboratory.A total of 1181 CSF samples collected in the period 1996 – 2003 from HIV patients attending at Luigi Sacco Teaching Hospital of Milan  affected by acute encephalitis or meningitis or encephalopathy or other neurological syndromes were considered.Particularly, they consisted of 684, 954 and 933 CSF samples previously tested for HSV-1, CMV and EBV, respectively, by means of nested PCR. Of these, all the positive samples and 150 negative samples randomly chosen were retested by means of nested PCR and real time PCR. Each sample was re-extracted and run in duplicate.CSF positive and negative samples were stored at -80°C until analysis.Clinical data of patients with positive samples were available only in the 20% of the total. These patients generally recorded meningoencephalitis signs such as central motor or sensory alterations, consciousness loss, seizures defects.Spin-column based QIAamp Mini Kit  protocol extraction for CSF was used as indicated by the manufacturer.This procedure allowed for the rapid purification of DNA from 200 μL of CSF and comprised four successive steps carried out using QIAamp Spin Columns in a standard microcentrifuge. Purified DNA was concentrated at a final volume of 20 μL.glycoprotein D gene of HSV-1 [gp58 of CMV [Bam HI-W region of EBV [Nested PCR was carried out in a 50 μl mixture containing 40 μl of first amplification mix -, 2U/μl Taq DNA polymerase (Roche Diagnostics-Germany) and 5 μl of purified DNA. Primer pairs selecting for of HSV-1 , for the8 of CMV , and forn of EBV  are showAfter an initial 2 min denaturation at 94°C, 35 cycles of 94°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec were carried out, followed by a 5 min extension at 72°C using a thermal cycler . The reaction mixture for the second amplification round was the same as for the first one, except for the "inner" primers used instead of the "outer" primers. In the second amplification round 44 μl of amplification mix and 1 μl of the first amplification round PCR product were used. The thermal cycling was repeated as for the first amplification round but using 30 cycles after the initial 2 min denaturation. Each amplification run contained a negative control, consisting of water and a positive plasmidial control.Analysis for the PCR products was performed by means of 4 % agarose gel electrophoresis followed by visualization with ethidium bromide (0.4 μg/mL) staining and UV illumination to confirm the expected products.For diagnostic real time PCR Taq polymerase RT PCR Kit  was used. Target regions for HSV and CMV were the same as in nested PCR, while EBNA-1 gene was amplified for EBV  as shownThe RT PCR was performed in 25 μl mixture containing 20 μl of amplification mix  and 5 μl of purified DNA.The amplification program included an initial decontamination with uracile N'-glycosilase at 50°C for 2 min, followed by denaturation at 95°C for 10 min and 45 two steps of 15 sec at 95°C and 1 min at 60°C.t) values were calculated by determining the point at which the fluorescence exceeded a background limit of 0.04. Each analytical session comprised also a negative control (distilled water).The RT PCR products were detected by measuring fluorescence with passive reference dye in Sequence Detection System ABI Prism 7000 (Applied Biosystem). Control threshold  and its concentration determined spectrophotometrically. Then, plasmidic DNA was serially diluted in a stabilizing buffer to the final desired concentration. Amounts of copies/mL in each sample were determined by means of a quantification software , by considering an extraction recovery of 80%.Quantification was carried out by analysing four positive plasmidial standards at 10Of 954 CSFs previously examined for CMV by means of nested PCR, 85 samples resulted positive. Among the 684 CSFs tested for HSV-1, 46 samples were found positive, while, 145 of 933 CSFs tested resulted positive for EBV.Reassessment of these positives and of 150 negative samples confirmed results previously obtained with the same method, with the exception of one HSV-1 positive sample, which resulted negative when retested, as shown in Table Results from real time PCR are reported in Table By considering nested PCR as gold standard ,13, sensIntroduction of PCR into routine diagnostic has rapidly gained a pivotal role for diagnosis of a wide range of diseases, supplanting, in many cases, other methods, such as the classical serodiagnosis. This is particularly true for diagnosis of herpes virus infections in immunocompromised patients, where diminished or suppressed virus-specific antibody responses do not reflect possible reactivated herpes mediated aethiologies .Real time PCR has represented a further step forward, since it allows for quantitative detection of target DNA in a single sample over a large range, remaining possible qualitative detection. Even if contradictory results have been found -21, quanIn the last years several real-time PCR methods have been developed for detection of herpesviruses in different biological specimens -27. RealIn the present work, we compared a real time PCR panel for detection of herpesvirus DNA in CSF with methods employing nested PCR. Our results indicate an overall agreement between the two methods, as reported by other authors ,27,28. DSince extraction panels and primers for HSV and CMV were uniform for the both types of assays, inhibitors present in the DNA preparations could not explain the different results obtained for these samples. Moreover, being nested PCR generally considered as the gold standard for diagnosis of herpes virus in CSF, we tried to use similar primers, chemistry and amplification conditions in order to limit differences for a better comparison of the performance of the two methods. Thus, discrepancies could be likely attributed to the different detection of amplification products, although real time PCR has been reported to be as sensitive as nested PCR . This suData obtained in this study confirms the validity of real-time PCR method for detection of herpesvirus DNA in CSF specimens of HIV patients, being sensitive, rapid and quantitative. Since specific and rapid diagnosis is the main target in the case of CNS infections, real time PCR could be considered the method of choice, due to its high specificity, sensitivity and rapidity, once proper quality controls will be available.The company Amplimedical SpA-Bioline Division-Italy will pay the article processing fees for the manuscript.LD conceived of the study and participated in its design and coordination, AL carried out real time PCR, EDV participated in data analysis and drafted the manuscript, GG performed statistical analysis, RB carried out nested PCR, MRG participated in design and coordination of the study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


As shown in previous investigations, in many primates and rodents this class of elements has remained transpositionally active, as reflected by increased copy number and high sequence diversity within and among taxa.Endogenous retrovirus-like elements  are a diverse group of reiterated sequences related to Here we examine whether proviral-like sequences may be suitable molecular probes for investigating the phylogeny of groups known to have high element diversity. As a test we characterized ERV-Ls occurring in a sample of extant members of superorder Uranotheria . The ERV-L complement in this group is even more diverse than previously suspected, and there is sequence evidence for active expansion, particularly in elephantids. Many of the elements characterized have protein coding potential suggestive of activity.In general, the evidence supports the hypothesis that the complement had a single origin within basal Uranotheria. From the perspective of evolutionary biology it is of great interest that some classes of ERVs are known to retain original functions, including the capacity to produce infectious viral particles [env gene [ERV-Ls are retroviral elements (retroelements) lacking the envelope gene  gene sequences from 22 mammalian species revealed ERV-Ls that have expanded in copy number and remained active over long periods of time [Comparison of ERV-L polymerase  fragment using degenerate primers tested in other mammalian orders. Extending our previous work [To investigate whether ERV-L and other retroelements may be useful in resolving phylogenetic questions involving uranotheres at multiple taxonomic levels, we utilized an ERV-L polymerase gene . No identical sequences were shared among taxonomic groups. All nine Asian elephant and all 10 manatee clones were unique. However, one Asian elephant clone, designated Max3 (accession number AY394573), was a recombination product of clone Max2 (accession number AY394572) and clone Max6 (accession number AY394576). Whether this represents a PCR artifact or is a genomic recombination event is not known. However, it is not expected that recombinational PCR would be observed in modern undamaged DNA .Procavia.Among the African elephant runs, four clones differed at 0–1 positions. As PCR errors probably account for these minor differences we assume only 6 unique ERV-Ls were discovered for this individual. Similarly, the hyrax sample yielded 3 groups comprised of 2 identical sequences, while two other sequences differed at 5 positions. Thus, 5 unique ERV-Ls were also obtained for gag and pol genes . Twelve clones were in frame with no stop codons. However, only 6 of the total 12 were unique  is known to be expressed in specific tissue types [The most important finding resulting from this study is that elements that have undergone expansion–i.e. have remained transpositionally active–are the ones that are most likely to group monophyletically and those that have not tend to be consistent in their higher-level taxonomic distribution . Thus, there are different elements within the same family demonstrating different evolutionary trajectories. It is reasonable to suppose that for some groups, such as rodents, primates, and uranotheres, continued expansion of active ERV-L elements was tolerated by the host. By contrast, in other ungulate and carnivore lineages active elements were not inherited or were silenced early during their evolution and ERV-L expansion did not occur. Older elements tend to evolve as typical orthologous sequences. An advantage of the great diversity of elements is that with a single PCR, cloning, and determination of multiple clone sequences one retrieves multiple independent sequences with which to do phylogenetic analysis. In these regards, the uranothere evidence is consistent with results previously reported for primates and rodents in which older elements and elements that have undergone bursts of transposition were found coexisting. In each of these groups there is now good sequence evidence for retroelements that have retained coding capacity, which is of some interest since HERV-L ue types . This inIn terms of our general results, elephants and the manatee were found to contain the most diverse sequences, while hyrax showed comparatively less diversity. This finding is consistent with results from our previous study involving extinct elephantids . ElementAlthough this study shows that the value of ERV-Ls for the narrow purpose of phylogenetic reconstruction is limited at higher taxonomic levels, ERV-L is only one class among many different groups of ERVs in mammalian genomes. Some regions of ERVs have been used successfully to reconstruct phylogenies at lower taxonomic levels . AdditioLoxodonta africana) DNA was supplied by N. Georgiadis of the M'Pala Research Centre, Kenya. Asian elephant (Elephas maximus) blood was provided by J. Hektor of the Tierpark Hellabrunn, Munich. Manatee (Trichechus manatus) blood was provided by D. Murphy of the Lowry State Park, Florida. Hyrax (Procavia capensis) muscle was provided by G. Amato of the Bronx Zoo, New York.African elephant (One ml of blood or approximately one gram of tissue was incubated in 1–2 ml 10 mM Tris-/Cl (pH 7.5), 10 mM EDTA (pH 8.0), 50 mM NaCl, 2% SDS, and 0.6 mg/ml Proteinase K overnight at 37 C, extracted with phenol and chloroform, and subsequently concentrated with 50 ul Millipore Ultrafee MC 30,000 NMWL columns or precipitated in 2.5 volumes ethanol and 1% NH4 Oac.pol gene are described in [PCR primers for the ERV-L ribed in . Three μribed in . Five μlRepresentatives of each clade determined by Bénit et al.  were incMaximum parsimony and neighbor joining analysis was performed using PAUP 4.0b . HeuristADG oversaw the molecular genetic studies, coordinated the study, participated in the sequence alignment and phylogenetic studies and co-drafted the manuscript. CCE participated in the phylogenetic analysis. RDEM participated in the design and coordination of the study and co-drafted the manuscript.


Defining and classifying disease is at the heart of medical practice, but the process is slow and laborious. A new "open source" approach could be faster and more democratic Defining and classifying disease is at the heart of medical practice. But the standard approach to classification is slow and laborious. A new approach promises to revolutionise the way in which we classify disease. It involves the free and public sharing of information via the Internet—the so-called open-source, or, perhaps more appropriately termed, “free community,” approach .http://www.ICDNS.org). The main characteristics of the ICDNS are free collaboration via the Internet, online access to all collaborative tools via the World Wide Web, global participation, and democratic decision making [The International Society for Neuropathology is the first worldwide professional medical organisation to adopt such an approach with its International Classification of Diseases of the Nervous System , are an example of a disease subset now defined by a common molecular pathology. The large field of pathology and the neurosciences are two areas where the translation of morphological phenotypes into molecularly defined entities is already well underway. With the pace of change accelerated by advances in molecular science, we need a much more effective way to develop the debate about medical classifications.One effective way to further develop this debate is to adopt the approach used by the free-software and open-source movements , which hhttp://www.opensource.org) is a nonprofit venture dedicated to managing and promoting the open-source idea (http://www.fsf.org). The creation of the GNU/Linux operating system (http://www.gnu.org and http://www.linux.org) has resulted from the work of both.The development of global computer networks and the World Wide Web, in particular, have fostered the evolution of free and global sharing of intellectual property. The Open Source Initiative (rce idea . A relathttp://www.opensource.org).“The basic idea behind open source is very simple: When programmers can read, redistribute, and modify the source code for a piece of software, the software evolves. People improve it, people adapt it, people fix bugs. And this can happen at a speed that, if one is used to the slow pace of conventional software development, seems astonishing… Open source software is an idea whose time has finally come. For twenty years it has been building momentum in the technical cultures that built the Internet and the World Wide Web. Now it is breaking out into the commercial world, and that's changing all the rules.”  SourceThe ICDNS was inspired by the free software and open-source approach to software development. The Council of the International Society for Neuropathology approved the ICDNS as a community activity at its last meeting in Turin, Italy, in September of 2003 . The imphttp://www.ICDNS.org, where the global community of neuropathologists can judge them  as well as expert interest groups propose core definitions, which are then posted so that the global consultation process begins via the World Wide Web. After an online discussion period, ICDNS members holding a specialist certification in diagnostic neuropathology are invited to vote. Comments made online by contributors become part of the history of a disease definition so that nobody is left out and divergent views are not forgotten. The discussion process is thereby open and democratic, allowing wide participation—including from individuals in developing countries who are often excluded in traditional academic discussions.Different countries around the world show variations in their use of diagnostic criteria and medical classifications, which, in turn, can lead to different treatment approaches. This can certainly create problems when trying to reach a global consensus. However, free access to the information held under the ICDNS open licence may help to minimise these problems by promoting and stimulating collaborative research and the exchange of scientific ideas across the globe.Certain diseases are far more common in particular parts of the world. In India, for example, neurotuberculosis, cerebral malaria, fungal infections of the central nervous system, human rabies, encephalomyelitis, and cerebrovenous thrombosis are more common than in most developed countries. While the conventional pathology of these diseases is well known—and in some cases we also have expert knowledge on their morphological phenotypes—their exact pathogenesis is not understood, and cellular as well as molecular knowledge is missing. The ICDNS is expected to stimulate local researchers to engage in collaborative international projects in which they can receive feedback via the global consultation process.Publication of ICDNS criteria occurs under a general public licence. This means that all text can be freely downloaded and republished, avoiding the need for defining basic facts over and over again. Both core definitions and comments may be used immediately for diagnostic purposes as outlined in the guidance section of the ICDNS Web site.In the future, definitions of histopathological phenotypes may be linked to clinical as well as molecular biological datasets, such as those obtained from microarrays and combined with imaging parameters. It is obvious that expert consensus on histological phenotypes is required before “genome matching” and similar procedures can be applied to complex diseases in a meaningful way. Most diseases are presently still defined on the basis of their histopathology. Online forums to find diagnostic consensus will provide a very effective means of correlating descriptive data with molecular data. Subsequently, statistical clusters representing “signatures of disease” may be extracted from multidimensional data spaces that will be available online. This opens new roads to link with diagnostic and therapeutic approaches.It seems reasonable to propose that the adoption of the ICDNS paradigm by other medical specialties would facilitate the development of a comprehensive, global body of medical knowledge. Free access to this knowledge would allow novel, collaborative approaches to be developed to address the most pressing medical problems through supranational concerted efforts. The rather lowly role traditionally ascribed to the exercise of defining and classifying diseases would give way to an appreciation for the key importance of this process as a potentially powerful driver of change.


The need for safe and effective treatment of dengue virus (DEN), a class A agent that causes dengue hemorrhagic fever/dengue shock syndrome, has been a critical global priority. An effective vaccine for DEN is not yet available. In this study the possibility of attenuating DEN infection using adeno-associated virus (AAV)-encoded short interfering RNAs (siRNA) was examined in Vero cells and human dendritic cells (DCs).A cassette encoding siRNA targeted to a 3' untranslated sequence common to all DEN serotypes was designed and tested for its ability to attenuate DEN infection by use of AAV delivery.Vero cells or DCs infected with AAV-siRNA showed a significant, dose-dependent reduction in DEN infection. Treatment of DCs with AAV-siRNA also decreased the DEN-induced apoptosis of DCs and did not induce significant inflammation.These results demonstrate that AAV-mediated siRNA delivery is capable of reducing DEN infection in cells and may be useful in decreasing DEN replication in humans. The need for a safe and effective prophylaxis or treatment for dengue virus (DEN) infection, a category A mosquito-borne human pathogen, is a critical global priority. DEN causes dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which is associated with heterologous secondary DEN infection and affects thousands of people worldwide. The incidence of DHF/DSS is increasing in the western hemisphere and, although many different approaches are being tried to develop prophylactic DEN vaccines, none have been licensed for public health and there are no specific antiviral treatments available.DEN belongs to the family of Flaviviruses and is an enveloped single plus-stranded RNA virus with four distinct serotypes. The DEN genome of approximately 11,000 nucleotides encodes a polyprotein (C-prM-E-NS1-NS2a-NS3-NS4a-NS4b-NS5) consisting of three structural proteins  and seven nonstructural proteins. The open reading frame is flanked by a 100 nucleotide-long noncoding region (NCR) at the 5' end and a 400 to 600 nucleotide-long NCR at the 3'end [in vivo gene-silencing approach using short interfering RNA (siRNA) to decrease DEN replication. RNA interference is triggered by dsRNA that is cleaved by an RNAse-III-like enzyme, Dicer, into 21–25 nucleotide fragments with characteristic 5' and 3' termini [argonaute2, that cleaves the target mRNA [We reasoned that an effective antiviral approach aimed at attenuating the DEN virus burden might protect infected subjects from DHF/DSS and, therefore, we examined the potential of an get mRNA ,5. This get mRNA ,7.in vivo leading to specific gene silencing. However, plasmids transfect mammalian cells poorly, and adenoviruses produce an acute inflammatory response and an immune response to viral vector-encoded antigens [Plasmid DNAs or adenoviruses encoding appropriate DNA sequences allow transient siRNA expression in cells and antigens ,8. We thNot I and the larger fragment was ligated to the synthetic adapter containing in order, Not I-Kpn I-Apa I-Xho I-Hind III-EcoR I-Bam HI-Sac II-Sac I-Cla I-Sal I-Bgl II-Not I. The U6 promoter was obtained by PCR amplification, using specific primers with the desired restriction sites from the template pSilencer 1.0-U6 (Ambion), and inserted into the adaptor at the Kpn I and Apa I sites to get a novel plasmid pCMV-U6.The pCMV-MCS plasmid (Stratagene) was digested with Apa I/Xho I and Xho I/EcoR I respectively. The modified pCMV-U6 plasmid was then redigested with Not I and the smaller fragment was ligated to the 2.9 kb fragment of pAAV-MCS (Stratagene) obtained following its Not I digestion to generate the corresponding si-vector for EGFP, RSV and DEN. HEK-293 cells were cotransfected with the helper plasmid and the si- plasmid to generate recombinant AAV.Pairs of oligos were synthesized to develop siRNA constructs. The nucleotide sequence for each siRNA is as follows: siEGFP: 5'-GGC GAT GCC ACC TAC GGC AAG CTT CTC GAT TCG AAG CTT GCC GTA GGT GGC ATC GCC CTT TTT G-3' ; siRSVNSHEK293 cells were cultured with DMEM (Cellgro) plus 10% FBS (Cellgro) and cotransfected with pSMWZ-siDEN, pHelper and pAAV-RC (Stratagene) by standard calcium phosphate transfection. Cells were harvested 48 hr post-transfection and the cell pellets were stored at -80°C.Cells were lysed by 5 cycles of freezing and thawing to release the virus. Crude viral lysate were collected by centrifugation at 27,000 × g for 30 min, and the supernatants were harvested and put onto a CsCl gradient (density 1.20/1.50) in fresh tubes and centrifuged for 16 h at 100,000 × g. Opalescent bands were collected after ultracentrifugation. Titers of purified AAVs were measured using an AAV titration ELISA kit .2 the nonadherent cells were removed and the adherent cells were cultured with fresh DMEM supplemented with 10% FBS (Cellgro), 200 ng/ml IL-4 (BD-Pharmingen) and 50 ng/ml GM-CSF (BD-Pharmingen) for 7 days prior to infection with DEN.Conditions were similar to those described previously . Buffy c5 Vero cells or DCs were seeded into six-well tissue culture plates and infected with different numbers of recombinant AAV carrying the DEN-siRNA silencing cassette. After 2 days the cells were infected with DEN-2 virus (strain16803) at an MOI of 0.1.1 × 10Cells were harvested and centrifuged for 10 min at 150 × g. The cell pellets were washed with PBS and resuspended with antibody to DEN-2 virus envelope protein,  on ice for 40 min. The cells were centrifuged and pellets were washed with PBS and then resuspended with secondary antibody conjugated with FITC (Sigma) for an additional 30 min. The number of infected cells was measured by flow cytometry 5 days post-infection. DCs were also stained with CD11c antibody conjugated with PE (BD-Pharmingen).2 for 5 days. Afterwards, 1% neutral red (Sigma) was added to each well and the plaques were counted 48 h later.The supernatants from DEN-2-infected DCs were collected at day 5 post-infection and 10-fold serial dilutions were allowed to adsorb to monolayers of Vero cells in six-well culture plates for 2 h. The medium was then removed and replaced by an agarose-containing overlay , 1% low melting-point agarose (Gibco BRL) and the plates were incubated at 37°C/5% COInfected-DCs were harvested on day 5 of infection with DEN-2 virus. Aliquots of DCs were put onto slides using a Cytospin and fixed with 4% paraformaldehyde. Apoptotic DCs were determined using the terminal dUTP nick end-labeling assay  and the annexin V apoptosis detection kit .Supernatants from infected-DCs were harvested at 24 h, 48 h, 72 h and 96 h post-infection. Cytokine concentrations were measured by cytometric bead array (CBA) and ELISA (BD-Pharmingen) following instructions in the manuals.t test. Values of p < 0.05 were considered statistically significant.Data were expressed as arithmetic mean ± SEM. Levels of significance of the differences between groups were determined by the student In order to develop an AAV-siRNA system, a plasmid pSMWZ-1 was engineered that comprised a mouse U6 promoter linked to a siRNA cassette Fig. . To testTo test whether AAV-siDEN-2 expression decreases DEN-2 virus infection in cultured Vero cells, cells were infected with recombinant AAV carrying siDEN-2 (MOI 10) or siEGFP (MOI ~1000) silencing cassettes. After 2 days the cells were infected with DEN-2 virus at an MOI of 0.1. Five days later, the numbers of DEN-2 virus infected cells were quantified by flow cytometry using anti-DEN-envelope protein. Cells pre-infected with AAV-siDEN3UT, but not AAV-siEGFP, showed a significant reduction in DEN infection, and the reduction was dose dependent  silencing cassette. After 2 days the cells were infected with DEN-2 at an MOI of 0.1. Five days later, the numbers of DEN-2 infected cells were quantified by flow cytometry using DEN-2 antibody. Cells preinfected with AAVsiDEN3UT showed a 50% reduction in the number of infected cells  and ELISA assays. As showed in Figure The significant findings of this report include the development of an adeno-associated virus-based siRNA approach for downregulating gene expression. A recombinant AAV-siDEN3UT was utilized to induce significant decreases in DEN infection compared to control in both Vero cells and human DCs. The results indicate that siRNAs may be used to attenuate DEN infection in human DCs and may have therapeutic value.in vitro. In this AAV system, we incorporated the mouse U6 promoter, which is important for transcription and folding of the suppressor RNA, into a plasmid pCMV-U6.Interference of gene expression by siRNAs is a novel strategy to knock down specific genes in cells or tissues, and the specific silencing of pathogen genes using siRNA is a very attractive approach for the clinical treatment of infectious diseases. Long dsRNAs (of >30 nt in length) activate a dsRNA-dependent protein kinase and 2', 5'-oligoadenylate synthetase in mammalian cells, which leads to a non-specific reduction in levels of mRNAs . The endin vivo study [The choice of appropriate target genes is necessary for the success of the siRNA strategy, and two siRNAs derived from either the pre-M or the 3' NCR region of DEN-2 were used in our study. An internal deletion of 3' NCR nucleotide sequences was found to be lethal for DEN virus replication in an vo study . The 3' vo study ,19. ThusAedes aegypti mosquito bite, and resident skin DCs are an early target of DEN infection [DEN is transmitted through nfection . Immaturnfection . Replicanfection , and we nfection . Increasin vivo.DCs are one of the most powerful of APCs. After infection with virus in the periphery, iDCs process viral antigens, then differentiate into mature DCs and migrate from peripheral tissues to lymph nodes where they prime naïve CD4 and CD8 T lymphocytes to maintain protective antiviral cytotoxic T cell memory ,24. ThusOne of the important features of AAV vectors is the lack of inflammation following infection. We failed to detect significant IFNγ or IL-12 production in the supernatants of AAV-siDEN-infected DCs. This is in accordance with previous data -28, whicIn conclusion, we developed a novel AAV-mediated siRNA delivery system. Our results demonstrate significant downregulation of DEN protein expression in Vero cells and human DCs, which strongly suggest that our AAV vector can be useful for siRNA delivery and that this AAV system may be applied in clinical settings to attenuate DEN infection.AAV, adeno-associated virus; DCs, dendritic cells; DEN, dengue virus; DHF/DSS, dengue hemorrhagic fever/dengue shock syndrome; MOI, multiplicity of infection; pEGFP, enhanced green fluorescent protein; siRNA, small interfering RNA; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.None declared.WDZ constructed the si-plasmids, performed cell culture, isolated and administered the virus and isolated and cultured dendritic cells. RS assisted with virus handling and cell culture, and performed flow cytometry. GH did TUNEL assays. XK did cytometric bead array assays and ELISAs. HSJ measured virus titer by plaque assay. RFL, SW, KP and SSM designed and implemented the experiments, performed troubleshooting, and did the analysis and interpretation of the data.


The sizes and structure of the particles were characterized using transmission electron microscopy (TEM). The size of the particles was in the range between 9.7 and 56.4 nm. Cholesterol oxidase (CHO) was successfully bound to the particles via carbodiimide activation. FTIR spectroscopy was used to confirm the binding of CHO to the particles. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of the enzyme were significantly improved upon binding to the nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy for free and bound CHO was 13.6 and 9.3 kJ/mol, respectively. This indicated that the energy barrier of CHO activity was reduced upon binding onto Fe3O4 nanoparticles. The improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound enzyme. The study indicates that the stability and activity of CHO could be enhanced via attachment to magnetic nanoparticles and subsequently will contribute to better uses of this enzyme in various biological and clinical applications.Magnetic nanoparticles (Fe Magnetic materials have been used with grain sizes down to the nanoscale for longer than any other type of material . This is3O4) nanoparticles can be attracted by a magnetic field and are easily separable in solution. Similarly, substances to which they have been attached can be separated from a reaction medium, or directed by an external magnetic field to site specific drug delivery targets [Due to their magnetic character, magnetite  that produces hydrogen peroxide according to the reaction 1.Cholesterol + O2 → 4 - Cholesten - 3 - one + H2O2   (1)3O4 magnetic nanoparticles will contribute to the basic understanding of its activity and function.The structure of cholesterol oxidase reveals deeply buried active sites occupied by water molecules in the absence of its substrate steroids . Cholest3O4 magnetic nanoparticles. The sizes and structure of the nanoparticles were characterized using TEM and FTIR spectroscopy. The stability, activity, and kinetic behavior of bound cholesterol were also examined.The present study proposes to investigate the direct binding of cholesterol oxidase to Fe3O4 particle. It could also be envisaged that CHO molecules formed aggregates to bind several magnetic nanoparticles. Another possible factor in the agglomeration process is the centrifugation process involved in the separation of the supernatant from the Fe3O4-CHO. It is obvious that the centrifugation tend to bring particles together as a compact material. The effect of agglomeration at this stage can be reduced by separating the Fe3O4-CHO by an external magnetic field. Since the particles are released after removal of the magnetic field, they may fall separately apart from each other, and are less likely to agglomerate.TEM micrographs of "bare" magnetic nanoparticles and CHO-functionalized magnetic nanoparticles are shown in Figure 3O4 nanoparticles used were 14.4, 17.2 and 20 mg/mL, corresponding to CHO/Fe3O4 weight ratios of 0.01, 0.08 and 0.007, respectively. These results show that in all the binding operations, there were sufficiently available amount of particles to bind the enzymes till complete saturation. In a previous study [3O4 nanoparticles, that is reducing the weight ratio of CHO to Fe3O4 below 0.033 caused an increase in lipase binding up to 100%. This was not observed in this study, possibly because of the difference in the binding mechanism, due to differences in the structure of the enzyme. However, the percentage of bound CHO (98–100%) shows that the binding process was successful.The unbound enzyme was determined by assaying the protein content in the supernatant. It was found that the percentage of cholesterol oxidase bound was between 98 and 100%, irrespective of the amount of particles. The amounts of Fe3O4, Fe3O4-CHO, and CHO in water, respectively. A characteristic band of NH2 was observed at 1618 cm-1 in the "bare" Fe3O4nanoparticles. The NH2 group can be associated with NH stretch at 3400 cm-1 which is not visible here, because of a possible hindrance by OH stretch from water. However, this band was not apparent in the spectra of Fe3O4-CHO suggesting that the binding of CHO to the nanoparticles involved this amino group and the carboxylic groups of CHO after being activated by Carbodiimide, as suggested by [-1 and 1445 cm-1 are more visible in Figure 3O4-CHO) and could be assigned to traces of residual ammonium hydroxide. The characteristic bands of proteins at 1647 and 1541 cm-1, and 1645 and 1541 cm-1, in the spectra of Fe3O4-CHO, and CHO, respectively shows that cholesterol oxidase was effectively present in the samples, confirming the binding of cholesterol oxidase to Fe3O4 nanoparticles. The negative peak at 3400-2799 cm-1 is possibly due to a reduced amount of water in the sample compared to the water used for background subtraction. The characteristic bands of proteins in the Fe3O4-CHO spectra were very weak compared to those in the spectra of cholesterol oxidase in water. The weakness of the peaks is due to the limited amount of CHO bound to the nanoparticles, in comparison to the amount dispersed in water.The binding of CHO to magnetic nanoparticles was confirmed by FTIR analysis. Figure Vmax and Km for CHO were determined to be 0.67 μmol/min mg and 2.08 mM for the free enzyme and 1.64 μmol/min mg and 0.45 mM for the immobilized enzyme, respectively. The Vmaxvalue of the bound CHO was 2.4 fold higher than that of the free, and the Km value of the bound CHO was 4.6 fold lower than that of the free CHO. The low Km reflects the high affinity to substrate [3O4 nanoparticles contributed to an enhancement of the activity of the enzyme.The kinetic parameters of the enzymatic reactions estimated by the Lineweaver-Burk plots of the initial rates of cholesterol oxidase from experimental data are presented in Figure ubstrate , the reaThe effect of pH on the activities of the free and bound CHO was investigated in the pH range of 6–8.5 at 25°C and presented in Figure k) at temperatures where the inactivation experiments were observed. The rate constants increased with increasing temperature and were higher for the free CHO than for bound CHO. As stated above, the binding to nanoparticles suggests a better resistance of the enzyme to temperature. We hypothesize that the bound enzyme could possibly undergo a conformational change and a spatial rearrangement that could slow down the folding process and denaturation of the enzyme.The thermal stability of free and bound CHO was investigated after 40 min of storage in the temperature range of 25–70°C Figure . There wThe effect of temperature on the activity of the free CHO was examined by measuring its relative activity when stored at various temperatures Figure . It can 3O4 nanoparticles might allow a better spatial orientation of the FAD prosphetic groups and the side chains of CHO providing a better stability to the enzyme.The stability and activity of the enzyme are naturally reduced during storage. Figure Nocardia sp. was purchased from VWR international . Carbodiimide-HCl (1-ethyl-3-(3-dimethyl-aminopropyl), ammonium hydroxide reagent, Triton X-100, TRIS (Hydroxymethyl) aminomethane HCL, 4-cholesten-3-one, bovine serum albumin (BSA), iron (II) chloride tetrahydrate 97 %, and iron (III) chloride hexahydrate 99% were obtained from Sigma-Aldrich, St Louis (USA). The Biorad Protein Assay Dye Reagent Concentrate was purchased from Biorad Laboratories . Acetonitrile was obtained from EMD Chemicals, .Cholesterol oxidase (EC 1.1.3.6), 3O4) were prepared by chemical co-precipitation of Fe2+ and Fe3+ ions in a solution of ammonium hydroxide followed by a treatment under hydrothermal conditions [4OH solution (30%), at a control pH (10–10.4). The suspensions were heated at 80°C for 35 min under continuous mixing and separated by centrifuging several times in water and then in ethanol at 2800 rpm. The purification step was used to remove impurities from Fe3O4 nanoparticles. The particles were finally dried in a vacuum oven at 70°C. The dried particles exhibited a strong magnetic attraction to a magnetic rod.Magnetic nanoparticles Fe3O were prenditions . Iron (I3O4 nanoparticles and Fe3O4bound cholesterol oxidase (Fe3O4-CHO) were washed with phosphate buffer pH 7.4 and 0.1 M Tris, pH 8.0, 0.1 M NaCl and then used for activity and stability measurements. NaCl was added to enhance the separation of the magnetic nanoparticles [50–70 mg of magnetic nanoparticles was added to 1 mL of phosphate buffer (0.05 M. pH 7.4). The mixture was sonicated for 15 min after adding 0.5 mL of carbodiimide solution (0.02 g/mL in phosphate buffer (0.05 M. pH 7.4). Following the carbodiimide activation, 2 mL of cholesterol oxidase (0.25 mg/mL) was added and the reaction mixture was sonicated for 30 min at 4°C in a sonication bath and the mixture was centrifuged at 3000 rpm . The preThe amount of protein in the supernatant was determined by a colorimetric method at 595 nm using the Biorad Protein Assay Reagent Concentrate with bovine serum albumin (BSA) as the protein standard. The amount of bound enzyme was calculated from:A = (Ci - Cs)*V   (2)A is the amount of bound enzyme, Ci and Cs is the concentration of the enzyme initially added for attachment, and in the supernatant, respectively (mg-mL-1), V is the volume of the reaction medium (mL).Where 3O4 nanoparticles and Fe3O4-CHO was characterized by transmission electron microscopy  and structure by Fourier Transform Infrared (FTIR) spectroscopy . The samples for TEM analysis were prepared by placing a drop of the magnetic nanoparticles dispersed in nanopure water onto a copper grid and evaporated in air at room temperature. Before preparing a sample onto the copper grid, the dispersed solution was sonicated for 4 min to obtain better particle dispersion. The binding of CHO onto the magnetic nanoparticles was investigated using FTIR. CHO and Fe3O4-CHO samples in phosphate buffer and Fe3O4 particles were dissolved in nanopure water for FTIR analysis.The size of Fe3O4-CHO, and mixed by vortex. A solution of free CHO of the same concentration was used to evaluate the activity of the free enzyme. The solution was incubated at various temperatures (25–70°C) at specific intervals of time (1 h) and centrifuged at 3000 rpm for 5 min to separate the supernatant from Fe3O4-CHO. 10 μL aliquots of the supernatant were then taken and the concentration of 4-cholesten-3-one was assessed. Before measuring the amount of 4-cholesten-3-one in a sample, the activity of the free enzyme was stopped by adding an equal volume of acetonitrile to the reacting solution [The activity of bound CHO was determined by measuring the initial oxidation rates of cholesterol by cholesterol oxidase at given temperature following the increase of 4-cholesten-3-one concentration at 240 nm, using a Beckman Du Spectrometer. A solution of cholesterol was prepared by dissolving 4.8 g of cholesterol in 10 mL of 2-propanol. A phosphate buffer solution (0.05 M. pH 7.4) containing 4% of Triton-100 was added to the mixture to result in a 0.26 M cholesterol solution. The mixture was gently heated until the solution was clear. To start the enzymatic reaction, 5 ml of cholesterol solution was added to 15 mL centrifuge test tubes containing Fesolution . Each ki3O4-CHO were determined by measuring the residual activity of the enzyme at 25°C, after being exposed to different temperatures (25–70°C) in phosphate buffer  for 40 min. Aliquots of the reacting solution were taken at time intervals (every 30 min for 7 hours) and assayed for enzymatic activity as described above. The first order inactivation rate constant, k was calculated from the equation:The thermal stability of free and FeA = In A0 - kt   (3)In A0 is the initial activity, A is the activity after a time t (min), k is the reaction constant.where 3O4-CHO was estimated by determining the concentration of 4-cholesten-3-one in samples at various temperatures. A solution of cholesterol was added to the various centrifuge test tubes containing bound or free enzymes. The test tubes were stored in a water bath at specific temperatures . At time intervals, the concentration of 4-cholesten-3-one was determined by spectrophotometric analysis.The effect of temperature on the free CHO and Fe3O4-CHO or free enzyme solution were stored at 25°C in phosphate buffer (0.05 M. pH 7.4) for 30 days. Thereafter, 5 mL of cholesterol was added. The storage stability of the free and bound cholesterol oxidase was determined by assaying for their residual activity.The storage stability was evaluated by determining the concentration of 4-cholest-en-3-one at room temperature at time intervals (5 days). Test tubes containing Fe3O4-CHO, Km and Vmax were determined by measuring initial rates of oxidation of cholesterol (1.3–5.2 mM) by CHO (0.25 mg/mL) in phosphate buffer pH 7.4 at 25°C.The kinetic parameters of free CHO and Fe3O4 magnetic nanoparticles reduced the energy barrier for CHO activity. As a result of the binding to the magnetic nanoparticles, the storage stability of CHO was considerably enhanced. This higher stability of the Fe3O4-CHO is attributable to its possible fixation on the surface of the particles preventing auto-digestion and thermal inactivity. In addition, the binding on Fe3O4 nanoparticles might allow a better spatial orientation of the FAD prosphetic groups and the side chains of CHO to provide better stability to the enzyme. The overall improvements observed in activity, stability, and functionality of CHO resulted from structural and conformational changes of the bound cholesterol oxidase. The study may be useful in improving the stability and activity of cholesterol oxidase, and will contribute to more efficient use of this enzyme.Magnetic nanoparticles were synthesized by thermal co-precipitation of ferric and ferrous chlorides. The binding of CHO to the particles was confirmed by FTIR spectroscopy and the size characterized by TEM. The binding efficiency was between 98 and 100% irrespective of the amount of particles used. Kinetic studies of the free and bound CHO revealed that the stability and activity of CHO were significantly improved upon binding to nanoparticles. Furthermore, the bound enzyme exhibited a better tolerance to pH, temperature and substrate concentration. The activation energy indicated that the binding of CHO onto FeCHO: Cholesterol oxidaseTEM: Transmission electron microscopyFTIR: Fourier Transform InfraredBSA: Bovine serum albuminDrs Gilles K Kouassi and Joseph Irudayaraj were the primary authors. They were responsible for the concept, experimental plan, and analysis. Dr Gregory McCarty was the secondary author and contributed to the overall effort.


In the microarray experiment, many undesirable systematic variations are commonly observed. Normalization is the process of removing such variation that affects the measured gene expression levels. Normalization plays an important role in the earlier stage of microarray data analysis. The subsequent analysis results are highly dependent on normalization. One major source of variation is the background intensities. Recently, some methods have been employed for correcting the background intensities. However, all these methods focus on defining signal intensities appropriately from foreground and background intensities in the image analysis. Although a number of normalization methods have been proposed, no systematic methods have been proposed using the background intensities in the normalization process.In this paper, we propose a two-stage method adjusting for the effect of background intensities in the normalization process. The first stage fits a regression model to adjust for the effect of background intensities and the second stage applies the usual normalization method such as a nonlinear LOWESS method to the background-adjusted intensities. In order to carry out the two-stage normalization method, we consider nine different background measures and investigate their performances in normalization. The performance of two-stage normalization is compared to those of global median normalization as well as intensity dependent nonlinear LOWESS normalization. We use the variability among the replicated slides to compare performance of normalization methods.For the selected background measures, the proposed two-stage normalization method performs better than global or intensity dependent nonlinear LOWESS normalization method. Especially, when there is a strong relationship between the background intensity and the signal intensity, the proposed method performs much better. Regardless of background correction methods used in the image analysis, the proposed two-stage normalization method can be applicable as long as both signal intensity and background intensity are available. After being reverse-transcribed into cDNA and labeled using red (Cy5) and green (Cy3) fluorescent dyes, two target mRNA samples are hybridized with the arrayed DNA sequences or probes. Then, the relative abundance of these spotted DNA sequences can be measured. The ratio of the fluorescence intensity for each spot represents the relative abundance of the corresponding DNA sequence.In cDNA microarray experiments, there are many sources of systematic variation. Normalization is the process of removing such variation that affects the measured gene expression levels. The main idea of normalization is to adjust for artifact differences in intensity of the two labels. Such differences result from differences in affinity of the two labels for DNA, differences in amounts of sample and label used, differences in photomultiplier tube and laser voltage settings and differences in photon emission response to laser excitation. Although normalization alone cannot control all systematic variations, normalization plays an important role in the earlier stage of microarray data analysis.et al.  = [log(R) - log(G)],     (2)In cDNA microarray experiments, there are red and green background intensities. It would be desirable to consider the background intensities that are more closely related with the signal intensities. We consider nine possible background measures from red channel, green channel, and both channels as follows:(a) Red channelY1 = log(rb),     (3)Y2 = log(rf / rb),     (4)Y3 = log(rf)/log(rb),     (5)(b) Green channelY4 = log(gb),     (6)Y5 = log(gf / gb),     (7)Y6 = log(gf)/log(gb),     (8)(c) Both channelsFor each category, there are three types of background measures. The first one is log-transformed background intensities. The second one is the log-transformed ratio of foreground and background intensities. The third one is the ratio of the log-transformed values of foreground and background intensities. Here we used log base 2, but any logarithmic base can be used as desired. Figure Yk  be an appropriate background intensity defined by red channel (a), green channel (b), or two channels (c). Then, fit the nonlinear LOWESS curve as follows:For simplicity, let Mk = C(1) (Yk),     (12)Mk(1) = Mk - C(1) (Yk), for k = 1,...,9     (13)C(1)(·) represents the LOWESS curve and Mk(1) is the residual from the curve. Note that Mk(1) is the log-ratio of relative intensities after removing the effect of background intensities. For these ratios, we can perform the usual MA normalization at the second stage.where In the second stage, we perform the normalization process as follows:Mk(1) = C(2) (A),     (14)Mk(2) = Mk(1) - C(2) (A),     (15)C(2) (·) is the LOWESS curve and Mk(2) is the residuals from the curve in the second stage.where Note that at the second stage any normalization method can be applied including a simple global normalization method.. The National Cancer Institute's Developmental Therapeutic Programs  has been studying a large number of anti- cancer drug compounds and molecular targets on the 60 cancer cell lines . In par [et al.  performeM versus Yk, where M = log(R/G) and Yk, k = 1,...,9 are background measures described in Methods section. The correlation coefficients between M and Yk's  are 0.2025, 0.6238, 0.6256, -0.0184, 0.5065, 0.5096, 0.1291, 0.5707, and 0.5729, respectively. Background measures Y2 and Y3 tend to have higher correlations than others.From ten slides we choose one slide to illustrate the proposed method. Figure M(1) versus Yk, where M(1) is the residual in equation (13) demonstrate some reduction in variability, which can be seen more clearly by comparing Figure M(1) and Yk have values close to zero. Using M(1), we carry out the second stage normalization.Figure quation 1 demonstrMA plots. The first row shows the MA plots for original , after global normalization, and after nonlinear LOWESS normalization, respectively (from left to right), The second row shows M(2) versus A plots after two-stage normalization for Y1, Y2, and Y3, respectively. The third row shows MA plots for Y4, Y5, and Y6 respectively, and the bottom row shows MA plots for Y7, Y8, and Y9, respectively. We can see that the two-stage normalization methods using Y2 and Y3 have the effect of reducing the variability among Ms and perform better than global and non-linear LOWESS normalization methods.Figure et al. [MSEl  which is the variance estimator for each gene derived from replicated slides. The main idea is that the better the normalization method, the smaller the variation among the replicated observations. Here, we use three different sets of microarray data: colorectal cancer data of NCI 60 , cortic [et al. ), and mo [et al. ).The goal of cortical stem cells study is to identify genes that are associated with neuronal differentiation of cortical stem cells. In this experiment, there are 3,840 genes in each slide from two experimental groups for comparison measured at six different time points . All experiments were replicated three times, thus we have 36 slides for the analysis.et al. [The objective of mouse gene expression study of Pritchard et al.  is to aset al. [et al. [In this comparative study, all five microarray data sets were used: colorectal cancer data set from NCI 60, cortical stem cells data set from Park et al. , and thr [et al. . Since rY2 and Y3 in two-stage normalization methods always yield better performances than the nonlinear normalization method, while the other background measures yield comparable results to those of the nonlinear normalization. Thus, we suggest either Y2 or Y3 as background measures in the two-stage normalization.In this study, the performances of two-stage normalization using nine background measures are compared to global normalization and intensity dependent nonlinear LOWESS normalization. Figure et al. [In microarray studies, many undesirable systematic variations are commonly observed. Normalization becomes a standard process for removing some of the variation that affects the measured gene expression levels. One major source of variation is the background intensities. Recently, some methods have been employed for adjusting for the background intensities. However, all these methods focus on defining signal intensities appropriately from foreground and background intensities during the image analysis . AlthouM in most subsequent analysis, the noise caused by background intensities may still remain in M even after normalization. The two-stage normalization may be quite effective especially when there is a high correlation between M and background measures such as Y2 and Y3.In this paper, we propose two-stage normalization for adjusting for the effect of background intensities systematically. The motivating idea is that the noise caused by background intensities may increase the variability in signal intensities. Although we use the log-transformed ratio of two channels denoted by Y2 and Y3 based on the results of the comparative studies. For these two background measures, we show that the two-stage normalization method always performs better than the global normalization methods and the nonlinear LOWESS normalization method.Among nine background measures, we recommend two background measures Y2 and Y3 is due to low intensities. We investigated this problem for NCI 60 data after removing spots with low intensities. The spots whose ratio of foreground and background intensities were smaller than 1.5 were removed in the analysis. This new data set also provided quite similar results.We wondered if the relative good performance of two-stage normalization using Y2 and Y3 perform better than other backgrounds are as follows. The background fluorescence might be relatively strong in the Cy5 channel due to interaction between the slide substrate and the hybridization materials. This effect is weaker in the Cy3 channel. It might be also possible that the background fluorescence in the Cy5 channel inflates the values of rb without correspondingly inflating the values of rf. This means that for weakly-responding spots, the rf and rb values are similar. This produces very low values in M, Y2 and Y3 for these spots. Note values under 5 for log(R) but not for log(G) in Figure M in all panes in Figure M are partially corrected by regressing against Y2 or Y3. However, the effectiveness of the proposed method for other data may depend on the background fluorescence artefacts.The main reasons why background measures The comparison is based on the variability measures derived from the replicated microarray samples. These variability measures can be easily derived from any replicated microarray experiment. Although we have studied only a limited number of data sets, our findings are quite consistent.Y2 and Y3 yielded better results than other background measures.For these data sets, we also conduct a similar analysis to see the effect of print-tips in normalization process. The results were consistent to those in Figure M: the original one (O), after the global normalization (G), after LOWESS normalization (L), and two-stage normalization using Y2 and Y3 . The density plot is in Figure M for T2 and T3 are quite similar to that of L. This simple empirical investigation shows that the proposed method does not always cause a bigger overcorrection than the LOWESS normalization. However, we must be careful for over-fitting which may overcorrects the biological findings of interest.One major concern for normalization is about over-fitting which may cause overcorrection of real biological significance. In fact, every normalization method has a possibility of overcorrecting the data and removing some existing biological significance in the data. For the NCI60 data, for example, it is not easy to find out whether such negatively low expression genes are biologically significant genes or not. We investigate the possibility of overcorrecting by drawing the density plot of In addition, we performed the hypothesis test for M equal to zero and counted the number of genes that have different expression level between two channels. The number of significant gene with different expression value is 909 for the original data before normalization, 326 for the data after global normalization, 302 for the data set after LOWESS normalization, and 297 for the data after two-stage normalization. The numbers of significant genes do not differ much among normalization methods.rf - rb) for the red channel. Our method was illustrated using the usual subtraction (rf - rb). Although our method starts with a most common approach based on the usual subtraction, it can be applied to any other models for defining spot intensity r = f, where r is the signal intensity, as long as a background intensity is available. Our method can be applicable as long as the image analysis provides the signal intensity and background intensity. For example, our method using Y2 builds on the relationship between r = f and rb.The proposed method can be applicable when both background and foreground intensities are available after the image analysis. Many image software provides both signal intensity as well as background intensity of spots. In most cases, the signal intensity is defined by simple subtraction such as (Y2 and Y3. The performance of the two-stage normalization method tends to depend on the correlations between background measures and M. That is, if there is a strong relationship between them, our method has a large effect. Thus, for the experimentalists it might be important to tell when to use the two-stage normalization method. In order to answer this question, we compute correlations between M and all background measures from Y1 to Y9. Figure M and Yks for each data set. For example, Figure Y2 and Y3 are relatively higher than those from other background measures. We also think that is why Y2 and Y3 have better performances than the other background measures.We recommend that experimentalists examine their data carefully and consider applying the two-stage normalization methods using Y2 and Y3 are higher than 0.5 for both CCD and SCD, while those for KD are smaller than 0.5. Note that CCD and SCD have more reduction in variability than KD in the two-stage normalization. For lower correlations of Yks do not reduce variability much compared to the usual LOWESS normalization.Figure


A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. However, there is very little known about how inputs across the body mid-line are processed within SII.Optical intrinsic signal imaging was used to evaluate the response of primary somatosensory cortex (SI and SII in the same hemisphere) to 25 Hz sinusoidal vertical skin displacement stimulation ("skin flutter") applied contralaterally, ipsilaterally, and bilaterally to the central pads of the forepaws. A localized increase in absorbance in both SI and SII was evoked by both contralateral and bilateral flutter stimulation. Ipsilateral flutter stimulation evoked a localized increase in absorbance in SII, but not in SI. The SII region that responded with an increase in absorbance to ipsilateral stimulation was posterior to the region in which absorbance increased maximally in response to stimulation of the contralateral central pad. Additionally, in the posterior SII region that responded maximally to ipsilateral stimulation of the central pad, bilateral central pad stimulation approximated a linear summation of the SII responses to independent stimulation of the contralateral and ipsilateral central pads. Conversely, in anterior SII , bilateral stimulation was consistently less than the response evoked from the contralateral central pad.The results indicate that two regions located at neighboring, but distinctly different A-P levels of the anterior ectosylvian gyrus process input from opposite sides of the body midline in very different ways. The results suggest that the SII cortex, in the cat, can be subdivided into at least two functionally distinct regions and that these functionally distinct regions demonstrate a laterality preference within SII. There is general agreement that in cats and monkeys (and presumably in humans) the spike discharge activity a mechanical stimulus sets up in rapidly adapting (RA), slowly adapting (SA), and Pacinian (PC) skin mechanoreceptors is projected centrally, at short latency and with relatively minor transformation, to primary somatosensory cortex (both SI and SII) in the contralateral hemisphere. There is no consensus, however, about the way in which the stimulus-evoked response in the ipsilateral hemisphere contributes to cerebral cortical somatosensory information processing and somatosensation. A distinctive property of SII is that it is the first cortical stage of the somatosensory projection pathway that integrates information arising from both sides of the body. Similar to SI, SII possesses a clear topographic organization , but unlFigure To more accurately characterize the spatial disparity between SII loci activated by contralateral vs. ipsilateral stimulation, the absorbance values were obtained and plotted along the posterior-anterior axis of SII. Figure A more comprehensive view of the SII response to the contralateral and bilateral stimulus conditions can be better appreciated with a multi-dimensional surface plot of the data. Figure To directly compare the time course of the response of the anterior and posterior regions of SII to the three stimulus conditions, we determined the time course of the absorbance changes in each region under each stimulus condition Figure . The ploCluster plots were used to directly compare the response of SII to the different conditions of ipsilateral and contralateral stimulation. In each plot in Figure 2 boxel surrounding the peak. In anterior SII, the majority of the responses to contralateral stimulation are stronger than the responses to bilateral stimulation – hence, the majority of the points plotted fall below the reference line (which has a slope of 1). In the posterior region, the majority of the points plotted are above the reference line – indicating that the response to the bilateral stimulus was greater than the response to the contralateral stimulus.The contralateral and bilateral stimulus conditions can also be compared using cluster analysis. However, because the difference between the contralateral and ipsilateral responses is more robust than the difference between the contralateral and bilateral responses, Figure Thus far, the results suggest that the anterior and posterior regions of SII are differentially activated by contralateral, ipsilateral and bilateral stimulation. To determine the across-subject consistency of these findings, the average absorbance values evoked by the 3 different stimulus conditions were determined for all 6 of the subjects Figure . ClearlyThe findings of this study demonstrated clearly that the anterior and posterior regions of SII process bilateral inputs very differently. At the locus of the maximal OIS response evoked in the posterior region by an ipsilateral stimulus, bilateral stimulation evoked a response that was, on average, about 25% larger than that evoked from the contralateral stimulus site. Conversely, at the locus of the maximal OIS response evoked by contralateral stimulation in the anterior region, bilateral stimulation evoked a response that was, on average, 35% lower than the activity evoked by a contralateral stimulus. This discrepancy between the optical responses of the anterior and posterior regions could be related to neurophysiological observations reported in earlier studies. For example, Carreras and Andersson  found inThe main, although not the only, route for ipsilateral input to SII is through the corpus callosum, from cells located in SI and SII of the opposite cerebral hemisphere ,7,8,11. The neurons in the distal limb regions of SII do receive substantial callosal connections, but these neurons have been reported to lack ipsilateral RFs , indicatA number of interactions between stimuli applied to both hands have been demonstrated in human psychophysical studies. Gilson  found thA recent report  demonstrOne question that the results suggest is whether or not SII can be segregated by laterality preference, in a manner similar to that observed in other sensory systems. Laterality has been demonstrated in the primary sensory cortex of both the visual system and the auditory system of both primates and cats, and the data in this report strongly suggest that there are cortical areas within SII that exhibit preference to ipsilateral or contralateral inputs. In terms of processing information from simultaneous contralateral and ipsilateral stimuli, there could be further similarities between the somatosensory, auditory and visual systems that have yet to be described. Future investigations will aim to further clarify the role of SII in integration of information from inputs across the body midline.The responses evoked by contralateral and ipsilateral flutter stimulation of the central pad of the cat forepaw define functional subdivisions in SII: the two modes of stimulation maximally activate cortical regions that are anterior and posterior to one another, respectively. Bilateral stimulation, or providing simultaneous contralateral and ipsilateral stimulation, reveals, additionally, that the two adjacent cortical areas process bilateral inputs differently. In the posterior region, where ipsilateral stimulation evokes a maximal response, bilateral stimuli evoke a response that is greater than the response evoked by either the individual ipsilateral or contralateral response. In the anterior region of SII, where the contralateral stimulus evokes a maximal response, bilateral stimuli evoke responses that are smaller in magnitude than the responses evoked by the contralateral stimulus.Adult cats  were subjects. All surgical procedures were carried out under deep general anesthesia . After induction of general anesthesia the trachea was intubated with a soft tube and a polyethylene cannula was inserted in the femoral vein to allow administration of drugs and fluids (5% dextrose and 0.9% NaCl). For each subject, a 1.5 cm diameter opening was made in the skull overlying somatosensory cortex, a chamber was mounted to the skull over the opening with dental acrylic, and the dura overlying anterior parietal cortex was incised and removed. Following the completion of the surgical procedures all wound margins were infiltrated with long-lasting local anesthetic, the skin and muscle incisions were closed with sutures, and each surgical site outside the recording chamber was covered with a bandage held in place by adhesive tape.2 between 3.0 – 4.0%; EEG and autonomic signs  were monitored and titrated (by adjustments in the anesthetic gas mixture) to maintain levels consistent with light general anesthesia. Rectal temperature was maintained (using a heating pad) at 37.5°C.Subjects were immobilized with Norcuron and ventilated with a gas mixture  delivered via a positive pressure respirator 1–3 hours prior to the data acquisition phase of the OIS imaging experiments. Respirator rate and volume were adjusted to maintain end-tidal COEuthanasia was achieved by intravenous injection of pentobarbital (45 mg/kg) and by intracardial perfusion with saline followed by fixative . Following perfusion fiducial marks were placed to guide removal, blocking, and subsequent histological sectioning of the cortical region studied. All procedures were reviewed and approved in advance by an institutional committee and are in full compliance with current NIH policy on animal welfare.Results were obtained during stimulation of the contralateral central pad of the forepaw and/or the ipsilateral central pad of the forepaw. The stimuli always consisted of sinusoidal vertical skin displacements  and were applied using a servocontrolled transducer  that is capable of delivering sinusoidal stimuli in the range of 1–250 Hz at amplitudes in the range of 0–1000 microns. The stimuli were delivered independently to the ipsilateral and contralateral skin sites, and also were applied simultaneously to both sites . The stimulus probes were positioned 500 microns beyond the point at which skin contact was detected (via force transducer on the Cantek). The bilateral stimulus protocols reported in this paper were synchronized to start and stop at the same time. The contralateral, ipsilateral and bilateral stimuli were interleaved on a trial-by-trial basis. This approach was used to control for temporal changes in cortical "state" unrelated to stimulus conditions which, if unrecognized, might obscure or modify any differences between the optical responses evoked by the contralateral, ipsilateral and bilateral stimulus conditions.Near-infrared  OIS imaging was carried out using an oil-filled chamber capped with an optical window . Images At the conclusion of the experiment, the imaged cortical region was removed immediately following intracardial perfusion with saline and fixative. The region then was blocked, postfixed, cryoprotected, frozen, sectioned serially at 30 μm, and the sections stained with cresyl fast violet. The boundaries between adjacent cytoarchitectonic areas were identified by scanning individual sagittal sections separated by no more than 300 μm and were plotted at high resolution using a microscope with a drawing tube attachment. The resulting plots then were used to reconstruct a two-dimensional surface map of the cytoarchitectonic boundaries within the region studied with optical and neurophysiological recording methods. The locations of microelectrode tracks and electrolytic lesions evident in the histological sections were projected radially to the pial surface and transferred to the map of cytoarchitectonic boundaries reconstructed from the same sections. As the final step, the cytoarchitectonic boundaries  identified in each brain were mapped onto the images of the stimulus-evoked intrinsic signal obtained from the same subject, using fiducial points (made by postmortem applications of india ink or needle stabs) as well as morphological landmarks . Locations of cytoarchitectonic boundaries were identified using established criteria .A-P = anterior-posteriorRA = rapidly adaptingSA = slowly adaptingPC = PacinianRF = receptive fieldOIS = optical intrinsic signalIR = near infraredEEG = electro-encephalogramMEG = magneto-encephalogramBW and OF participated in the design of the experiments, the data collection, and drafting of the manuscript. SS, JC, and VT made significant contributions to the data collection and the analysis of the data. MT played a major role in all aspects of the development of the manuscript.