Contrast echocardiography has an established role for enhancement of the right heart Doppler signals, the detection of intra-cardiac shunts, and most recently for left ventricular cavity opacification (LVO). The use of intravenously administered micro-bubbles to traverse the myocardial microcirculation in order to outline myocardial viability and perfusion has been the source of research studies for a number of years. Despite the enthusiasm of investigators, myocardial contrast echocardiography (MCE) has not attained routine clinical use and LV opacification during stress has been less widely adopted than the data would support. The purpose of this review is to facilitate an understanding of the involved imaging technologies that have made this technique more feasible for clinical practice, and to guide its introduction into the practice of the non-expert user. Two aspects of micro-bubbles are important – their gas content and the nature of their shell. Recently-approved micro-bubbles almost universally involve encapsulation of a high molecular weight gas, which improves the persistence of the bubble, optimising the number available in the left heart chambers. Air has a greater propensity to dissolve into solution and although currently unattractive because of loss of gas before arrival on the right side of the heart, better encapsulation may allow its resurgence – the benefit would be more rapid disappearance when the bubble bursts. The nature of the shell or surface modifying agent, which improves stability and prevents dissolution, may become important for new targeted imaging approaches.Even when optimal microbubble delivery to the myocardium is achieved with invasive coronary  or aortiMicro-bubbles oscillate (expand and contract) in the ultrasound field. The pattern and nature of their oscillation, and thus the nature of the backscatter signal, differs, depending on the acoustic power of the transmitted ultrasound field, which is expressed on modern ultrasound machines as the mechanical index (MI). In general echocardiography, the amplitude of returning backscatter depends on the nature of the insonated structure, and is represented by brightness on the formed image. Most backscatter returns at the same frequency as the transmitted ultrasound .very low mechanical index imaging (MI < 0.1), micro bubbles demonstrate linear oscillation, where the contraction and expansion of the micro bubble are equal. All returning micro bubble backscatter remains within the range of the frequency transmitted by the transducer  – as for surrounding structures , generates nonlinear oscillation of the micro bubble whereby expansion is greater than contraction. In addition to the usual backscatter of a particular amplitude within the range of the fundamental frequency, the bubbles also produce backscatter of (lesser) amplitude at harmonic frequencies [quencies ,5. For ehigh mechanical index imaging (MI > 0.6, ie the MI used for standard imaging) the bubbles oscillate wildly and burst. Upon destruction, micro-bubbles produce a brief, high amplitude signal, with good resolution, which is rich in harmonic signals, containing backscatter at the second third and fourth harmonics etc , myocardial micro-bubbles are destroyed continuously, causing apical swirling on LVO images and never replenishing the myocardium within the beam, and thus not returning significant myocardial backscatter. This exacerbates the underlying difficulty of differentiating on grey scale, between tissue backscatter and micro-bubble backscatter.Changing to standard harmonic imaging  significantly improves LVO, but offers little benefit for myocardial perfusion, with ongoing bubble destruction and sub-optimal differentiation between contrast and tissue, because both produce harmonics at high power. Contrast specific imaging modalities, with the aim of enhancing the contrast to tissue backscatter signal ratio (CTR) are required for optimal performance of a myocardial contrast study.Porter demonstrated that intermittent high power imaging demonstrated significantly better myocardial opacification than continuous imaging, with the best opacification obtained using intermittent harmonic imaging . During Moreover, by incrementally increasing the triggering intervals (continuous → 1:1 → 1:2 → 1:4 → 1:8 etc), the rate of replenishment of the ultrasound beam over time can be assessed both qualitatively and quantitatively.The qualitative assessment of perfusion using intermittent harmonic imaging can be improved by digital subtraction of the myocardial signal, and optimized by colour coding techniques to allow better extraction of bubble signals  Figure . The firPower Doppler technology is designed to detect motion of blood or tissue, and is used in myocardial contrast echo to overcome the need for off line digital subtraction . As withThis method is ideally suited to high mechanical index destructive imaging, as the first pulse destroys the myocardial micro-bubbles, generating a brief, high amplitude echo rich in harmonics. The second pulse finds that the bubbles have 'moved', and thus colour is overlaid on the echo image over the areas of myocardium that contained micro-bubbles. In an area with no micro bubbles, there is no 'movement' recognised and there is no colour overlay applied to that region. Note that the technique is not actually detecting movement of the bubbles in the circulation, but rather their destruction. This technique has been correlated in human subjects with SPECT and has The most important limitation of this technique is motion artefact from tissue, because tissue motion will be expressed like bubble destruction, potentially showing perfusion when none is present . If Doppler frequency is increased, pulse separation is reduced, so tissue movement between pulses can be minimized. However, if the pulses are too close, not all the gas within the bubble will have dissipated before arrival of the next pulse, so the difference between pulses is reduced, possibly leading to false positive perfusion defects. Air-filled micro-bubbles are optimal for this technique because of rapid dissipation of the gas, allowing closely spaced pulses.Another grey scale high MI technique is pulse-inversion imaging whereby two beam mode pulses are sent in rapid succession into the myocardium. The second pulse sent is a mirror image of the first (i.e. 180° phase shift). The scanner processes the consecutive returning pulses by adding them together. Tissue generates a linear echo, thus the addition of one pulse to the other should cancel out to zero and no signal is generated. Micro-bubbles produce non-linear echo signals and the summation of returning pulses will not equal zero and a signal will be registered Figure .Using this technique, processing can theoretically be limited only to signals generated by bubbles. As well as being a grey-scale technique, tissue motion artefacts are a major limitation, as movement of tissue also creates non-linear signals. Nevertheless the theory behind this technology has led to the development of successful real time imaging.With the development of increased bandwidth transducers, another intermittent high MI technique has evolved to improve the contrast to tissue ratio of backscatter. While intermittent harmonic imaging demonstrates myocardial perfusion, it is limited by the presence of tissue signals at the second harmonic. However, bubble destruction causes backscatter at 3rd 4th and sub-harmonics. At these higher harmonics the tissue signal is negligible, thus processing only backscatter from these further harmonics enables selective enhancement of the contrast signal. Interestingly, current ultra-harmonic imaging involves processing signals from between the second and third harmonic.The strength of the high MI approaches are their sensitivity for the presence of contrast, because bubble destruction results in the highest amplitude backscatter. The disadvantages are that they lack simultaneous assessment of function, require reliable ECG triggering and image acquisition, and can be both technically challenging and time consuming (of particular importance for stress imaging). While the difficulty maintaining image position with long triggering intervals has been aided by the use of low MI localization images, respiratory movement is almost inevitable.In recent years, technologies have been developed which so successfully exploit the non-linear responses of micro-bubbles, that even low amplitude micro-bubble backscatter can be isolated from tissue signals for processing. This allows continuous low power imaging to be performed, with limited bubble destruction, enabling simultaneous assessment of wall motion and perfusion in real time   The bubbles undergo stable non-linear oscillation emitting continuous fundamental and harmonic signals and (2) the tissues themselves do not generate harmonic signals at low MI. Like incremental triggered imaging, low MI imaging enables assessment of micro-bubble replenishment of the myocardium over time after bubble destruction. With low MI imaging, myocardial bubble destruction is achieved by transmission of a series of a high MI pulses ("flashes"), after which replenishment can be observed in real time . For MCE, bubbles should be infused with the aim of LV opacification and adequate myocardial perfusion with minimal/no attenuation of the basal segments. The amount of contrast required for this varies from bubble to bubble, machine to machine and technique used (intermittent or continuous), as well as showing patient to patient variability. Ideally all studies, particularly those assessing perfusion would involve a continuous infusion of micro-bubbles, with a mechanically controlled infusion preferable to a manually controlled one (slow continuous injection). This enables establishment of a true steady state for optimal imaging and in particular quantification.Accurate evaluation of regional and global left ventricular function by echocardiography is dependent on adequate endocardial border resolution. Using fundamental imaging, approximately 20% of resting echos demonstrate inadequate endocardial definition , definedTechniques that enhance discrimination between myocardial tissue and the blood pool may therefore improve the clinical utility and diagnostic accuracy of echo. Left ventricular opacification (LVO) by contrast echo enhances this discrimination to better define the endocardial surface.Contemporary LVO involves administration of perflurocarbon micro-bubbles , and inWhile there is clear evidence that contrast LVO can improve endocardial border definition, there are theoretical reasons why this may not necessarily translate into more accurate assessment of LV cavity size, volume and function. Attenuation artefacts sometimes obscure the endocardial surface, particularly in the basal segments and myocardial contrast may confuse distinction of the border between the blood pool and the endocardium. Most importantly, the use of any 2-D echo technique to assess left ventricular volumes and ejection fraction (with or without contrast), requires a standard imaging plane. Fore-shortened views or views not oriented through the centre of the ventricular cavity will lead to an underestimation of volume no matter how good the endocardial border definition .Notwithstanding these potential technical limitations, contrast LVO using fundamental imaging improves the correlation between LV volumes obtained with echo and MRI . In addiEnhanced endocardial border definition can improve the accuracy and inter-observer agreement for assessment of regional wall motion at rest . CompareStress echocardiography is an established clinical tool with a high sensitivity and specificity for the diagnosis of coronary artery disease (CAD). During stress echo the diagnosis of CAD is based on detection of regional contractile dysfunction, and requires visualization of all myocardial segments to document or exclude abnormalities definitively. Reduced endocardial border definition is exacerbated during stress because of chest wall movement during hyperventilation and cardiac translational movement during tachycardia. With fundamental imaging, inadequate endocardial definition has been reported in up to 30% of stress echos . In addiWall motion scoring and reproducibility during stress echo were even improved with air filled contrast agents and fundamental imaging. PerflurDespite these clear advantages, the critical clinical question of whether LVO actually improves the accuracy of stress echo for diagnosis of CAD remains unanswered – Dolan  came cloThus, left ventricular opacification by contrast echo:(1) improves the visualization of myocardial endocardial border definition;(2) improves the accuracy of ventricular volume assessment and estimation of ejection fraction compared with standard fundamental and harmonic imaging.(3) improves the ability to identify and grade resting wall motion abnormalities(4) provides superior endocardial visualization while imaging at peak dobutamine stress and can enable the accuracy of a dobutamine stress echo in a technically difficult patient to be at least as good as that of a patient with good resting images;(5) reduces the inter-observer variability for all of the above.From an economic standpoint, the use of contrast agents during stress echo has been calculated to be cost effective  with theIn summary, the use of contrast for LVO is justified for standard or stress imaging of technically difficult patients, and possibly, for calculation of ventricular dimensions in patients whom accurate quantitative serial follow up is critical, eg. chemotherapy or valvular heart disease. Other clinical uses of contrast for left ventricular opacification include confirming or excluding the presence of left ventricular thrombus and delineating other left ventricular structures like pseudoaneurysm, apical hypertrophic cardiomyopathy Figure ,25, and In the ischaemic cascade, hypoperfusion precedes wall motion abnormalities, which precede ECG changes and the onset of chest discomfort . Stress 1) Interpretive. Wall motion analysis is fundamentally subjective , leading2) Dependence on the induction of ischaemia  to provide diagnostic information. Many problems spring from this – dependence on performance of adequate stress, influence by beta blockade, limited sensitivity for mild (eg single vessel) CAD, poor capacity to identify the extent of CAD (eg. 50% sensitivity for the detection of multi vessel disease).SPECT perfusion imaging is the best-established clinical method to address perfusion directly and theoretically should be more sensitive than stress echo because hypoperfusion occurs earliest the ischaemic cascade. However, the difference between the methods is marginal because of the technical limitations of SPECT scanning, which also relies on subjective interpretation.If technically feasible there is clear role for echocardiography to assess myocardial perfusion. Ultrasound is more widely available, portable, avoids radiation and has a better spatial resolution than SPECT scanning (up to 1 mm, compared with >10 mm). An ideal technique would allow assessment of myocardial perfusion and wall motion simultaneously.Understanding the pathophysiology of normal myocardial perfusion is essential for understanding detection of CAD with MCE. Contrast micro-bubbles act as pure intravascular tracers, traversing the myocardial vasculature, and contrasting with MRI tracers (which escape into the extravascular space) and radionuclides (which enter myocytes).The coronary blood volume (CBV) encompasses the entire coronary system, which includes the epicardial arteries, the arterioles, the capillary network, venules, veins and the coronary sinus. Approximately one-third of the CBV resides within the ventricular myocardium. This myocardial blood volume (MBV) includes "microvessels" of < 300 microns in diameter, with approximately 90% of the total myocardial blood volume lying within the capillaries (6–7 microns in diameter). Assessmmyocardial blood flow appreciated with MCE i) the intensity of the backscatter signal from the micro-bubbles within the myocardium (brightness of its appearance on echo) – related to the myocardial blood volume, ii) the rate of increase in intensity after bubble destruction reflects the red blood cell velocity. Their product, represents myocardial blood flow. The response of MBF myocardial blood flow and it's components to stress is central to the application of perfusion imaging.There are 2 components of this Because of the near complete extraction of O2 from red cells by the myocardium under basal conditions, any increase in myocardial O2 demand must rapidly translate into increases in coronary blood flow. In the absence of a coronary stenosis, when myocardial O2 demand is increased, there is sufficient vasodilator reserve to allow coronary flow to increase by a factor of 4–6 fold above resting levels.increased red blood cell velocity across the capillary network, and opening dormant capillary networks in order to maintain mean trans-capillary pressure, thus increasing the overall myocardial blood volume. Hence overall myocardial blood flow is increased. Importantly, pure vasodilator stress tends to increase red cell velocity without marked changes in overall myocardial blood volume.The work of Kaul, Wei and Lindner at the University of Virginia has shown that the ability to increase coronary flow in response to increased demand is achieved through a process of coronary autoregulation, controlled predominantly by the arterioles  epicardial artery stenosis at rest, completely Thus identification of perfusion defects in the setting of a non-critical coronary stenosis requires stress imaging to induce either ischaemia  or hyperaemia . During stress we expect a 4–6 fold increase in flow to areas supplied by non-stenotic arteries, mediated by arteriolar vasodilation, increased red cell velocity and increased myocardial blood volume (opening of dormant capillary networks). Flow in the perfusion bed subtended by a significantly stenosed artery is not augmented, as resting arteriolar vasodilation is present, so limited augmentation of flow can be achieved with hyperaemia. In some cases the pre-capillary pressure drops significantly due to low distal coronary pressure and steal. With reduced pre-capillary pressure the only means of attempting to maintain normal trans-capillary pressure is to shut down capillary networks which were previously open (capillary de-recruitment). The net result is that direct comparison between regions subtended by a significant stenosis and normal territories reveals perfusion mismatch.In the presence of a critical stenosis, maximal arterial vasodilation maintains perfusion at rest. Any further reduction in distal coronary pressure directly translates into reduced precapillary pressure and reduced myocardial blood flow. Patients with critical lesions commonly have collateral vessels, making assessment of resting perfusion a complex phenomenon.There are three aspects to myocardial contrast opacification – signal intensity , pattern of filling and rate of filling.Myocardial blood volume is the easiest parameter to interpret qualitatively, and most studies have addressed differences in MBV between rest and stress in myocardial segments. Perfusion has traditionally been scored using a graded scale; 1 for homogenous perfusion, 0.5 for reduced perfusion and 0 for absent perfusion . The preFalse positive defects often reflect technical limitations Figure . FailureWhile most infarctions are shown as a transmural perfusion defect, stress-induced defects are often restricted to the subendocardium Figure . The detThe location of a defect has an important relationship to its likelihood of being a true or a false positive. Generally, defects on the left hand side of the image are most likely to be true positives and those restricted to the right side  should be considered very critically before they are identified as abnormal, as these are the most common sites of false positives. Fortunately, in our experience the apex is a more sensitive marker of LAD disease than the anterior wall, and is usually well seen. Likewise, a lateral segment should be matched with a posterior wall finding before circumflex disease is reported. The basal segments are also problematic – especially with low MI imaging – and in our experience, the basal inferior wall should be matched to an adjacent abnormal segment  to reduce false positive interpretations.The shape of the LV cavity is a clue to true positive findings. A "C-shaped" perfusion defect in the inferior wall Figure  is a comThe rate of replenishment after bubble destruction is dependent on coronary flow, and even if this is not quantified, rate of refill is a reliable and sensitive marker of a true positive defect. Using low MI techniques, micro-bubbles are infused until adequate LVO and myocardial opacification are achieved Figure . SeveralAs the mean myocardial microbubble velocity is 1 mm/sec, and the beam elevation is approximately 5 mm, it would take up to 5 seconds (ie 5–6 heart beats at a resting heart rate from 60–80) for homogenous opacification of the myocardium at rest. With hyperaemia in the absence of stenosis, myocardial blood flow should increase 4–6 fold. Thus, images taken within 1–2 seconds following bubble destruction (ie 2–3) heart beats at a peak heart rate of 140) should demonstrate complete homogenous refill within this time frame, and failure to fill in a perfusion defect thus represents reduced myocardial blood flow. Perfusion defects noted on the post stress images, not evident on the resting images suggest ischaemia . -see fiHigh MI techniques apply the same principles, with infusion until homogenous myocardial opacification. Imaging is then changed to a sequential intermittent mode, beginning with continuous high MI imaging (which results in bubble destruction and empties the myocardium). Images are then recorded intermittently, gated from the ECG  with an incrementally lengthening triggering interval, ie 1:1 an image every cardiac cycle, then 1:2, and image every two cardiac cycles, then 1:3, 1:4 etc. The same principles are used for assessment of perfusion defects. A perfusion defect evident on 1:2 or 1:3 triggering at peak stress not seen at 1:5/6 triggering at rest is significant and reflects an ischaemic response to stress.Qualitative assessment of perfusion with MCE was validated in open chest dogs using graded coronary stenosis . The oriWhile numerous studies examining various stress and imaging modalities have demonstrated concordance between MCE and SPECT, there remains a paucity of data using quantitative coronary angiography (QCA) as the gold standard. The presence and extent of CAD by QCA has traditionally been used as the reference standard for the assessment of CAD, but few studies have compared MCE and angiography Table  42]. In. In42]. Like stress echo and nuclear perfusion imaging, MCE is also limited by the qualitative nature of the interpretation. Subtle differences in video intensity between vascular beds may not be visually evident, potentially reducing the sensitivity for the detection of stenosis. Quantitative methods may help to alleviate this limitation and possibly reduce intra-observer and inter-observer variability in assessment.The process of quantitative myocardial contrast echo was validated in open chest dogs using intermittent imaging, and the same destruction-replenishment approach is used for contemporary qualitative assessment . At a stplateau myocardial video intensity (A) represents the myocardial blood volume. The rate of rise of the slope (β) represents the mean myocardial red blood cell velocity and their product (A*β) represents myocardial blood flow. Wei demonstrated an excellent linear relationship between absolute myocardial blood flow measured with radiolabelled microspheres and myocardial contrast echo derived myocardial blood flow. While ECG gated intermittent triggered harmonic imaging was used to validate these measurements, both power Doppler and real time techniques have subsequently demonstrated similar results in animal experiments  where y is the video intensity at the pulsing interval or time t, A is the plateau video intensity and β represents the rate of rise of the slope. In this model the riments -46)..plateau More recently, Wei evaluated the ability of MCE to calculate flow reserves from these measurements in humans , using It is now well recognised that regional or global ventricular dysfunction does not necessarily imply irreversible necrosis. Hypokinetic, akinetic or dyskinetic, yet viable myocardium may be stunned or hibernating. Myocardial stunning occurs after a period of acute ischaemia, despite restoration of completely normal blood flow. The natural history of stunning is of spontaneous improvement in the viable myocardium over time. Hibernating myocardium is the term used to describe the presence of significant ventricular dysfunction in patients with chronic CAD, which recovers after revascularisation. Improvement in function of sufficient numbers of viable but hibernating segments is associated with symptomatic benefit and improved survival. Sadly, Radionuclide scanning, dobutamine echocardiography, MRI and PET scanning are currently available modalities, each with various advantages and disadvantages but similar efficacy for prediction of myocardial functional recovery after revascularization. MCE may also have an important role in this clinical setting. Viable myocardium is associated with preservation of the micro-vasculature, and as micro-bubbles act as pure intra-vascular tracers, the presence of myocardial perfusion by any MCE technique at rest implies viability  has been very low. There have been particular theoretical concerns raised about histologic abnormalities and cardiac marker elevation in animals with high MI imaging, and whiIn recent months the agent SonoVue (a phospho-lipid shell encapsulating sulphur hexafluoride gas), approved in Europe, was withdrawn from clinical use because of adverse events including fatalities related to idiosyncratic hypersensitivity reactions. At this stage, specific details about the dose of agent and imaging modality used in these cases is not available.Advances in micro-bubble development, combined with the development of contrast specific imaging modalities have enabled not only excellent LVO, but reliable qualitative and quantitative assessment of myocardial perfusion by ultrasound, following intravenous injections of micro-bubbles. Use of this technology during stress echo increases sensitivity and improves the non-invasive evaluation of CAD.Power Doppler 4CV at rest in normal subject.Click here for fileA4CV standard harmonic imagingClick here for fileContrast LVO with standard harmonic imagingClick here for fileReal time MCE with LVO and myocardial perfusion.Click here for fileContrast LVO with ideal machine settings to optimize image quality and frame rateClick here for file3-D non contrast imagingClick here for file3-D imaging with contrastClick here for fileReal time 3-D without contrastClick here for fileReal time 3-D with contrast.Click here for fileMr CC PLAX view restClick here for fileMr CC PLAX view stressClick here for fileMr CC PSSAX view restClick here for fileMr CC PSSAX view stressClick here for fileMr CC A4CV restClick here for fileMr CC A4CV stressClick here for fileMr CC A2CV restClick here for fileMr CC A2CV stressClick here for fileMr CC ALAX view restClick here for fileMr CC ALAX view stressClick here for fileMr CC contrast 4CV restClick here for fileMr CC contrast 4CV stressClick here for fileMr CC contrast 2CV restClick here for fileMr CC contrast 2CV stressClick here for fileApical HCM standard 4CVClick here for fileApical HCM contrast restClick here for fileMr JD Rest PSAXClick here for fileMr JD Rest A4CVClick here for fileMr JD Rest PLAXClick here for fileMr JD Rest A2CVClick here for fileMr JD Rest ALAX viewClick here for fileMr JD Rest contrast 4CVClick here for fileMr JD Rest contrast A2CVClick here for fileMr JD Stress PLAXClick here for fileMr JD Stress PSSAXClick here for fileMr JD Stress A4CVClick here for fileMr JD Stress A2CVClick here for fileMr JD Stress ALAX viewClick here for fileMr JD Stress contrast 4CVClick here for fileMr JD Stress contrast 2CVClick here for fileMr JD angiogram.Click here for fileMr T 4CV restClick here for fileMr T 4CV stressClick here for fileMr T 2CV restClick here for fileMr T 2CV stressClick here for fileMr T 2CV contrast restClick here for fileMr T 2CV contrast stressClick here for fileMr T cath LCAClick here for fileMr T cath RCAClick here for fileMr JD curve fits A4CVClick here for file


Proteins may be the workhorse of the cell, but when a cell can synthesize one protein in a matter of minutes, chances are some will become obsolete. Though many proteins put in years of productive service, others quickly outlive their usefulness and can even damage the cell. Proteins that help form bone and muscle, for example, function for years while regulators of mitosis and cell proliferation might finish their jobs in seconds. Such short-timers are soon tagged as superfluous by a chain of small proteins called ubiquitin, which marks the proteins for degradation in an enzyme called the proteasome. Once in the proteasome, these proteins are broken down and can then be recycled for more productive ventures.A massive structure by cellular standards, the proteasome consists of multiple subunits, including a cylindrical core particle called 20S, which catalyzes degradation, and regulatory complexes called 19S caps, which form lid and base structures at both ends of the core. While the structure and biomechanics of the 20S core have been well characterized, much less is known about the functional mechanics of the regulatory complexes. The lid--base complex recognizes only ubiquitin-tagged proteins, which are then unfolded so they can enter the proteasome. But first ubiquitin chains must be detached from the protein, a task performed by an enzyme in the proteasome called Rpn11 isopeptidase. How the lid–base complex removes the ubiquitin tag, unfolds the protein, and shuttles it into the proteasome's core is not clear. Now Raymond Deshaies and colleagues present the structure of a homolog of the 19S lid's isopeptidase enzymatic center and provide new insights into these questions.The proteasome Rpn11 subunit contains a key region called the JAMM motif, which Deshaies' lab has shown previously is required for the proteasome to remove ubiquitin tags. For the work discussed in this paper, the researchers set out to understand how the proteasome strips off ubiquitin tags from proteins about to be destroyed by determining the three-dimensional structure of the JAMM motif.Archaeoglobus fulgidus. After determining the structure of the JAMM protein , the researchers discovered that AfJAMM looks nothing like the well-known deubiquitinating enzymes. But the arrangement of a set of amino acids that binds a zinc ion and forms the proposed active site of AfJAMM does resemble that found in a well-known protein-degrading metalloprotease called thermolysin, even though in other respects AfJAMM and thermolysin have very different features. The researchers mutated amino acid residues in another JAMM protein called Csn5  and found that the residues are indeed important for Csn5 function. These results suggest that JAMM does indeed represent a novel family of metalloproteases.The researchers tested many genes to look for a JAMM-containing protein that would crystallize properly and found one in the heat-loving prokaryote As for the wider function of JAMM proteins, the researchers speculate that these proteins are likely to be involved in a variety of important regulatory systems since they appear in life forms that lack ubiquitin and ubiquitin-like proteins. The crystal structure reported in this paper will provide a valuable tool for investigations into the underlying structural and functional mechanisms of these enzymes. And it may have important therapeutic implications. Proteasome inhibitors are promising anticancer therapies—fighting cancer by blocking machinery required by rapidly dividing cells. In the hopes of developing more targeted therapies, scientists are trying to fine-tune their control of the ubiquitin system and the proteasome. Inhibiting the JAMM domain of enzymes like Csn5, which remove ubiquitin-like tags from proteins upstream of the proteasome, for example, might just do the trick.


With some exceptions, mitochondria within the class Insecta have the same gene content, and generally, a similar gene order allowing the proposal of an ancestral gene order. The principal exceptions are several orders within the Hemipteroid assemblage including the order Thysanoptera, a sister group of the order Hemiptera. Within the Hemiptera, there are available a number of completely sequenced mitochondrial genomes that have a gene order similar to that of the proposed ancestor. None, however, are available from the suborder Sternorryncha that includes whiteflies, psyllids and aphids.COIII)-tRNAgly-NADH dehydrogenase subunit 3(ND3)-tRNAala-tRNAarg-tRNAasn from the ancestral position between genes for ATP synthase subunit 6 and NADH dehydrogenase subunit 5. Based on the position in which all or part of this fragment was inserted, the mitochondria could be subdivided into four different gene arrangement types. PCR amplification spanning from COIII to genes outside the inserted region and sequence determination of the resulting fragments, indicated that different whitefly species could be placed into one of these arrangement types. A phylogenetic analysis of 19 whitefly species based on genes for mitochondrial cytochrome b, NADH dehydrogenase subunit 1, and 16S ribosomal DNA as well as cospeciating endosymbiont 16S and 23S ribosomal DNA indicated a clustering of species that corresponded to the gene arrangement types.We have determined the complete nucleotide sequence of the mitochondrial genomes of six species of whiteflies, one psyllid and one aphid. Two species of whiteflies, one psyllid and one aphid have mitochondrial genomes with a gene order very similar to that of the proposed insect ancestor. The remaining four species of whiteflies had variations in the gene order. In all cases, there was the excision of a DNA fragment encoding for cytochrome oxidase subunit III(COIII-tRNAgly-ND3-tRNAala-tRNAarg-tRNAasn can be transposed from its ancestral position to four different locations on the mitochondrial genome. Related species within clusters established by phylogenetic analysis of host and endosymbiont genes have the same mitochondrial gene arrangement indicating a transposition in the ancestor of these clusters.In whiteflies, the region of the mitochondrial genome consisting of genes encoding for  Bemisia tabaci, the order of some of the mitochondrial genes was quite different from the frequently found order of genes in the mitochondria of the class Insecta. This observation led us to obtain the full sequence of the mitochondrial genome of representatives of the suborder Sternorrhyncha. Due to the observed differences in the order of genes in the mitochondrial genome of whiteflies, we obtained additional mitochondrial sequences from species representative of the major phylogenetic clusters previously established on the basis of whitefly mitochondrial and endosymbiont genes [Whiteflies, psyllids, and aphids correspond to superfamilies within the suborder Sternorrhyncha (Hemiptera) . These int genes . Previount genes -9. TheseCOI, COII, COIII), two subunits of ATP synthase (atp6, atp8), seven subunits of NADH dehydrogenase (ND1, ND2, ND3, ND4, ND4L, ND5, ND6), and cytochrome b (cytB). Two genes encode for the large subunit of ribosomal RNA (16S) and the small subunit of ribosomal RNA (12S). In addition, there are 22 tRNAs, two for leucine and two for serine, and one tRNA each for the remaining eighteen amino acids. In general, there is conservation of the gene order within phyla but variation between phyla [12S rDNA. Such a region most often has a reduced G+C content and all or some of the following properties: a) direct repeats, b) inverted repeats, c) stretches of "T"s, "A"s, or "TA"s. By analogy with other well-studied mitochondria, such a region is considered to be a putative origin of DNA replication and a region from which transcription is initiated [In animals, the mitochondrial genome is generally circular 14–17 kb), is maternally transmitted and has a relatively simple genetic structure, and a rapid rate of sequence change -12. Of ten phyla ,13-17; t kb, is mnitiated ,12. Varinitiated ,18. Howenitiated . The comnitiated . The latnitiated . The avacytB , ND1, and 16S rDNA is presented in Fig. Neomaskellia andropogonis which becomes part of the cluster containing Bemisia tabaci, Tetraleurodes acaciae, Aleurochiton aceris, and Trialeurodes vaporariorum. Whiteflies, psyllids and aphids have associations with different primary endosymbionts that are transmitted vertically and are essential for the survival of the insect host [Table ect host -6. The tect host . The repPachypsylla venusta (psyllid), Schizaphis graminum (aphid), as well as Aleurodicus dugesii and Trialeurodes vaporariorum (whiteflies)  Fig. . In pyslA-Y Fig. , 3, 4, 6COIII-(tRNA-G)-ND3-(tRNAs-A-R-N). In most cases in which these genes are removed, there is a change in the direction of transcription of the adjacent downstream tRNA-S1 from clockwise to counter clockwise -ND3-(tRNA-G)-COIII), the minimal insertion involves ND3-(tRNA-G)-COIII -ND3-(tRNAs-A-R-N). However, the DNA that is inserted always contains COIII-(tRNA-G)-ND3 and may contain all or only some of the tRNA genes.A number of whitefly mitochondria had transpositions of DNA fragments containing ise Fig. , 6. TherIII Fig. , 5. In aIII Fig. ,5,6. In COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) is removed from the inferred ancestral position and placed between 12S rDNA and tRNA-I. Additional changes involve the position of tRNA-D, tRNA-Q and the direction of transcription of tRNA-S1 and tRNA-E. There is a total of 5 difference between the A type gene arrangement and the ancestral whitefly gene order. Sequence determination of smaller DNA fragments from two related species (cytB-COIII) were consistent with the same gene order -COIII is inserted into a location downstream of 12S rDNA and is bounded by tRNAs that have also changed locations (tRNAs-Q-V and tRNAs-R-D). In addition, the position of tRNA-A is changed as compared to the ancestral position. There were 6 differences from the putative ancestral gene order. No tRNA genes for N, S1, and I were detected. Sequence determination of a smaller DNA fragment (cytB-COIII) from two related species was consistent with the same gene order.Transposition of the B type is shown in Fig. ND3-(tRNA-G)-COIII is inserted downstream of 16S rDNA between tRNA-P and tRNA-C. Another major difference is the change in the direction of the transcription of tRNA-V and 12S rDNA. Other differences include the change in the order and the position of tRNA-Y and tRNA-C, the change of position of tRNA-P, and the direction of transcription of tRNA-S1. Putative tRNA-W is transcribed clockwise. A small change in the span of the DNA fragment resulted in a putative tRNA-S2, transcribed counter clockwise. The initially adjacent tRNAs-A-R-N as well as tRNA-I were not detected. There was a total of 7 differences between the whitefly ancestral gene order and the C type gene order. Sequence determination of a fragment of mitochondrial DNA from a related whitefly species was consistent with the C type gene order.Transposition of the C type is shown in Fig. (tRNAs-R-A)-ND3-(tRNA-G)-COIII is found after tRNA-S2 and before tRNA-N. Additional differences from the ancestral gene order involve the change in position of tRNA-N and tRNA-Q and the direction of transcription of tRNA-S1. tRNA-I was not detected. The total number of differences between the ancestral gene order and the D type gene order is 4. The sequence of a mitochondrial DNA fragment from a related species indicated a gene order of the D type -ND3-(tRNAs-A-R-N) between COII and ND5  for tRNA-S1 for B. tabaci, A. aceris, N. andropogonis and T. vaporariorum. The latter codon maybe the usual tRNA-S1 anticodon in whiteflies; this tRNA was not detected in T. acaciae and A. dugesii.In general, the anticodons found in the tRNAs of the mitochondria of whiteflies, psyllids, and aphids were those expected of insect mitochondria . Some exCOIII-(tRNA-G)-ND3-(tRNAs-A-R-N) that is prone to excision followed by insertion as a unit or as fragments in different parts of the mitochondrial genome. Based on the collection of whiteflies we have examined, this event occurred three or four different times in the ancestors of the studied species. These conclusions are summarized in Fig. COIII-(tRNA-G)-ND3)  arrangement. From the 16S*-23S* rDNA sequence divergence of Portiera (the primary endosymbiont of whiteflies) and the estimated rate of endosymbiont sequence change [Aleyrodes species. This value corresponds to 30–60 million years ago which is the maximum time for the occurrence of the transposition in an ancestor of cluster C.The novel aspect of this study is the finding that whitefly mitochondria contain a region of their genome spanning D3) Fig.  but diffe change , it is ptRNA-S1 -12S and the genes are transcribed in the counterclockwise direction [Thrips imagines where these two genes are distant from each other and transcribed in opposite orientations [12S-(tRNA-V) that is possibly associated with the insertion of ND3-(tRNA-G)-COIII between 16S and 12S rDNA -ND3-(tRNAs-A-R-N), there are also substantial rearrangements involving single tRNAs. The physiological significance (if any) of these rearrangements is not known. Genes that are highly expressed (16S, 12S rDNA) when separated and transcribed in opposite orientations would have to become part of different transcription units. In addition, we are not certain of the validity or significance of our inability to find a few of the tRNAs. In some cases, this may stem from our inability to recognize them. In other cases, such as tRNAs-A-R-N that are absent in the type C gene order, there would not appear to be any room for these genes on the mitochondrion and it might be that the tRNAs for these amino acids are provided by the host [The excision of the same mitochondrial fragment at least four times during the evolutionary history of whiteflies suggests that this fragment is prone to transposition. In spite of the apparent similarity of the excisions, we have not been able to find any conserved sequence properties either adjacent to the region of the excised fragment or adjacent to its insertion site. The excision appears to be associated with a change in the direction of transcription of the previously adjacent -S1 Fig. , 5, 6 anirection ,18. A maDNA Fig. . This sithe host .A. dugesii has a G+C content of 13.8 moles %  we would have concluded that the whiteflies have the ancestral mitochondrial gene order and not pursued further studies of mitochondria within this group of insects. Previously, evidence was found of a correlation between the rate of nucleotide sequence change and the rate of gene rearrangement [Our work points to the uncertainty inherent in making generalizations from one or a few organisms assumed to be representative of a group. We were fortunate that in the whiteflies the first mitochondrion we chose to study was that of angement . If thisCOIII-(tRNA-G)-ND3-(tRNAs-A-R-N) from the ancestral position, between atp6 and tRNA-S1, and the insertion of all of these genes or fragments containing COIII-(tRNA-G)-ND and tRNAs into different locations on the mitochondrial genome. On the basis of the insertion positions, four gene arrangement types were identified. A phylogenetic analysis of 19 whitefly species involving mitochondrial and endosymbiont genes showed that each arrangement type was characteristic of a cluster of related whitefly species indicating that the transposition occurred in a common ancestor of the related species. The reason for the "restlessness" of this DNA segment in whiteflies and the physiological significance of these rearrangements are not known.Psyllids, aphids, and many whiteflies have mitochondria in which the order of the genes resembles the proposed Insecta ancestral gene order. However, in a variety of whitefly species there is a change in the gene order. In these organisms, there is an excision of a DNA segment containing COI using previously described primers [In all cases, the starting material was whole insect DNA that was prepared and used in a previous study . In our  primers , followeT. acaciae was obtained in three overlapping fragments. Using primers F-CYTB and R-12S-2 (Table 2, 0.2 mM dNTP, 10 pmoles of each primer, 0.6 U Bio-X-Act DNA polymerase, in Opti-Buffer (Bioline, London, United Kingdom). The PCR program was 94°C for 3 min, 30 cycles of, 94°C 30 sec, 55.0–65.0°C  30 sec, 70° 5 min, followed by 70°C 10 min. For the 5.5 and 7.3 kb DNA fragments, the PCR reaction mixture was modified by the increase of dNTPs to 0.3–0.4 mM and Bio-X-Act to 0.8 U. The PCR program was 94°C for 2 min, 10 cycles of 92°C 20 sec, 55.0–65.0°C  30 sec, 68° 10 min, followed by 20 cycles of 92°C 20 sec, optimal annealing temperature 30 sec, 68°C 10 min with increases of 15 sec each cycle, followed by 68°C 10 min. The DNA fragments were purified by means of the Wizard SV gel and PCR clean-up system (Promega, Madison, Wisconsin) as directed by the manufacturer. Following digestion with restriction enzymes the mitochondrial DNA fragments were cloned into pBluescript (Stratagene). In some cases where difficulty was experienced with using this vector due to possible toxicity of the inserts, the low copy number plasmid pWSK130 was used [We will illustrate the approach by describing how the full genome of the mitochondrion of was used . The DNACytB-12S mitochondrial DNA fragments were amplified and cloned into pBluescript as previously described [CytB-COIII DNA fragments were obtained using oligo WF-CYTB-3  and WF-COIII-3 (KpnI, 5'-GTGCGGTACC TTCWATTTGR TATTGRCATT TYGTTGA-3') and cloned into pBluescript. COII-ND5 DNA fragments (indicative of the presence or absence of COIII-(tRNA-G)-ND3-(tRNAs-A-R-N) were obtained by use of oligo WF-COII (5'-TGYTCAGAAA TYTGTGGRGT TAATCAYAGR TTTATRCC-3') and WF-ND5 (5'-TCAGCMTTAG TYCAYTCWTC AACAYTAGTW ACAGCAGG-3'). CytB-COIII fragments (size diagnostic of the arrangement type) were obtained by use of WF-CYTB-1 (5'-TTTATRGGBT ATATYTTRCC TTGRGG-3') and WF-COIII-1 (5'-TATTCWRTWT GATATTGACA TTTYGT-3'). The PCR reaction mixture (10 ul) differed from those above in containing 0.1 mM dNTP, and 0.8 U Bio-X-Act DNA polymerase. The PCR program was 94°C for 5 min, 30 cycles of, 94°C 30 sec, 56.0–63.1°C  30 sec, 70° 5 min, followed by 70°C 10 min.escribed . CytB-COcytB-ND1-16S was based on 2730 characters; the analysis in Fig. cytB-ND1-16S also included cospeciating endosymbiont 16S*-23S* rDNA [The protein-coding and rRNA genes were identified by BLAST searches  of GenBatRNA-A , tRNA-C (cys, GCA), tRNA-D (asp, GTC), tRNA-E (glu, TTC), tRNA-F (phe, GAA), tRNA-G (gly, TCC); tRNA-H (his, GTG), tRNA-I (ile, GAT); tRNA-K (lys, TTT or CTT), tRNA-L1 (leu, TAG), tRNA-L2 (leu, TAA), tRNA-M (met, CAT), tRNA-N (asn, GTT), tRNA-P (pro, TGG), tRNA-Q (gln, TTG), tRNA-R (arg, TCG), RNA-S1 (ser, TCT or GCT), RNA-S2 (ser, TGA), tRNA-T (thr, TGT), tRNA-V , tRNA-W (trp, TCA), and tRNA-Y (tyr, GTA).tRNA-one letter amino acid abbreviation (parenthesis three letter amino acid abbreviation followed by anticodons): atp6 (ATP synthase, subunit 6), atp8 (ATP synthase, subunit 8), COI (cytochrome oxidase, subunit I), COII (cytochrome oxidase, subunit II), COIII (cytochrome oxidase, subunit III), ND1 (NADH dehydrogenase, subunit 1), ND2 (NADH dehydrogenase, subunit 2), ND3 (NADH dehydrogenase, subunit 3), ND4 (NADH dehydrogenase, subunit 4), ND4L (NADH dehydrogenase, subunit 4L), ND5 (NADH dehydrogenase, subunit 5), ND6 (NADH dehydrogenase, subunit 6), 12S , 16S , 16S* , 23S* (large subunit of primary endosymbiont rDNA).%G+C (moles percent guanine+ cytosine in DNA).MLT cloned and sequenced the mitochondrial genomes of whiteflies. LB cloned and sequenced the mitochondrial genome of a psyllid and an aphid as well as smaller fragments of mitochondrial DNA from psyllids and aphids. PB directed the research and in collaboration with MLT and LB performed the data analysis and wrote the paper.


A highly non-random distribution of nucleotides is observed in the immediate vicinity of edited sites (within 20 nucleotides 5' and 3'), but no precise consensus motif has been identified.RNA editing is the process whereby an RNA sequence is modified from the sequence of the corresponding DNA template. In the mitochondria of land plants, some cytidines are converted to uridines before translation. Despite substantial study, the molecular biological mechanism by which C-to-U RNA editing proceeds remains relatively obscure, although several experimental studies have implicated a role for Arabidopsis thaliana, Brassica napus, and Oryza sativa; additionally, a combined data set of observations across all three genomes was generated. We selected datasets based on the 20 nucleotides 5' and the 20 nucleotides 3' of edited sites and an equivalently sized and appropriately constructed null-set of non-edited sites. We used tree-based statistical methods and random forests to generate models of C-to-U RNA editing based on the nucleotides surrounding the edited/non-edited sites and on the estimated folding energies of those regions. Tree-based statistical methods based on primary sequence data surrounding edited/non-edited sites and estimates of free energy of folding yield models with optimistic re-substitution-based estimates of ~0.71 accuracy, ~0.64 sensitivity, and ~0.88 specificity. Random forest analysis yielded better models and more exact performance estimates with ~0.74 accuracy, ~0.72 sensitivity, and ~0.81 specificity for the combined observations.Data for analysis were derived from the the complete mitochondrial genomes of Simple models do moderately well in predicting which cytidines will be edited to uridines, and provide the first quantitative predictive models for RNA edited sites in plant mitochondria. Our analysis shows that the identity of the nucleotide -1 to the edited C and the estimated free energy of folding for a 41 nt region surrounding the edited C are the most important variables that distinguish most edited from non-edited sites. However, the results suggest that primary sequence data and simple free energy of folding calculations alone are insufficient to make highly accurate predictions. RNA editing is the process whereby an RNA sequence is modified from the sequence corresponding to the DNA template. A particular form of RNA editing in plant mitochondria, by which some cytidines are converted to uridines before translation, occurs in many land plant lineages. Although cytidine to uridine conversion is most common, the reverse conversion is sometimes observed -4. In plThe majority of plant mitochondrial RNA editing occurs in coding sequences, and editing frequently changes codons, resulting in changes of amino acids, or, in some cases, creation of entirely new open reading frames ,9,10. Thcis-recognition [The precise biochemical basis for C-to-U editing in plant mitochondria is unknown, although experimental evidence suggests a deamination reaction -18. Despognition -21. The ognition . A highlognition ,16. Addiognition .Arabidopsis thaliana, Brassica napus and Oryza sativa. The objective for the current research was to identify sequence features that may provide insights into C-to-U editing of plant mitochondrial RNA. We address the following specific questions. Is there evidence that sufficient information exists within sequence regions flanking edited sites to accurately predict editing? Is there an association between estimated free energy of folding for short sequence regions containing edited sites and C-to-U editing? We report tree-based statistical analysis of three complete mitochondrial genomes and show that relatively simple models provide moderately accurate prediction of C-to-U edited sites.Identifying edited sites thus remains an open problem, one to which we have applied tree-based statistical models and an extension of such models. When applied to a similar problem (predicting peptide binding to major histocompatibility complex (MHC) class I molecules ), tree-b = 0.640, and specificity [the proportion of observations correctly identified as non-edited]  = 0.883) across the three species.Analysis of each of the three species-specific mitochondrial genome data sets yielded substantially similar results Table . Using fAs an additional classification tree analysis, we examined a dataset generated by combining the data from the three species. These results were generally similar to those described above for the mean of the individual genome datasets. The classification tree model is shown in Figure  = 0.717, specificity  = 0.809). Analysis of the larger, combined data set yielded a model better than any of the single genome models with an accuracy of 0.848  each of which was built using random subsamples of the data. This sub-sampling has the effect of reducing the variance through averaging and also reducing the correlation among models.One of the advantages that random forests have over single classification trees is that they provide quantitative measures of variable importance, whereas with a simple classification tree, one is primarily limited to inferring variable importance from the frequency and location of the occurrence of variables in the model. One measure of variable importance is the decrease in the Gini index (a measure of impurity of observations at a particular node) induced by splitting on the variable, averaged over all trees .Arabidopsis thaliana, which noted the -1, and -2 positions had highly non-random nucleotide distributions [Arabidopsis in that we find no indication that the -17 position has much importance in edited site recognition. Also previously noted was that for 93.1% of the time [In order to infer the relative importance of the predictor variables, we considered the measure of variable importance produced during the random forest run on the combined dataset, which is the most broadly representative dataset considered here. A plot of the variable importance measure for this dataset is shown in Figure ibutions . HoweverThe free energy results contrast with previous studies indicating that secondary structure was not important in edited site recognition ,19. Our Simple models based on nucleotides surrounding edited/non-edited sites and on estimated folding energies of those regions provide moderately accurate prediction of C-to-U RNA edited sites. More nuanced representation of secondary or higher-order structure in combination with variables based on the nucleotide positions found important here might improve models. Overall, the results strongly suggest that the C-to-U editing mechanism in plant mitochondria does not depend exclusively on the primary sequence immediately in the vicinity of the edited site.Arabidopsis thaliana (L.) Heynh. (mouse-ear cress), 455 edited sites, GenBank accession number NC_001284 [Brassica napus L. (rapeseed), 425 edited sites, GenBank accession number AP006444 [Oryza sativa L (rice), 486 edited sites, GenBank accession numbers AB076665 and AB076666 [We obtained complete mitochondrial genome sequences and information regarding edited sites from GenBank  for threC_001284 ; BrassicAP006444 ; and OryAB076666 . None ofArabidopsis, 444; Brassica, 422; Oryza, 481. In total, 19 edited sites in the GenBank files were not included across all three species.Incomplete annotations in the GenBank sequences required us to algorithmically determine on which strand an edited site fell (the GenBank files sometimes supplied only a position number, with no strand information). The algorithm, implemented in a Perl script, scanned the entire GenBank file and built an in-memory representation of the layout of all genes and coding sequence regions in the genome. The strand with which an edited site was associated could then be determined by consulting the resultant genome map and checking which strand at the edited site contained a gene region. In no case were genes on both strands at an edited site, so strand localization was always unambiguous. In a few cases, however, a gene containing an edited site could not be located, or a site marked as a C-to-U edit did not contain a C in either strand. In these cases, the supposed edited site was eliminated from further consideration. Final numbers of included sites were as follows: We also constructed a set of null observations of cytidines that are not edited to uridines. In constructing a null-set, it is important to ensure that the observations are as alike as possible to the edited observations (differing only in the trait to be measured), or the resulting model may be fictive. Here, our null-set observations were non-edited cytidines chosen at random from within gene regions of the genome. Additionally, we chose cytidines such that the null set had exactly the same distribution of codon positions as did the edited set, because the distribution of edited sites within the three possible positions of a codon is highly non-random with a bias to the first two positions  Table 33.For each observation, we recorded 40 nucleotide state variables: one variable for each of the 20 nucleotides sites 5' and 3' of the edited C (on the same strand). We chose a value of 20 for the number of nucleotides 5' and 3' so as to encompass the entire range of semi-conserved positions previously suggested, the most extreme of which occurs 17 bases 5' of the edited site . In someFinally, we created a combined data set to use alongside the species-specific datasets. The combined dataset is the result of combining all edited sites from all three species , and then randomly selecting negative examples from the set of those already chosen for the three individual datasets. Negative examples were chosen to exactly match the positive examples in distribution over both species and codon position. The combined dataset comprises 2,694 observations.We used the R language for statistical computing , versioni ∈ A? Where A is a region of the variable space defined by some criterion of a single predictor variable. Answering such a question for all observations produces two groups: those observations for which the answer is yes (those in region A) and those for which the answer is no (xi ∉ A, those in ). Subsequent binary partitioning continues until stopping criteria (variously defined) are met [SE of the best tree.Tree-based statistical models , also kn are met . The res4 permutations is the estimate of the probability associated with the observed result.We assessed the significance of our tree-based statistical models through permutation where the predictor variables are randomized with respect to the response variable . The fre4 individual models constructed by sub-sampling seven predictor variables at each node.If one tree-based statistical model is good, then an ensemble (forest) of appropriately constructed tree models should be even better, which is the principal idea of random forests. A random forest attempts to improve upon a simple tree-based statistical model by generating a collection of such models and using them in aggregate ,32. EachSeveral model summary statistics were calculated, including sensitivity, which is the proportion of observations correctly identified as edited, specificity, which is the proportion of observations correctly identified as non-edited, and accuracy, which is the total proportion of observations correctly identified. More formally, these definitions are:sensitivity = true positives/;specificity = true negatives/; andaccuracy = (true positives + true negatives)/total.MPC conceived, designed and coordinated the study. DSM carried out the programming and statistical analyses. Both authors wrote and approved the final manuscript.Arabidopsis thaliana data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation.Click here for fileBrassica napus data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation.Click here for fileOryza sativa data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation.Click here for fileCombined data file File is plain text, space delimited. First row is column headings with variable names: edit; + site is edited, - site is not edited; -20 through 20, nucleotide position relative to edited site; cp, codon position; fe, estimated folding energy; dfe, difference in estimated folding energy between pre-edited and edited sequences; and loc, location of focus site (position 0) in GenBank file. Each subsequent line represents a observation.Click here for file


The Icelandic population is now a part of a unique epidemiological study, which has involved investigating the genetic heritage of many of them. The reason that this experiment can be done is because of the remarkable records that exist in Iceland. Not only is there almost complete genealogical information dating back to the 18th century on all current (288,000) and many previous Icelanders , but in addition the country has an almost complete cancer registry dating from 1955. A company, deCODE Genetics, was set up to mine health-care data in Iceland, and to use it to assess the effect of genetics on health. Initially, the company attracted criticism, with some questioning the ethics of providing access to health-care data for many disease projects to a for-profit company. But the company has been supported by many Icelanders themselves, demonstrated by Icelanders donating blood samples with informed consent for research on multiple diseases, and now the project's scientific value is becoming apparent.PLoS Medicine that assesses how much genetic factors contribute to cancer risk across the whole Icelandic population.One such analysis is the subject of a paper by Laufey Amundadottir and colleagues in this month's The paper looked at 27 different types of cancers  that had been registered between 1955 and 2002 and analysed the frequency of close and distant relatives also having that cancer, or another kind of cancer. Of the 27 cancers, 16 showed significant “familiality,” and for some this risk even extended to distant (that is, third- to fifth-degree) relatives. The seven cancers with the highest increased familial occurrence both in close and distant relatives were breast, prostate, stomach, lung, colon, kidney, and bladder cancers. And, interestingly, three cancers—stomach, lung, and colon cancer—were also seen more frequently in mates of patients, indicating a shared environmental risk factor. And for some cancers there was a familial association with other cancers, for example, relatives of individuals with stomach, colon, rectal, or endometrial cancer were more likely to have any of these cancers.Cathryn Lewis, the academic editor for the paper comments on the study's strengths. “This level of family relationship and clinical diagnosis is rarely available from interviewing patients and family members. The size of the study  and the high quality of data enables the authors to detect subtle effects across distant relationships.”How robust are these data, and what do they mean for the biological understanding of cancer? As Lewis says, “Although the current study is impressive in its size and scope, even here, the sample size becomes an issue, with the most convincing results seen in the most common cancers.” Certainly not all the findings are surprising; some rare cancers are already known to be associated with particular genetic defects, and syndromes that predispose to multiple cancers have been described, for example, that of Hereditary Nonpolyposis Colorectal Cancer. Other associations are more intriguing—the cluster of related cancers that include prostate, kidney, and bladder could possibly have a developmental origin, since all arise from the same part of the embryo.So, by highlighting these subtle links, the study's particular value may become apparent: deciding future avenues of investigation in the complex interrelationships that interact to produce cancer.


Microarray technologies are rapidly becoming available for new species including teleost fishes. We constructed a rainbow trout cDNA microarray targeted at the identification of genes which are differentially expressed in response to environmental stressors. This platform included clones from normalized and subtracted libraries and genes selected through functional annotation. Present study focused on time-course comparisons of stress responses in the brain and kidney and the identification of a set of genes which are diagnostic for stress response.Fish were stressed with handling and samples were collected 1, 3 and 5 days after the first exposure. Gene expression profiles were analysed in terms of Gene Ontology categories. Stress affected different functional groups of genes in the tissues studied. Mitochondria, extracellular matrix and endopeptidases  were the major targets in kidney. Stress response in brain was characterized with dramatic temporal alterations. Metal ion binding proteins, glycolytic enzymes and motor proteins were induced transiently, whereas expression of genes involved in stress and immune response, cell proliferation and growth, signal transduction and apoptosis, protein biosynthesis and folding changed in a reciprocal fashion. Despite dramatic difference between tissues and time-points, we were able to identify a group of 48 genes that showed strong correlation of expression profiles (Pearson r > |0.65|) in 35 microarray experiments being regulated by stress. We evaluated performance of the clone sets used for preparation of microarray. Overall, the number of differentially expressed genes was markedly higher in EST than in genes selected through Gene Ontology annotations, however 63% of stress-responsive genes were from this group.1. Stress responses in fish brain and kidney are different in function and time-course. 2. Identification of stress-regulated genes provides the possibility for measuring stress responses in various conditions and further search for the functionally related genes. Until recently multiple gene expression profiling was applied almost exclusively to human and a few model organisms. At present cDNA microarrays are being constructed for new species including teleost fishes -6. SinceFunctional annotation of genes, especially Gene Ontology  is increStress is closely associated with many diverse issues in fish biology and environmental research (reviewed in ). StressThe rainbow trout cDNA microarray was composed of EST and selected genes. The cDNA libraries were prepared from tissues of stressed fish using suppression subtractive hybridization, SSH  and a moFish were stressed with netting and samples were collected 1, 3 and 5 days after the first exposure. We used plasma cortisol as a stress marker . The horGSM22355). Two genes were up-regulated in both tissues at all time-points  increased steadily, which implied possible degradation of tissue with prolonged stress. We could expect abrupt fluctuations in the rainbow trout brain, since transient induction and up-regulation of gene expression was observed in response to cold in the brain of channel catfish . In our Grouping of individual differentially expressed genes by the functional classes reduced noise and enhanced cluster and factorial analyses. This helped to identify stress-responsive genes that showed correlated expression in 35 microarray experiments  and "fishes NOT stress" (10069 abstracts). Abstracts were split into separate words and a list of non-redundant terms was composed. The numbers of abstracts including each term were estimated. The terms were ranked by the Z-scores of hypergeometric distribution and enrichment was analysed with exact Fisher's test (p < 0.05).One year old rainbow trout were stressed with netting for 2 min, this treatment was repeated once a day for a duration of 5 days. Fish were killed with over-dose of anaesthetic (MS-222) and blood was taken from the caudal vein. The kidneys and brains were snap-frozen in liquid nitrogen. Plasma cortisol was determined with RIA using Orion Spectra Cortisol kit.EcoRI and NotI sites: 5'-ACGAGGCGAATTCACAGAGAGGAG(T)VN-3', 5'-GAGAGAGAGTGGTGCGGCCGCGGTGTATGGGG-3'). Double-stranded cDNA was generated using Advantage DNA polymerase mix (Clontech) and PCR primers: 5'-ACGAGGCGAATTCACAGAGAGGAG-3' and 5'-GAGAGTGGTGCGGCCGCGGTGTA-3'. The PCR products were purified with QIAquick kit (Qiagen), precipitated with ethanol and dissolved to 1 μg/μl in hybridization buffer (1 M NaCl, 50 mM HEPES (pH 8.3), 1 mM EDTA). DNA was denaturated for 5 min at 94°C. Following re-association at 72°C for 16 hours, DNA was ethanol precipitated and digested with 150 U of exonuclease III (MBI Fermentas) for 15 min at 37°C. This treatment eliminates re-associated double-stranded DNA [®-11Zf (+) (Promega). Normalized and subtracted cDNA libraries were prepared from the stressed fish (whole fry, brain, kidney and spleen of 1-year old fish). A number of clones were from the rainbow trout and Baltic salmon cDNA libraries constructed in University of Turku. The sequences were analysed with stand-alone blastn and blastx [RNA was extracted with Trizol reagent (Invitrogen) and mRNA was purified with Dynabeads kit . SSH cloning was performed as described . For prended DNA . Single-d blastx  Microarrd blastx  and the I/B ≥ 3 and (I-B)/(SI+SB) ≥ 0.6, where I and B are the mean signal and background intensities and SI, SB are the standard deviations. After subtraction of mean background, lowess normalization [Total RNA was extracted with Trizol reagent (Invitrogen) and 4 individuals were pooled in each sample. Stressed fish was compared with time-matched control. Labeling with Cy3- and Cy5-dCTP (Amersham Pharmacia) was made using SuperScript III (Invitrogen) and oligo(dT) primer; cDNA was purified with Microcon YM30 (Millipore). We used a dye swap experimental design ,15 and elization  was perfPrimers Table  were desAK designed microarray, carried out experiments with fish and data analyses and drafted the manuscript. HK conducted the microarray analyses. PP developed software for annotation of genes and analyses of Medline abstracts. CR constructed the multi-tissue cDNA library and provided the selected genes. SA developed software for management of microarray data and performed the statistical analyses. HM coordinated research. All authors read and approved the final manuscript.


A diaphragmatic hernia is defined as a defect in part of the diaphragm through which abdominal contents can protrude into the thorax. It may be congenital or acquired. In this case report, we aim to demonstrate a congenital diaphragmatic hernia in an adult marfanoid patient which required emergency treatmentA 43 year old woman was admitted with classical appendicitis requiring surgery. She incidentally had Marfan's clinical features with a positive family history for the syndrome. At operation she had grossly abnormal abdominal anatomy. Radiological investigations demonstrated a large right congenital diaphragmatic hernia with an intrathoracic hernial sac containing a perforated gangrenous appendix. The hernial sac was opened surgically and the appendix excised. The patient made a full recovery.Diaphragmatic hernias are usually congenital in nature often requiring early corrective surgery for future survival. We have demonstrated the presence of an unusually large diaphragmatic defect, almost a hemidiaphragmatic defect, of unknown direct etiology, but of some possible association with Marfan's syndrome in an adult patient presenting with an acute perforated gangrenous appendix requiring emergency life-saving surgery. A diaphragmatic hernia is defined as a defect in part of the diaphragm through which abdominal contents can protrude into the thorax. It may be congenital or acquired, usually through trauma .Congenital diaphragmatic hernias usually occur in the posterolateral portion of the diaphragm  and are on the left side in 90% of cases. Loops of bowel, even most of the abdominal contents, may protrude into the hemithorax on the involved side ,5.Most congenital diaphragmatic hernias are detected in the newborn whereby, after delivery, as the infant cries and swallows air, the loops of bowel quickly fill with air and rapidly enlarge, causing acute respiratory embarrassment as the heart and mediastinal structures are pushed to the right, compressing the right lung. Respiratory distress is immediate in severe cases; a scaphoid abdomen  is likely. Bowel sounds (and an absence of breath sounds) may be heard over the involved hemithorax. In less severe cases, mild respiratory difficulty develops a few hours or days later as abdominal contents progressively herniate through a smaller diaphragmatic defect . Urgent Acquired diaphragmatic hernias are relatively rare and result from either blunt or penetrating trauma. Blunt trauma typically produces large radial tears measuring 5–15 cm, most often at the posterolateral aspect of the diaphragm.Penetrating injuries to the diaphragm can follow accidental trauma, knife or gunshot wounds. Typically, the defect is small, less than two centimeters in size and may present late after years of gradual herniation and enlargement. Occasionally a shotgun blast causes a large defect ,4.et al [et al [et al [Diaphragmatic hernias are usually congenital in nature often requiring early corrective surgery for future survival. There have been three recent publications -7 in theet al  describel [et al  recentlyA 43 year old female district nurse presented to the accident and emergency department with a 6 hour history of initially generalized abdominal pain which was localizing to the right. This was associated with nausea and loss of appetite, made worse by movement and not relieved with intramuscular morphine injection. There was no history of recent or past trauma to the chest or abdomen. On examination, she was tender in the right upper quadrant and right iliac fossa with rebound and guarding, with a Rovsing positive sign and normal bowel sounds. She was incidentally found to have some marfanoid features including the long span of upper limbs, a high arched palate and the very soft early diastolic murmur of aortic regurgitation. The patient had a positive family history of Marfan's syndrome but had never undergone genetic testing to confirm the diagnosis.A likely diagnosis of appendicitis was made on the clinical picture associated with a pyrexia and raised white cell count and C – reactive protein. Her abdominal X-ray at that time showed absence of gas in the right side of her bowel. Her chest X-ray did not show any obvious abnormality Figure She was taken to theatre the same day for appendicectomy through a standard right iliac fossa incision. There was a small amount of free fluid but the right iliac fossa was filled with a normal looking gall bladder and small bowel only with no sign of the caecum, appendix, ascending or proximal transverse colon. The terminal ileum was found to run up under the right lobe of the liver towards the hilum. The right iliac fossa wound was closed and we proceeded to an exploratory laparoscopy.At laparoscopy, the liver was retracted to see under the right liver lobe. The proximal transverse colon was found to be running up towards the liver hilum were it felt to be tethered. This suggested a possible herniation of the right colon into the chest. The procedure was abandoned and a CT thorax and abdomen were performed to define the anatomy involved.This Figure , 3 demonThe symptoms and signs persisted and a laparotomy was performed. At laparotomy, there was some free fluid. There was definitely a large defect in the right hemidiaphragm. The right liver lobe had grown right up into the right intrathoracic space. There was a large hernial sac beneath the right lobe of the liver between the common bile duct, duodenum and liver, displacing the right kidney medially and containing transverse colon, terminal ileum, caecum, appendix and free fluid. The contents of this sac were successfully reduced and a perforated gangrenous appendix with pus was found within the intrathoracic hernial sac. A standard appendicectomy was performed. The right colon was formally mobilized, fully reduced and the caecum was fixed within the right iliac fossa.The patient was admitted to the intensive care unit postoperatively where she made a good recovery. She had instant relief of her abdominal symptoms and post-operative contrast study demonstrated the presence of the right colon within the abdomen Figure . She wasWe have demonstrated a new case of an adult patient, with Marfan's syndrome clinical features and a positive family history of the syndrome, presenting with a large congenital diaphragmatic hernia, compatible with life and undiagnosed into adulthood, requiring an emergency admission into hospital with a perforated gangrenous intrathoracic appendix.CT = computerized tomographyThe author(s) declare that they have no competing interests.MJB designed the case report, researched the article and drafted the manuscript.Both MJB and JHV carried out the surgery and were involved in all investigations.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The major damaging factor during and after the ischemic/hypoxic insult is the generation of free radicals, which leads to apoptosis, necrosis and ultimately cell death. Rubia cordifolia (RC), Fagonia cretica linn (FC) and Tinospora cordifolia (TC) have been reported to contain a wide variety of antioxidants and have been in use in the eastern system of medicine for various disorders. However, their mechanism of action was largely unknown. We therefore selected these herbs for the present study to test their neuroprotective ability and the associated mechanism in rat hippocampal slices subjected to oxygen-glucose deprivation (OGD).2) in the supernatant and protein assays were performed in the respective groups at various time intervals. EPR was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion (O2.-), hydroxyl radicals (. OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-NO and Sin-1 respectively. RT-PCR was performed for the three groups for GCLC, iNOS, Cu-Zn SOD and GAPDH gene expression.Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) and divided into 3 groups: control, OGD and OGD + drug treated. Cytosolic Cu-Zn superoxide dismutase (Cu-Zn SOD), reduced glutathione (GSH), glutathione peroxidase (GPx), nitric oxide (NO) was measured as nitrite (NOAll the three herbs were effective in elevating the GSH levels, expression of the gamma-glutamylcysteine ligase and Cu-Zn SOD genes. The herbs also exhibited strong free radical scavenging properties against reactive oxygen and nitrogen species as studied by electron paramagnetic resonance spectroscopy. In addition all the three herbs significantly diminished the expression of iNOS gene after 48 hours which plays a major role in neuronal injury during hypoxia/ischemia.RC, FC and TC therefore attenuate oxidative stress mediated cell injury during OGD and exert the above effects at both the cytosolic as well as at gene expression level and may be an effective therapeutic tool against ischemic brain damage. It is generally believed that a major portion of post-traumatic neuronal necrosis after brain injury does not result from diffuse primary injury, but rather from a secondary process. The injury appears to trigger a cascade of molecular events that lead to gradual vascular and neuronal tissue degeneration, thus destroying the anatomical substrate necessary for the neurological recovery. A large body of evidence obtained from a wide variety of experimental studies of acute CNS injury strongly suggest that various reactive oxygen species (ROS) and nitrogen species (RNS) have been implicated in the progressive secondary degeneration that follows the injury -3. ROS aAll reagents unless stated otherwise were obtained from Sigma Chemical Co. USA, Merck, India and Loba Chemie, India. All animals used were as per the institutional animal ethics committee approval.2 – 2.0, MgSO4 – 1.0, NaHCO3 – 26, Na2HPO4 – 1.25 and D-glucose – 10 mM. The chilled brain was removed and the hippocampal regions were dissected, slices of 0.6 mm were obtained on a modified Stadie – Riggs microtome and were immersed in oxygenated aCSF between 22–28°C for 120 min to allow the tissue to get stabilized.Method for preparation of slices was similar to those of Taylor & Weber. Normal 2, until the partial oxygen pressure was less then 20% as that of normoxic aCSF . Slices were incubated in oxygen glucose deficient aCSF for 30 min at 37°C. Transferring the slices to reperfusion chamber containing aCSF started reperfusion. The extent of injury was estimated by assaying LDH release in the medium using an LDH kit.Hippocampal Slices were subjected to OGD (oxygen glucose deprivation) .  The overall control group consisted of the slices immediately stabilized for 2 hours at 22–26°C in normoxic aCSF.b) The experimental group consisted of stabilized slices subjected to OGD without any drug treatment.c) The experimental treated group consisted of OGD slices reperfused in the medium (aCSF) containing1) 2 mg/ml concentration of RC, TC and FC for 30 min.2) Ascorbic acid and Reduced glutathione equivalent to their content in RC, FC and TC for 30 min.In all the above study groups the following biochemical parameters were monitored.1) Free Cu-Zn superoxide dismutase (Cu-Zn SOD) was assayed using themethod of Beyer et al.2) Reduced Glutathione (GSH) was assayed by the method of Teitz .3) Glutathione peroxidase (GPx) was assayed by the method of Paglia et al. .2) in the supernatant by the method of Green et al. [4) Nitric oxide (NO) was measured as nitrite  Protein was estimated by the method of Lowry et al. .2.), hydroxyl radicals (.OH), nitric oxide (NO) radical and peroxynitrite anion (ONOO) generated from pyrogallol, menadione, DETA-No and Sin-1 respectively. All the free radical donors (100 μM – 500 μM) were incubated in phosphate buffered saline (pH 7.4, 37°C) containing the spin-trap, Tempone-H (1 mM), oxidation of which generates 4-oxo-tempo with a characteristic three-line EPR signal centred at 3365 G. Development of this signal was monitored for 60 min from addition of the oxidising species and compared to parallel incubations containing RC, FC and TC (10 or 50 μM, n = 3). The amplitude of the first line of the spectrum was measured; data are expressed in arbitrary units. The EPR parameters for these experiments were as follows: Microwave frequency – 9.4 GHz; microwave power – 20 mW; modulation frequency – 100 kHz; modulation amplitude – 1500 mG; centre field 3365 G; sweep width 50 G; sweep time 20 sec; No. of passes – 1; receiver gain 3E1.EPR (Magnettech X-band Miniscope MS-100, Berlin Germany) was used to establish the antioxidant effect of RC, FC and TC with respect to superoxide anion , iNOS (inducible nitric oxide synthase) and Cu-Zn SOD mRNA was isolated using TriZOL reagent from the hippocampal slices and reverse transcribed to study the effect of RC, FC and TC on the expression status of the above genes in OGD untreated slices after a period of 24 and 48 hours post OGD. Gene expression in treated groups was studied after 24 hours. GAPDH served as the house-keeping gene. After an initial reverse transcription, the cDNAs obtained were amplified using the following respective primers and PCR conditions:for' 5'gtggtactgctcaccagagtgatcct and rev' 5'tgatccagtaactctgggcattcaca. iNOS was amplified at 94°C for 1 min, 60°C for 1 min, and 72°C for 1 min for a total of 27 cycles followed by a 10-min extension at 72°C using primers: for'5'-gtgttccaccaggagatgttg-3' and rev' 5'-tggggcagtctccattgcca-3'. Cu-Zn SOD was amplified at 94°C for 45 s, 56°C for 30 s, and 72°C for 45 s for a total of 23 cycles followed by a 10-min extension at 72°C using primers: for'5'-tctaagaaacatggcggtcc-3' and rev'5'-cagttagcaggccagcagat-3'. GAPDH was amplified using 20 thermal cycles of 94°C for 45 s, 60°C for 45 s, and 72°C for 1 min 30 s, followed by final extension for 10 mins at 72°C, primer used were for'5'ccacccatggcaaattccatggca and rev 5'tctagacggcaggtcaggtcaacc.GCLC was amplified by 32 thermal cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 2 mins followed by an extension at 72°C for 10 mins using primers: Figure Figure 2.-, .OH, NO and ONOO using specific donors. EPR spectroscopy revealed that addition of RC, FC or TC (10 μg/ml) to the O2.- generator, pyrogallol , which requires GSH as one of the co-substrates for the reaction ,31. This2- and NO and acts as a direct scavenger of these extremely potent hazardous species. We therefore investigated whether the antioxidant properties exhibited by the three herbs also involve their modulatory effect on the expression of the Cu-Zn SOD expression. The increased expression of the Cu-Zn SOD gene in RC, FC and TC treated OGD slices clearly confirm the positive modulatory effect the three drugs have on the cellular antioxidant system. Increased expression of Cu-Zn SOD in response to these herbs is a crucial finding especially since this enzyme is directly implicated in the scavenging of not only O2- but also the more potent toxicant ONOO [A large body of evidence suggests that another important intracellular enzyme Cu-Zn SOD is found to be neuroprotective in nature along with GPx and GSH ,33. We oant ONOO . Thus th2- to form a more toxic ONOO, which is extremely deleterious to the cells [2-. Furthermore the three herbs were found to repress the expression of the iNOS gene, which is considered to be an important damaging factor during hypoxia/ischemia [The mechanism of antioxidant and neuroprotective effects reported above for RC, FC and TC are new findings. However, as to what components of the drugs bring about such cell-protective effects could not be fully ascertained. Preliminary analysis has revealed that all the three herbs have significant amounts of GSH and Vit C and another important antioxidant, polyphenol. In addition inductively coupled plasma spectroscopic analysis have also revealed the presence of important trace elements in the three herbs . We therefore thought that the three herbs exert their ameliorative properties due to their antioxidant and trace element contents. In order to test this hypothesis we investigated the effect of GSH and Vit C on NO generation in OGD treated and untreated hippocampal slices. The amount of GSH and Vit C used were equivalent to that found in the three herbs. Our results indicate that GSH and the Vit C components of the herbs are at least partly responsible for the attenuation of NO generation in the treated OGD slices. The effects of RC, FC and TC were almost similar to those recorded for GSH and Vit C. NO is an important neurotransmitter in the brain and also can be rendered harmful due to unprecedented generation during hypoxic/ischemic/inflammatory conditions ,36. In ahe cells . Hence oischemia . Thus thIn conclusion, RC, FC and TC exert cell/neuroprotective properties via preventing the depletion and increasing GSH levels by inducing GCLC expression, by reducing oxidant levels via direct scavenging, decreasing iNOS expression and by increasing the antioxidant gene Cu-Zn SOD. Further protective ability may be attributed to the enhanced activity of GPx brought about by the herbs. The antioxidant contents of the herbs appear to be important components for the observed effects. However, further investigations are required to ascertain the role of individual constituents in the efficacy of the above described properties of the herbs in order to ascribe potential pharmacological applications to these herbs.Authors do not have any competing interest with anyone whatsoever.AKR – This work is a part of AKR's PhD thesis and he has done all the experiments, data collection and processing and manuscript preparation.MGM – He is co-supervisor for AKR and was involved in the ideology, manuscript preparation and providing facilities for the work.SKB – He is the supervisor for AKR and was involved in the main ideology and study design of the work, manuscript preparation and performing EPR studies and training AKR for the respective techniques.The pre-publication history for this paper can be accessed here: