Many protein-protein interactions (PPIs) are compelling targets for drug discovery, and in a number of cases can be disrupted by small molecules. The main goal of this study is to examine the mechanism of binding site formation in the interface region of proteins that are PPI targets by comparing ligand-free and ligand-bound structures. To avoid any potential bias, we focus on ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) techniques and deposited in the Protein Data Bank, rather than on ensembles specifically generated for this study. The measures used for structure comparison are based on detecting binding hot spots, i.e., protein regions that are major contributors to the binding free energy. The main tool of the analysis is computational solvent mapping, which explores the surface of proteins by docking a large number of small “probe” molecules. Although we consider conformational ensembles obtained by NMR techniques, the analysis is independent of the method used for generating the structures. Finding the energetically most important regions, mapping can identify binding site residues using ligand-free models based on NMR data. In addition, the method selects conformations that are similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. This agrees with the conformational selection model of molecular recognition, which assumes such pre-existing conformations. The analysis also shows the maximum level of similarity between unbound and bound states that is achieved without any influence from a ligand. Further shift toward the bound structure assumes protein-peptide or protein-ligand interactions, either selecting higher energy conformations that are not part of the NMR ensemble, or leading to induced fit. Thus, forming the sites in protein-protein interfaces that bind peptides and can be targeted by small ligands always includes conformational selection, although other recognition mechanisms may also be involved. Many protein-protein interfaces (PPIs) are biologically compelling drug targets. Disrupting the interaction between two large proteins by a small inhibitor requires forming a high affinity binding site in the interface that generally can bind both peptides and drug-like compounds. Here we investigate whether such sites are induced by peptide or ligand binding, or already exist in the unbound state. The analysis requires comparing ligand-free and ligand-bound structures. To avoid any potential bias, we study ensembles of ligand-free protein conformations obtained by nuclear magnetic resonance (NMR) rather than generated by simulations. The analysis is based on computational solvent mapping, which explores the surface of the target protein by docking a large number of small “probe” molecules. Results show that ensembles of ligand-free models always include conformations that are fairly similar to some peptide-bound or ligand-bound structure in terms of the properties of the binding site. The analysis also identifies the models that are the most similar to a bound state, and shows the maximum level of similarity that is achieved without any influence from a ligand. While forming the binding site may require a combination of recognition mechanisms, there is preference for the spontaneous formation of bound-like structures. Many protein-protein interactions (PPIs) are involved in disease pathways where therapeutic intervention could bring widespread benefit, and hence are biologically compelling targets for drug discovery To evidence conformational selection we need to show that some of the conformations in the free state resemble the ligand-bound structure While simulations tools are extremely useful for the characterization of molecular motion, they do not necessarily represent the best starting point for the analysis of recognition mechanisms, as the goal of increasing the number of conformational transitions for more significant results may unintentionally influence the conclusions. Thus, it is useful to consider conformational ensembles that are fully independent of the particular study. With this motivation in mind, in this work we consider already existing conformational ensembles, obtained by nuclear magnetic resonance (NMR) methods. The structures in such ensembles are low energy models that satisfy the highest number of NMR derived restraints. The ensembles are well documented as they have been deposited in the Protein Data Bank (PDB) Once ensembles of free and ligand-bound conformations are available, we need an appropriate measure for comparing ligand-free and ligand-bound conformations in order to assess their similarity. The overall RMSD is clearly not suitable, since we are interested only in the changes around the ligand binding site. While one can calculate RMSD for the binding site residues, the latter are generally selected on the basis of their proximity to a bound ligand, which makes the results specific to a particular compound. The unique feature of this paper is that the measures used for structure comparison are based on binding hot spots, i.e., regions that are major contributors to the binding free energy. Binding hot spots are good binding sites in the general sense, i.e., without reference to any ligand As the primary tools of our analysis, we rely on two algorithms called FTMap and FTSite. FTMap is a direct computational analog of the fragment screening experiments We have studied the five proteins listed in Residues with any atom closer than 4 Å to any atom of the ligand in the bound structure were defined as binding site residues. Although for the five test proteins in As discussed, the residues in the binding site are likely to interact with probes in many structures. This is shown in n residues, for each unbound structure the mapping fingerprint define a vector of n-dimensional space, X = , and the ligand fingerprint of the bound structure is also an n-dimensional vector, Y =   is an important drug target for its role in binding and negatively regulating the tumor suppressor p53 The three PDZ domains of the postsynaptic density protein 95 (PSD-95) regulate signaling in glutaminergic neurons by modulating protein-protein interactions The PDZ domains of membrane-associated guanylate kinase with inverted domains 1 (MAGI-1) interact with the E6 proteins of human papillomaviruses (HPVs) The like-SM (LSm) domain of the enhancer of decapping 3 (EDC3) activator protein modulates the activity of the DCP1:DCP2 decapping complex, which catalyzes the removal of the mRNA 5′ cap B-cell lymphoma-extra large (Bcl-xL). In several tissues, DNA damage induces apoptosis via the stabilization of p53. Bcl-xL is an antiapoptotic protein, which sequester p53 The main tools of our analysis are the FTMap Using the FTSite results BSSC for a ligand-free structure is the correlation coefficient between the mapping fingerprint, defined as the number of probe-protein interactions for each residue divided by the total number of interactions, and the ligand fingerprint of a bound structure, which is the number of ligand-protein interactions for each residue divided by the total number of interactions. If the bound structure is an NMR ensemble, BSSC is based on the average ligand fingerprint. As shown by the Figure S1Mapping fingerprints of MDM2 from unbound model 9 (blue), in comparison to ligand fingerprint calculated from a piperidinone bound structure (red). Horizontal axes list residues of MDM2 from Glu25 to TYR104 . Vertical axis shows the fraction of atom-atom interactions each protein residue makes with probe or ligand atoms.(TIF)Click here for additional data file.Table S1Validity of averaging fingerprints over bound structures solved by NMR. Pairwise correlation coefficients between the fingerprints for models 1–5 and the average fingerprint of the five ligand-bound MDM2 structures (PDB ID 2lzg).(DOCX)Click here for additional data file.Table S2Validity of averaging fingerprints over bound structures solved by NMR. Correlation coefficients between each fingerprint for models 1–22 and the average fingerprint of the 22 peptide-bound PSD-95 PDZ1 structures (PDB ID 1rgr).(DOCX)Click here for additional data file.Table S3Validity of averaging fingerprints over bound structures solved by NMR. Correlation coefficients between each fingerprint for models 1–20 and the average fingerprint from the ensemble of the 20 peptide-bound MAGI-1 PDZ1 structures (PDB ID 2kpl).(DOCX)Click here for additional data file.Table S4Validity of averaging fingerprints over bound structures solved by NMR. Correlation coefficients between the fingerprint for models 1–22 and the average fingerprint from the ensemble of the 22 peptide-bound EDC3 structures (PDB ID 4a54).(DOCX)Click here for additional data file.Table S5Binding site hit rates (HRs) and bound state similarity coefficients (BSSCs) for the ensemble of ligand-free MDM2 structures (PDB ID 1zlm). The BSSC values are calculated using the three ligand-bound structures with PDB IDs shown. The models are sorted based on the hit rate. The maximum value in each column is shown in bold.(DOCX)Click here for additional data file.Table S6Binding site hit rates and bound state similarity coefficients (BSSCs) for the ensemble of ligand-free PSD-95 PDZ1 structures (PDB ID 1iu2). The BSSC values are calculated using the ligand-bound structure with PDB IDs 1rgr. The models are sorted based on the hit rate. The maximum value in each column is shown in bold.(DOCX)Click here for additional data file.Table S7Binding site hit rates and bound state similarity coefficients (BSSCs) for the ensemble of ligand-free MAGI-1 PDZ1 structures (PDB ID 1kpk). The BSSC values are calculated using the ligand-bound structure with PDB IDs 2kpl. The models are sorted based on the hit rate. The maximum value in each column is shown in bold.(DOCX)Click here for additional data file.Table S8Binding site hit rates and bound state similarity coefficients (BSSCs) for the ensemble of ligand-free EDC3 structures (PDB ID 4a53). The BSSC values are calculated using the ligand-bound structure with PDB IDs 4a54. The models are sorted based on the hit rate. The maximum value in each column is shown in bold.(DOCX)Click here for additional data file.Table S9Binding site hit rates and bound state similarity coefficients (BSSCs) for the ensemble of ligand-free Bcl-xL structures (PDB ID 2m03). The BSSC values are calculated using the two ligand-bound structures with PDB IDs shown in the table. The models are sorted based on the hit rate. The maximum value in each column is shown in bold.(DOCX)Click here for additional data file.Table S10Predicted and observed binding site residues.(DOCX)Click here for additional data file.


Fine-grained influenza surveillance data are lacking in the US, hampering our ability to monitor disease spread at a local scale. Here we evaluate the performances of high-volume electronic medical claims data to assess local and regional influenza activity.We used electronic medical claims data compiled by IMS Health in 480 US locations to create weekly regional influenza-like-illness (ILI) time series during 2003–2010. IMS Health captured 62% of US outpatient visits in 2009. We studied the performances of IMS-ILI indicators against reference influenza surveillance datasets, including CDC-ILI outpatient and laboratory-confirmed influenza data. We estimated correlation in weekly incidences, peak timing and seasonal intensity across datasets, stratified by 10 regions and four age groups . To test IMS-Health performances at the city level, we compared IMS-ILI indicators to syndromic surveillance data for New York City. We also used control data on laboratory-confirmed Respiratory Syncytial Virus (RSV) activity to test the specificity of IMS-ILI for influenza surveillance.Regional IMS-ILI indicators were highly synchronous with CDC's reference influenza surveillance data . Seasonal intensity estimates were weakly correlated across datasets in all age data (rho≤0.52), moderately correlated among adults (rho≥0.64) and uncorrelated among school-age children. IMS-ILI indicators were more correlated with reference influenza data than control RSV indicators . City-level IMS-ILI indicators were highly consistent with reference syndromic data (rho≥0.86).Medical claims-based ILI indicators accurately capture weekly fluctuations in influenza activity in all US regions during inter-pandemic and pandemic seasons, and can be broken down by age groups and fine geographical areas. Medical claims data provide more reliable and fine-grained indicators of influenza activity than other high-volume electronic algorithms and should be used to augment existing influenza surveillance systems. The last decade has seen dramatic developments in influenza surveillance systems at the regional and national scales. In the US however, despite intensified surveillance for influenza-like-illness (ILI) and laboratory-confirmed virus activity Electronic medical claims data provide a unique source of information on diagnoses made by physicians and are routinely used by pharmaceutical companies to monitor disease incidence and anticipate drug or vaccine sales. So far however, this resource has remained largely untapped by epidemiologists and public health researchers. A few promising studies have suggested that electronic claims data may be useful to monitor disease patterns of diarrheal and respiratory viruses in the US and evaluate pediatric vaccine coverage in Germany http://www.imshealth.com/portal/site/imshealth. In keeping with similar epidemiological analyses of large-scale insurance administrative databases, no institutional board review was sought. Further, all statistical analyses were based on aggregated incidence time series rather than individual patient-level information.All patient records and information were anonymized and de-identified prior to being handed over to researchers; all records were part of routinely collected information for health insurance purposes. Dr Farid Khan, Director of Advanced Analytics, IMS Health, granted access to the patient data. The database is not accessible online but researchers interested in gaining access to the data should refer to the IMS Health website: Our general approach is to compare weekly influenza indicators derived from electronic medical claims data against reference influenza surveillance time series, and against control time series unrelated to influenza (such as RSV surveillance data). Our statistical measures include correlations in weekly incidences, peak timing and seasonal estimates of epidemic intensity. Additionally we use permutation tests to show that the estimated correlations are stronger than those expected by chance between incidence time series that share common winter seasonality, so as to confirm that medical claims data capture signals truly specific of influenza activity. Analyses are conducted at the national, regional, and local scales, and stratified by age group.We used data maintained by IMS Health, a private data and analytics business that collects de-identified electronic CMS-1500 medical claim forms from full-time office-based active physicians throughout the US. Claims data are sourced from the practice management software vendors directly from the physician's office, or from the intermediary billing systems that coordinate the insurance claim transactions. In 2009, there were 560,433 active physician practices in the US of which IMS Health collected data from 354,402, or an approximate coverage rate of 61.5%. IMS Health receives the records within 1–2 weeks of the patient's visit. For validation purposes, we focused here on historic IMS Health data from July 2003 to June 2010. Claims data were kindly compiled by IMS Health for research purposes under a collaborative agreement with the authors.th revisions (ICD-9). We extracted visits for different outcomes, including ILI and RSV, as well as the total number of visits for any reason for denomination purposes. We created weekly time series based on the date of office visit. Several ILI case definitions were tested with the expectation that the most appropriate definition would produce a large and geographically heterogeneous spike in disease rates during the 2009 A/H1N1 influenza pandemic period, as observed in other surveillance datasets Diagnoses are coded in the physician offices using international classification of diseases, 9Based on preliminary analyses and previous work exploring the spatial dynamics of the 2009 influenza pandemic Weekly incidence time series were compiled and broken down by 10 administrative regions  and 4 agAll patient records and information were anonymized and de-identified; all records were part of routinely collected information for health insurance purposes. In keeping with similar epidemiological analyses of large-scale insurance administrative databases Publicly-available influenza surveillance data from 2003–2010 were obtained from two separate reference systems maintained by the CDC: (1) The Outpatient Influenza-like Illness (ILI) Surveillance Network and (2) the US Influenza Virologic Surveillance System We also compiled weekly national data on laboratory-confirmed RSV activity during 2003–2010 from the CDC's National Respiratory and Enteric Virus Surveillance System To evaluate the performances of IMS-Health at a local level, we focused on New York City, where disease surveillance is particularly well-established We also document 2009 pandemic disease patterns in 21 cities or county regions of New York State based on medical claims data, as there was important spatial heterogeneity in pandemic activity in this state We compared weekly ILI and RSV indicators based on medical claims with weekly reference surveillance data from July 2003 to June 2010. This period included 6 pre-pandemic seasons , and the spring and fall 2009 A/H1N1 pandemic waves (May–Aug 2009 and September 2009–June 2010).The spatial scale of most of our analyses was the region or city, except for comparisons with RSV laboratory-confirmed surveillance, for which retrospective data were available only nationally.t and region i, we defined the IMS-ILI incidence indicator as the ratio of all ILI visits in the IMS dataset to the total number of IMS visits that week, per 100,000 population, as in For week t,i/IMS_visitst,i)]*.IMS_ILI_incidence  = [ = flu_positivest,i, is the number of samples testing positive for influenza in week t and region i, total_specimens_testeds,i is the total number of samples tested in influenza season s and region iWhere flu_positivesTo investigate whether the IMS-ILI indicator provided accurate measurement of influenza epidemic patterns and following earlier work A complementary test of the specificity of medical claims for influenza surveillance was obtained by computing the correlation between the IMS-ILI indicators and reference RSV surveillance time series. These indicators share common winter seasonality but are presumably prone to independent yearly and weekly fluctuations specific to influenza and RSV.We compared the peak timing of disease activity each season . We computed the difference in peak timing per season, and report the average and range of differences by region.To obtain a summary measure of influenza intensity by season, we applied Serfling seasonal regression model to both medical claims and reference ILI time series From laboratory-confirmed influenza time series, we defined influenza seasonal intensity as the total virus percent positive each respiratory season , as in CDC summary reports All analyses where performed in R; scripts are available from the authors upon request.Weekly regional influenza time series are displayed in All three influenza surveillance datasets were strongly synchronous, as evidenced by high average week-to-week correlation across the 10 regions ; Permutations of respiratory seasons resulted in much lower week-to-week correlation between the regional incidence time series than in the original analysis . These results indicate that winter seasonality alone was insufficient to explain the high level of synchrony observed between these influenza surveillance time series.Results were robust to national aggregation of the data .Peak timing provides a complementary measure of synchrony between influenza surveillance systems. All three systems exhibited high agreement in estimated influenza peak week across all regions , with only 2 regions showing significant correlation .Next, we repeated the previous analyses stratified by four age groups . NeverthP<0.05, To check the specificity of medical claims data to monitor influenza activity, we compared IMS-ILI indicators against national reference surveillance data for RSV. The IMS-ILI indicator was significantly less correlated with reference RSV data than with reference influenza indicators on a national scale  compares favorably to that reported for Google Flu Trends , to the localized spring 2009 pandemic wave, and the very spiky and unusual fall 2009 pandemic wave. Further, we did not test the performances of IMS Health data for real time surveillance in a prospective manner, which would require a careful study of the dynamics of accumulation of claims into the IMS Health data warehouse.In summary, we have shown that the medical claims-based surveillance is a very promising tool to study influenza and RSV activity at regional and local scales in inter-pandemic and pandemic seasons. While there has been great progress in the last decade in building sophisticated spatial simulation models of pandemic influenza spread in the US and globally Figure S1Synchrony in peak timing between IMS-ILI indicators and reference surveillance systems, including CDC-ILI (A), CDC laboratory-confirmed influenza virus activity (B), and CDC laboratory-confirmed RSV activity (C). Comparisons are based on nationally-aggregated data. Red circles represent 6 pre-pandemic seasons, 2003–04 to 2008–09; green dots represent the spring 2009 pandemic wave and blue dots the main pandemic wave in fall 2009.(TIF)Click here for additional data file.Figure S2Age-specific ILI time series in the Boston region (region 1) based on CDC surveillance and IMS Health.(TIF)Click here for additional data file.Figure S3Comparison of IMS-ILI, CDC-ILI and IMS-RSV indicators in the Boston region (region 1) for children under 5 yrs. Note the semi-annual pattern of activity in IMS-ILI data , which is most pronounced in the North-East. The bottom panel suggests that IMS-ILI does not align with RSV activity but instead accurately captures unusually early influenza activity in fall 2003 (severe A/H3N2 Fujian season) and fall 2009 Click here for additional data file.Figure S4Comparison of IMS-ILI, CDC-ILI and IMS-RSV indicators in the Atlanta region (region 4), for children under 5 yrs. In this region, RSV is known to display very early timing of activity and predates influenza in most years, except for the 2003–04 influenza season and 2009 fall pandemic wave. The bottom panel confirms that the pediatric IMS-ILI indicator does not capture RSV activity patterns Click here for additional data file.Text S1Description of supplementary text.(DOC)Click here for additional data file.


Plasmodium falciparum infection and is a common cause of death from malaria. In addition, more than 10 % of children surviving CM have neurological and long-term cognitive deficits. Understanding the pathogenesis of CM enables design of supportive treatment, reducing neurological morbidity and mortality. Vaso-occlusion and brain swelling appear to be leading to clinical features, neuronal damage and death in CM. It is proposed that parasitized red blood cells (pRBC), due to cytoadhesion to the endothelium and vasospasm induced by reduced bioavailability of nitric oxide, are causes. Stasis of blood flow and accumulation of pRBC may allow, after schizont rupture, for high concentration of products of haemolysis to accumulate, which leads to localized nitric oxide depletion, inducing adhesion molecules and cerebral vasospasm. Features consistent with an involvement of vasospasm are rapid reversibility of neurological symptoms, intermittently increased or absent flow in medium cerebral artery detectable on Doppler ultrasound and hemispheric reversible changes on cerebral magnetic resonance imaging in some patients. Clinical trials of treatment that can rapidly reduce cerebral vasospasm, including nitric oxide donors, inhaled nitric oxide, endothelin or calcium antagonists, or tissue plasminogen activators, are warranted.Due to delay in treatment, cerebral malaria (CM) remains a significant complication of  Plasmodium falciparum parasitaemia is detectable in peripheral blood and without an alternative cause. Even with optimal care CM was reported to have a mortality of 15–25 % in children and 20 % in adults [A significant proportion of the 584,000  malaria deaths in 2013 [Mouse models have been used to investigate potential treatments for human CM. The cerebral pathology in the mouse model of CM ) is fundamentally different from human CM in the sense that in Plasmodium berghei (ANKA strain) infection of C57BL/6 or CBA mice, the CM is characterized by sequestration of leukocytes in the brain, while in human CM mainly parasitized red blood cells (pRBC) are sequestered. Conclusions drawn from findings in this model may not apply to humans [Neurological features of acute CM and long-term neurological deficits were comprehensively reviewed previously . FollowiElectroencephalography during seizures showed that the origin of seizures was often the temporo-parietal region (a watershed area). The review authors suggested that this may indicate that ischaemia and hypoxia are involved in its aetiology.Neurological deficits in children with CM summarized from previous studies  were ideIn adults neurological deficits as sequelae were observed in less than 5 % and characteristically included extrapyramidal tremor and other cerebellar signs, cranial nerve palsies, mononeuritis multiplex, and polyneuropathy . In the In a recent study, magnetic resonance imaging (MRI) was performed in 38 paediatric survivors of CM. Abnormalities found were focal cortical defects (16 %), sub-cortical T2 signal changes (18 %), atrophy (47 %), and periventricular T2 signal changes (53 %). Acute papilloedema occurred in patients with sub-cortical T2 signal changes. Periventricular T2 signal changes were found in patients with peripheral retinal whitening [A large, detailed MRI study on 152 children with CM was conducted in Malawi and compared findings in retinopathy-positive and -negative patients; MRI features, which were more common in children with retinopathy were brain swelling, basal ganglia involvement, cortical abnormalities on T2, periventricular white matter changes, brainstem abnormalities on T2, thalamic involvement and corpus callosum changes on T2 and diffusion-weighted images, as well as cerebellar changes. In children with retinopathy-positive CM, it is important to note that some had unilateral hemispheric predominance of oedema, predominance in the globus pallidus and cortical involvement was noted to be “occasionally markedly asymmetrical hemispheric” . In a caRecent in vivo studies on retinas in 34 Malawian children with CM by fluorescein angiography showed zones of capillary non-perfusion in 76 % of patients, involving the macula in 56 % and the retinal periphery in 65 %, indicating the presence of obstructed retinal capillaries and postcapillary venules. The blood vessels affected were matched by retinal whitening in fundal photography. In 26 % of patients, angiography showed large occluded vessels with resulting ischemic areas in the supplied peripheral retina downstream. There were vessels with abrupt pruning of larger arterioles with normal perfusion right up to the point of non-perfusion. Normal angiograms in six patients, including two with neurological sequelae, indicated a lack of long-standing vaso-occlusion .An observational study was performed in 42 adults with falciparum malaria, of which 27 patients were suffering from complicated malaria. The study used bedside, portable, high-resolution, digital retinal photography and compared patients with complicated and uncomplicated falciparum malaria.Malarial retinopathy was present in 63 % of adults with severe malaria, in 70 % with CM and in 43 % with non-cerebral severe malaria. The severity of retinal findings correlated with disease severity. Admission blood lactate levels correlated significantly with the severity of retinal and macular whitening in patients with severe malaria. Haematocrit on admission correlated negatively with the quantity of white-centred haemorrhages. The severity of retinal haemorrhages correlated with anaemia. Differences between retinopathy in adults and children evident from this study were that blood vessel discolouration detected in about 30 % of children with CM was not found in any of the adults. The vessel discolouration was compatible with depletion of stationary RBCs infected with mature parasites of haemoglobin.Retinal whitening and discolouration of retinal blood vessels, but not retinal haemorrhages, were associated with occluded blood vessels on fluorescein angiography .Peripheral arterial tonometry measures the change in digital pulse wave amplitude in reaction to hyperaemia resulting in a reactive hyperaemia peripheral arterial tonometry (RH-PAT) index, which provides a measurement of reactive vasodilation. The arterial pulsatile volume is measured in the index finger at rest and compared with that obtained after an increase induced by 5 min of forearm ischemia (sphygmomanometer inflated to 200 mmHg) and with values obtained in the contralateral index finger without reactive hyperaemia. It is known that the RH-PAT index is partially (≤50 %) reflecting endothelial nitric oxide (NO) production.Patients with severe malaria had a significantly reduced RH-PAT index in the peripheral circulation compared to uninfected controls or patients with uncomplicated malaria .l-arginine concentrations [In another study using RH-PAT, patients with severe malaria had a significantly lower RH-PAT index and cell-free haemoglobin, and plasma arginase levels were inversely correlated to RH-PAT indexes. In patients who survived >24 h after enrolment, there were significant associations between increase in RH-PAT index and both decreasing cell-free haemoglobin and increasing trations .Cerebral vasospasm has been defined by Lindegaard using blood velocity measurements by transcranial Doppler ultrasounds. He found that a narrowing of vessel diameter on angiography is associated with increased velocity with a ratio of >3 in middle cerebral artery to extracranial internal carotid artery flow diagnostic of vasospasm .In a pioneering study, Newton et al. investigated cerebral haemodynamics by transcranial Doppler . On inveOver recent years the role of NO in the pathogenesis of CM including reduction of pRBC sequestration and reduction of vasospasm has been increasingly recognized. NO is an endogenous vasodilator produced in endothelium by the endothelial NO synthase (eNOS) enzyme. Without the presence of RBCs NO has a half-life of up to several minutes. It can therefore diffuse across cell membranes and act as an intercellular messenger. NO diffuses from the endothelium to nearby smooth muscle. There it binds to the haem group of the guanylate cyclase. This causes this enzyme to convert guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP) and cGMP then activates cGMP-dependent protein kinases and produces vasodilatation .NO can down-regulate transcription of endothelial adhesion molecule genes, including E-selectin, VCAM-1, ICAM-1 and P-selectin. NO increases the expression of endothelin B receptors (a receptor mediating vasodilatation) and decreases expression of endothelin-1, the most powerful endogenous vasoconstrictor .P. falciparum pRBC adhesion, while addition of a NO-donor reduced adhesion. In vitro studies showed that the NO donor 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PPN) treatment reduced the number of adherent pRBCs on resting and TNF stimulated human microvascular endothelial cells (HMECs) by down-regulating basal ICAM-1 expression [P. falciparum infection by inhibition of cyto-adherence. Patients with CM were found to have decreased plasma and cerebrospinal levels of the nitric oxide metabolites nitrite and nitrate [Inhibition of NO production by exposure of human microvascular endothelial cell to the NOS inhibitor L-N(G)-nitro-arginine-methyl-ester (L-NAME) increased pression . NO effe nitrate .l-Arginine, the substrate for NO production by nitric oxide synthase. Hypoargininaemia was strongly associated with mortality [Patients with CM also had reduced levels of ortality .3−) in a rapid and irreversible reaction [Replication of schizonts inside RBCs leads to haemolysis and therefore to production of cell-free haemoglobin which after exposure to reactive oxygen species is oxidized. It is established that during haemolysis, cell-free oxyHb in the plasma acts as a NO scavenger leading to production of metHb and nitrate  can both bind ET-1 [To gather evidence for a role of vasospasm in CM it is essential to examine data on the association of vasoconstrictors with features of CM. Endothelin-1 is one of the most potent vasoconstrictors. Two studies analysing ET-1 during malaria infection in humans have been reported to date , 30. Theind ET-1 . The autpost-mortem studies of patients in Thailand and Vietnam, it was demonstrated that adults with CM had more pRBCs sequestered in the brain than patients who died of other complications of malaria. In addition, pRBC sequestration in the brain of CM patients was also more pronounced compared to other organs [In landmark quantitative analyses of brain sections in r organs , 33. Adur organs . Inflammr organs , 36. In r organs . There wr organs .The most extensive retinal histopathological examination in CM reported so far included findings of the eyes of 64 children, including 13 cases with a clinical diagnosis of CM but an alternative cause of death. All patients with systemic pathological features of CM had prominent sequestration of early or late stage parasites in the retinal vasculature. Haemorrhages were significantly more common in CM patients compared to patients with non-malarial disease. There were significantly more thrombi seen in the retinal vessels of patients with CM than in the non-CM group. Thrombi were found to often only partially occlude blood vessels and seen without surrounding haemorrhage .In a more recent study on the same population in Malawi  in 13 chl-Arginine (l-Arg) was associated with an increase in the RH-PAT index and led to an increase in exhaled NO levels in patients with moderately severe malaria [l-Arg infusion in Indonesian adults with severe malaria, no effect on clearance of lactate or RH-PAT index was detected [There has been, so far, only been one published trial in humans addressing the bioavailability of the vasodilator nitric oxide. Intravenous infusion of  malaria  and was  malaria , 43. In detected . Currentdetected , or are detected  explorinThis review shows that previous studies have not demonstrated direct evidence of vasospasm in human CM. Reason for this may be that studies conducted so far have not used internationally recognized criteria, such as the Lindegaard criteria of flow ratio of >3 in middle cerebral artery to extracranial internal carotid artery flow, or cut-offs for definition of significantly increased flow on Doppler ultrasound , to defiThe rapid reversibility of coma and neurological deficits in the majority of patients with CM is consistent with a role of transient vasospasm and against a vascular obstruction by thrombi or sequestered pRBCs;post-mortem studies of patients who died of CM;The unilateral asymmetrical hemispheric changes on cerebral MRI and transient hemispheric changes on single-photon emission computed tomography. described in some patients are compatible with a transient vasospasm in a large supplying artery and cannot be explained by thrombi or sequestration. Obstruction of a large artery by thrombus or sequestered pRBCs has not been found on Transient increase in Doppler flow during seizures in CM cannot be explained by sequestration or thrombi but by vasospasm. The lack of detection of middle cerebral artery by Doppler in patients associated with transient neurological deficits is also most likely due to transient vasospasm;Normal angiograms in six patients, including two with neurological sequelae, indicated a lack of long-standing vaso-occlusion more compatible with vasospasm than other forms of vasocclusion;In a sub-group of patients, features of fatal CM were found without sequestration of pRBCs or thrombi in cerebral blood vessels on autopsy. The absence of inducible NO synthase (iNOS) expression in endothelial cells in these patients was against this absence of pRBCs being due to recent simultaneous bursting of sequestered pRBCs, which would have induced iNOS expression;RH-PAT shows changes during malaria compatible with changes of nitric oxide bioavailability, which could predispose to and cause vasospasm.Data presented above provide the following lines of indirect evidence of cerebral vasospasm in CM:To assess the specific contribution of vasospasm to the pathophysiology of CM, a comparison with a condition characterized by pure and isolated cerebral vasospasm without an underlying inflammation or haemorrhage is helpful. Such a condition is the so-called reversible cerebral vasoconstriction syndrome  , 51. PreAnother proposed explanation of brain swelling in CM is pooling of blood in congested cerebral veins from parasite sequestration. Against this hypothesis is the finding of unilateral brain abnormalities on scanning found in some patients with CM, which is unlikely to occur with the disseminated nature of parasite sequestration affecting both cerebral hemispheres.Cerebral vasospasm is likely to be more reversible than sequestration. Resolution of vasospasm may be an essential requirement for reversal of sequestration by increasing shearing forces by increased flow through widening blood vessels. Vascular resistance is crucially dependent on diameter. The rapid reversibility of vasospasm makes it the ideal target for rapid treatment of this potentially fatal complication of falciparum malaria.To find therapeutic options for any vasospasm contributing to the pathogenesis of CM it is essential to be aware of all treatment that has been shown to reduce cerebral vasospasm rapidly in humans and has been demonstrated to be safe in randomized controlled trials. Such trials have been conducted in patients with cerebral vasospasm associated with sub-arachnoid haemorrhages. A similar mechanism to sub-arachnoid haemorrhage-associated vasospasm caused by haemolysis of extravasated RBCs in the sub-arachnoid space may mediate vasospasm through depletion of nitric oxide and induction of endothelin-1 release in CM because of schizont rupture-related haemolysis as well as the breakdown of extravasated blood from the frequently encountered intracerebral microhaemorrhages associated with more severe disease. Treatments that were found to rapidly reverse cerebral vasospasm in sub-arachnoid haemorrhage and have been summarized previously  include The relevance of erythropoietin in CM was demonstrated by the fact that high levels of erythropoietin (EPO) were measured in African children with severe malarial anaemia and were associated with a 80 % reduction in the risk of neurological sequelae during CM . EPO is It has recently been described that the non-haematopoietic and haematopoietic functions of EPO are transmitted through different receptors. To reduce side effects of EPO, non-haematopoietic EPO derivatives have been generated, such as asialoEpo and carbamylated Epo (CEPO), that maintain their tissue-protective activities but do not increase the concentration of RBCs . EPO hasFuture research needs to find supportive evidence for the role of haemolysis in CM. This can be gained indirectly by investigating the role of the HO-1 in CM: ferritin, bilirubin and iron could be measured in the cerebrospinal fluid of patients with CM compared to controls with uncomplicated and complicated falciparum malaria, as they are end-products of haem metabolism by HO-1 .Prevention of the effect of free haemoglobin in reducing nitric oxide bioavailability has been achieved by use of haptoglobin . Capturil-arginine, nitrite or nitroglycerin for reducing cerebral vasospasm are established, side effects could be minimized by using combinations of these agents, potentially enabling dose reductions of the individual agent.Inhaled nitric oxide (iNO) could be particularly useful as supportive treatment of CM: unlike nitric oxide donors, such as nitroglycerine and sildenafil, iNO has not been associated with anaemia or systemic vasodilatation causing hypotension. Unlike NO donors, for iNO there is no requirement for a functional endothelial cell NO synthase (eNOS). eNOS may be dysfunctional in some patients. NO gas manufacture is affordable, and can be administered by mask without significant infrastructure requirements  and HawkCM remains a significant clinical problem associated with delayed recognition and treatment of falciparum malaria in endemic areas. CM is associated with significant mortality occurring rapidly after admission to hospital. Survival of CM can be associated with permanent neurological and neurocognitive deficits. The key events in the pathophysiology of CM may be vaso-occlusion by sequestration of pRBCs and vasospasm. Haemolysis resulting from rupture of adherent schizonts may cause depletion of nitric oxide by binding of nitric oxide to free haem released by this haemolytic process. Nitric oxide depletion causes vasospasm and increases pRBC adhesion by up-regulation of intercellular adhesion molecule-1 expression. Strategies relieving cerebral vasospasm are most likely to rapidly reverse coma and reduce the risk of ischemic neurological deficit in CM.


Oryza sativa L.), we previously analyzed the expression profile of stably transformed rice in which an ER stress sensor/transducer OsIRE1 was knocked-down, using the combination of preliminary microarray and quantitative RT-PCR. In this study, to obtain more detailed expression profiles of genes involved in the initial stages of the ER stress response in rice, we performed RNA sequencing of wild-type and transgenic rice plants produced by homologous recombination in which endogenous genomic OsIRE1 was replaced by missense alleles defective in ribonuclease activity.The endoplasmic reticulum (ER) stress response is widely known to function in eukaryotes to maintain the homeostasis of the ER when unfolded or misfolded proteins are overloaded in the ER. To understand the molecular mechanisms of the ER stress response in rice . Furthermore, we successfully identified 17 genes from the set of 380 ER stress-responsive genes that were not included in the probe set of the currently available microarray chip in rice. Notably, three of these 17 genes were non-annotated genes, even in the latest version of the Rice Annotation Project Data Base .Therefore, RNA sequencing-mediated expression profiling provided valuable information about the ER stress response in rice plants and led to the discovery of new genes related to ER stress. The endoplasmic reticulum (ER) is an organelle in which the synthesis of secretory proteins and the folding and assembly of newly synthesized premature proteins occurs. When these functions are perturbed by the accumulation of unfolded or misfolded proteins in the ER, the cells incur ER stress conditions. ER stress then induces countermeasures in cells referred to as the ER stress response. The ER stress response is a mechanism that maintains ER homeostasis by balancing the folding capacity and folding demands imposed on the ER through the induction of genes encoding chaperones and protein folding-related enzymes, the attenuation of translation, ER-associated degradation, or regulated IRE1-dependent decay (RIDD) . Severe Arabidopsis also play key roles in inducing the ER stress response ).Raw sequences in FASTQ format obtained from the Illumina platform were analyzed using publicly available tools. Low-quality bases (Q < 15) were trimmed from both ends of the sequences using a customized program, and the adapters were trimmed using Cutadapt  (http:// mapping  and TopH mapping . Referenmd.edu/) . The exp® 480 system  using transcript-specific primers  and treated with DNase I . The first-strand cDNA was synthesized using the Transcriptor First Strand cDNA synthesis kit  according to the manufacturer’s protocol. The resulting cDNAs were amplified in the LightCyclerThe authors declare that they have no competing interests.YW and YO contributed equally to this research. YW, YO, TY and SH conducted the experiment. YW, YO and SH drafted the manuscript with edits from KO, HH, TM and FT. All authors read and approved the final manuscript.Quantification of gene expression levels by RNA-Seq analysis in rice roots under TM treatment. Scatter plot shows FPKM (Fragments Per Kilobase of transcript per Million fragments sequenced) values of RNA-Seq data from wild type treated with DMSO and WT treated with TM. The abscissa and ordinate show the FPKM of each treatment.Click here for fileDifferentially expressed genes between untreated and DMSO-treated samples.Click here for fileList of ER stress-responsive transcripts identified by statistical analysis . The expression levels are expressed at the FPKM (Fragments Per Kilobase of transcript per Million fragments sequenced) value. Fold changes (TM/DMSO) were calculated based on FPKM values (see Methods).Click here for fileDifferential gene expression heat map from Table Click here for fileDifferential gene expression heat map from Table Click here for fileDifferential gene expression heat map from Table Click here for fileOriginal gene list of the basis of Table Click here for fileList of transcripts exhibiting RIDD-like changes in expression under ER stress conditions.Click here for filehttp://plantgrn.noble.org/psRNATarget/)] was used for this prediction.Prediction of micro(mi)RNA target gene. Website [miRbase (Click here for filePrimer sets used for qRT-PCR.Click here for file


Background: Major depressive disorder (MDD) is a public health problem worldwide. There is increasing interest in using non-invasive therapies such as repetitive transcranial magnetic stimulation (rTMS) to treat MDD. However, the changes induced by rTMS on neural circuits remain poorly characterized. The present study aims to test whether the brain regions previously targeted by deep brain stimulation (DBS) in the treatment of MDD respond to rTMS, and whether functional connectivity (FC) measures can predict clinical response.Methods: rTMS (20 sessions) was administered to five MDD patients at the left-dorsolateral prefrontal cortex (L-DLPFC) over 4 weeks. Magnetoencephalography (MEG) recordings and Montgomery-Asberg depression rating scale (MADRS) assessments were acquired before, during and after treatment. Our primary measures, obtained with MEG source imaging, were changes in power spectral density (PSD) and changes in FC as measured using coherence.Results: Of the five patients, four met the clinical response criterion (40% or greater decrease in MADRS) after 4 weeks of treatment. An increase in gamma power at the L-DLPFC was correlated with improvement in symptoms. We also found that increases in delta band connectivity between L-DLPFC/amygdala and L-DLPFC/pregenual anterior cingulate cortex (pACC), and decreases in gamma band connectivity between L-DLPFC/subgenual anterior cingulate cortex (sACC), were correlated with improvements in depressive symptoms.Conclusions: Our results suggest that non-invasive intervention techniques, such as rTMS, modulate the ongoing activity of depressive circuits targeted for DBS, and that MEG can capture these changes. Gamma oscillations may originate from GABA-mediated inhibition, which increases synchronization of large neuronal populations, possibly leading to increased long-range FC. We postulate that responses to rTMS could provide valuable insights into early evaluation of patient candidates for DBS surgery. Major depressive disorder (MDD) is characterized by depressed mood, anhedonia, irritability, poor concentration, feelings of hopelessness, thoughts of self-harm, inappropriate guilt, and abnormal appetite and sleep patterns. About a sixth of the American population will suffer from clinical depression in their lifetime. Because of its significant societal and economic impact on the general population, both the underlying mechanism(s) of MDD and markers of individual response to treatment are important areas of ongoing research.Previous studies have proposed that MDD is a circuit-based disorder in which several regions of the brain  are functionally aberrant  and ventromedial prefrontal cortex (VMPFC) are two of the main targets of neuromodulation therapy. These two targets exhibit opposite metabolic activity in the pathological depressed state  as a target for stimulation remains ambiguous due its broad anatomical definition. In a resting-state functional magnetic resonance imaging (fMRI) study  Which depressive circuits previously targeted for DBS are modulated by successful rTMS therapy for MDD? (2) Can changes in FC predict clinical response? Our hypothesis is that modulation of the L-DLPFC will be reflected through longitudinal changes in the power spectrum of local neural oscillations, along with changes in FC with other regions of the depressive circuit.The major contribution of this study is to provide a bridge between physiological markers of MDD (neuroimaging) and clinical response to treatment. In the “Materials and Methods” Section, we describe the acquisition of high-temporal resolution MEG before, during and after delivery of high-frequency rTMS. Next, we present the approach of combining MEG source imaging with neuronavigated-rTMS to quantify how changes in depressive symptoms correlate with modulating specific circuits. In the results section, we report the significance of engaging specific functional connections in the fronto-limbic circuit for effective MDD improvement demonstrated through frequency-dependent connectivity analysis. Lastly, in the discussion section, we highlight the advances in source-imaging analysis techniques that have enabled the current study along with the limitations of those methods. We also discuss the relevance of our findings in the context of the field of neuromodulation and MDD.n = 5). Patient selection and the decision to treat using rTMS was made through standard care, independent of this study. In tandem with rTMS treatment, we enrolled patients into a research study approved by the Institutional Review Board (IRB) at the Medical College of Wisconsin. During treatment, a total of 1000 biphasic pulses were delivered in 25-s cycles  over 20 trains per session using a Magstim Rapid stimulator . Treatment was delivered for five 15-min sessions per week over 4 weeks. Stimulation was applied at 110% of RMT, which was determined by stimulating the hand-knob of motor cortex. Periodic behavioral assessments were performed with the Montgomery-Asberg depression rating scale (MADRS).High frequency (10 Hz) rTMS was administered to the L-DLPFC (Brodmann areas 9 and 46) in five MDD patients (High-resolution (3T) structural magnetic resonance imaging (MRI) was acquired before treatment to assist with targeting and navigation. It was used for definition of TMS targets and registration with MEG imaging using FreeSurfer , at the end of the treatment course (4 weeks), and 2 months after the end of treatment. Empty-room recordings were also collected at each acquisition for noise modeling in source imaging. During MEG, the subject was supine under the MEG sensor array. A total of 24 min of data was collected in four 6-min runs. Each condition (eyes-open and eyes-closed) was repeated once.MEG was recorded at 2000 Hz using a 306-channel VectorView system . EOG and EKG were also recorded simultaneous to MEG for MEG data for the eyes-closed condition were analyzed for cortical source modeling, power spectral density (PSD), and FC using Brainstorm, with default imaging parameters , a subtype of distributed source models, was used to generate the source image maps, with default imaging parameters in Brainstorm. Elementary dipole current sources were constrained perpendicularly to the individual cortex; additive sensor noise was modeled with a Gaussian distribution with known covariance statistics estimated from empty-room recordings. The Welch periodogram was used to obtain the PSD of each individual source time series on the cortex. Signals from the L-DLPFC were averaged over the stimulation area to determine the power at that region  previously investigated for DBS therapy: pregenual anterior cingulate cortex (pACC) and sACC, NAc, and amygdala. The Desikan-Killiany atlas  to investigate the relationship between change in behavior (MADRS) and change in brain activity, controlling for time and subject effects which were treated as categorical variables. We performed this regression analysis for each frequency band in the source analysis (L-DLPFC) and the connectivity analysis . We did not correct for multiple comparisons given the exploratory nature of this study and its small sample size. All tests were two-tailed and statistical significance was assessed at the 0.05 level.Data from five MDD patients were analyzed in this study. Of them, four were responders and one was a non-responder using the clinical response criterion of a 40% reduction in MADRS scores from baseline. Stimulation was applied to the L-DLPFC using a predefined target coordinate on an anatomical MRI. By midpoint, an improvement in depressive symptoms was observed in responders Table . These pp(γ) = 0.01) and beta  power (p(β) = 0.04) at the L-DLPFC was significantly correlated with an improvement in depressive symptoms  power  = 0.04; Table Changes in FC of L-DLPFC and several ROI in the depressive circuit were assessed. An increase in mean coherence in the delta band 2–4 Hz) correlated with a better clinical outcome for depression in the connectivity analysis between L-DLPFC and amygdala  for depression  with source analysis and FC as determined from MEG. We postulate that this approach could fill two important gaps in our current knowledge. First, it could improve identification of specific regions and functional connections to target in order to maximize the therapeutic response. Second, this approach could provide insights about common mechanisms for different types of neuromodulation therapy including rTMS, cortical stimulation and DBS.YP, OS, SB, ZL, and CRB participated in manuscript preparation and editing; YP and CRB conducted the analysis to determine the effect of repetitive transcranial magnetic stimulation on changes in the power spectral density and functional connectivity; OS was the psychiatrist for the clinical study and assisted with the administration of therapy; ZL assisted with the acquisition of neuroimaging data; SB guided the analysis and interpretation of the neuroimaging data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CP and the handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Prenatal immune challenge has been associated with alteration in brain development and plasticity that last into adulthood. We have previously shown that prenatal activation of toll-like receptor 4 by lipopolysaccharide (LPS) induces IL-6-dependent STAT-3 signaling pathway in the fetal brain. Whether this IL-6-dependent activation of fetal brain results in long lasting impact in brain plasticity is still unknown. Furthermore, it has been shown that prenatal LPS heightens the hypothalamic–pituitary–adrenal (HPA) response in adulthood. In the present study we tested whether LPS administration during pregnancy affects neurogenesis in adult male offspring. Because corticosterone, the end-product of HPA axis activity in rats, alters neurogenesis we tested whether this enhanced HPA axis responsiveness in adult male offspring played a role in the long lasting impact of LPS on neurogenesis during adulthood. Pregnant rats were given either LPS, or LPS and an IL-6 neutralizing antibody (IL-6Ab). The newly born neurons were monitored in the subventricular zone (SVZ) and the dentate gyrus (DG) of the hippocampus of adult male offspring by monitoring doublecortin and T-box brain protein-2 expression: two well-established markers of newly born neurons. Prenatal LPS decreased the number of newly born neurons in the DG, but not in the SVZ of adult offspring. This decreased number of newly born neurons in the DG was absent when IL-6Ab was co-injected with LPS during pregnancy. Furthermore, administration of a corticosterone receptor blocker, RU-486, to adult offspring blunted the prenatal LPS induced decrease in newly born neurons in the DG. These data suggest that maternally triggered IL-6 plays a crucial role in the long lasting impact of LPS on adult neurogenesis. Birth of new neurons (neurogenesis) persists into adulthood in two discrete areas of the adult brain, the subventricular zone (SVZ) and dentate gyrus (DG) of the hippocampus. The newly born neurons functionally integrate into neuronal networks and have been shown to play a role in cognitive functions such as learning and memory . SeveralIt has been shown that LPS does not cross the feto-placental barrier but it rather exerts its effect through inducible pro-inflammatory cytokines . Indeed,ad libitum. Female rats were mated with proven breeder male rats and vaginal smear were monitored daily for signs of copulation. Positively identified females with vaginal deposition of sperm were separated from the males and housed individually. On gestation day 15 (GD15), pregnant rats received intraperitoneal injections of either LPS  or saline in the presence or the absence of an IL-6 neutralizing antibody . The IL-6Ab is an IgG polyclonal antibody made in goat and purified by rat IL-6 affinity chromatography. We have previously shown that this antibody has no pyrogenic activity and that goat serum per se does not impact LPS-activated STAT-3 signaling pathway in the fetal brain , where food and water were available al brain . Pregnanal brain . Each raPregnant rats gave birth to normal size litters. Pups were weaned when they reach the age of 21 days. Male rats were group-housed as four animals per cage until they were 45 days old. After this time, animals were housed two per cage for the remainder of the experiment. This study included only male offspring because ovarian hormones affect neurogenesis and the magnitude of neurogenesis varies as a function of the phase of the estrous cycle in female offspring , 2005.Adult rat offspring (70 day old) of dams given either LPS or pyrogen-free saline in the presence or the absence of IL-6Ab on GD15 were anesthetized with urethane , transcardially perfused with ice cold saline followed by a 10% neutral-buffered formalin solution. The rat brains were removed from the skull and post-fixed for at least 2 consecutive days. They were subsequently embedded in paraffin and processed for immunofluorescent staining as previously described . Serial + and Tbr2+ cells in both left and right of SVZ and DG areas of the brain were counted by an experimenter blind to the rat’s treatment using a 20x objective . Iba1+ were counted in the hilus of the hippocampus and expressed as number of cells per area (in mm2). The number of Iba1+ along the granular cell layer (GCL) of the DG were counted. A line was drawn at the base of the GCL, its length was measured and the data were presented as the number of Iba1+ per line length (in mm). The area of the hilus and length of GCL base were measured using imageJ software . The DCXsoftware . All immIn a different series of experiments, male rats born to dams given either saline or LPS during pregnancy were injected with LPS  during adulthood. These rats were given either 50 mg/kg (i.p.) of the glucocorticoid receptor antagonist RU486 (Sigma–Aldrich) dissolved in dimethyl sulfoxide (DMSO) or DMSO alone as previously described . Twenty post hoc test whenever possible. The difference between groups was declared statistically significant at p < 0.05. Results are shown as mean ± SEM.All data were compared using 2-way ANOVA followed by Student–Newman–Keuls Figure 1 shows that body weight gains in rats born to dams injected with pyrogen-free saline or LPS in the absence or the presence of IL-6Ab. Injection of either LPS  or IL-6Ab during pregnancy did not affect the offspring’s body weight gain. Previous study has also shown that prenatal LPS or IL-6Ab does not affect the litter size . Administration of LPS to pregnant dams did not result in any significant effect on the number of DCX containing cells in either the SVZ or the RMS of adult offspring. Similarly, there was no significant effect of prenatal administration of IL-6Ab on the number of DCX containing cells in either areas.Because DCX expression in adult brains reflects neurogenesis , we usedFigure 3). Interestingly, co-administration of IL-6Ab and LPS during pregnancy halted this prenatal LPS-induced reduction in the number of newly born neurons in adults . Experimental evidence suggests that prenatal immune challenge dampens neurogenesis was associated with enhanced microglial activation  or along the basal area of the DG .In contrast, prenatal administration of LPS led to a reduced number of DCX containing cells in the DG of the hippocampus .In addition to DCX labeling of newly born neurons, we also assessed whether prenatal LPS affects Tbr2 containing cells within the DG. Tbr2 is a transcription factor expressed by intermediate neuronal progenitors which are largely committed to become glutamatergic neurons and is indispensable for neurogenesis in the DG during adulthood . PrenataPrenatal activation of TLR4 has been associated with increased HPA responsiveness to LPS in adult offspring . BecauseFigure 5A) and Tbr2 . However, such programing effect of LPS on neurogenesis was absent when adult offspring were given RU486.In the absence of adult treatment with RU486 (DMSO rat group), the prenatal injection of LPS led to a significant reduction in the number of both DCX  was relatively higher in the experiment involving DMSO injection during adulthood. This peculiar observation hold true in rats that were either saline-injected (Supplementary Data S1) or immune-challenged  during adulthood. It is possible that the anti-inflammatory/neuroprotective effect of DMSO contributes to this enhanced neurogenesis (One of the most explored mechanism underlying the long lasting impact of maternal immune activation on offspring’s brain function and plasticity is the HPA axis activation. It is plausible that IL-6 mediated activation of fetal brain during pregnancy alters the developmental trajectory of the HPA axis and thus alters the HPA “set point” in utero . Indeed,in utero . Severalin utero . This rein utero . This obin utero , we asseogenesis .Tbr2 gene resulted in abolished neurogenesis in the DG during adulthood but not in the sub-ependymal zone at the lateral ventricles (We observed that prenatal immune challenge resulted in a specific alteration of neurogenesis in the DG, but not in the SVZ. This observation is akin to previous studies  and suppntricles . It is pExperimental evidence suggests that prenatal exposure to LPS induces a long lasting activation of microglia within the hippocampus , 2013, wThis study gives strong evidence for IL-6 involvement in prenatal LPS induced depressed neurogenesis in adult offspring. While the heightened HPA axis activity could, at least in part, explain the reduced neurogenesis in immune or behaviorally stressed adult offspring, the reduced neurogenesis in basal condition is less likely related to the HPA axis activity. The present study opens new research avenues for exploring the long lasting impact of maternal IL-6 on transcriptional genes involved in brain plasticity of adult offspring.AM designed the research, performed research, analyzed data, and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we confirmed that the infertility of ap female moths is a result of the degradation of the bursa copulatrix. Positional cloning of ap locus and expression analyses reveal that the Bombyx mori sister of odd and bowl (Bmsob) gene is a strong candidate for the ap mutant. The expression of Bmsob is down-regulated, while the corresponding Hox genes are up-regulated in the ap mutant compared to the wild type. Analyses with the dual luciferase assay present a declined activity of the Bmsob promoter in the ap mutant. Furthermore, we demonstrate that Bmsob can inhibit Hox gene expression directly and by suppressing the expression of other genes, including the BmDsp gene. The results of this study are an important contribution to our understanding of the diversification of insect body plan.The morphological diversity of insects is important for their survival; in essence, it results from the differential expression of genes during development of the insect body. The silkworm  Drosophila, genes involved in the formation of body segments can be divided into four conceptual categories; maternal effect, gap, pair-rule and segment polarity45Hox) genes, whose expression patterns largely determine the morphology and development of tissues and organs78Hox genes can lead to changes in insect body plan and morphology1Drosophilia insects remain obscure.The morphological diversity of insects makes them able to adapt rapidly to environmental changes and provides good models for studying body plan diversity2Drosophila, the odd-skipped gene family consists of odd-skipped (odd), brother of odd with entrails limited (bowl), sister of odd and bowl (sob) and drumstick (drm), which have key roles during embryogenesis1112odd gene, which was identified as a member of the pair-rule genes, encodes a C2H2 zinc finger transcription factor regulating the development of multiple tissues14151617bowl and drm genes are required for leg, notum and hindgut pattern formation1719odd-skipped family. Although recent studies suggested the sob gene might be involved in the development of antennae, legs and wings212223In Bombyx mori, the apodal (ap) mutant exhibits marked multiple morphological variations, including degraded thoracic legs and wings as well as female sterility, which is an ideal subject for the study of the molecular mechanisms for morphogenesis. The spontaneous ap mutant was identified by the presence of degraded thoracic legs throughout development and the ap adults exhibited smaller or deformed wings seriously affecting their ability to mate  on the third linkage in the silkworm genetic linkage map and controlled by a single recessive geneHox genes, display ectopic legs, abnormal wings and genital system25ap mutant and Hox genes.Wings, ventral limbs and the reproductive system have pivotal roles in insects; hence, the identification of the corresponding developmental genes is of particular importance. In the silkworm  to mate . For allap female infertility, and then identified and characterized the strong candidate gene responsible for the ap mutant. We confirmed that the infertility of ap female moths was due to the degradation of the bursa copulatrix, and demonstrated that the abnormal expression of the Bmsob gene encoding C2H2 zinc finger-containing transcriptional factor should be probably responsible for ap mutant. Finally, we found that the Hox genes in the ap mutant are abnormally expressed and verified the Hox genes can be regulated by the Bmsob gene.In this study, we investigated the physiological cause of ap mutant  of Drosophila melanogaster, which is one member of the odd-skipped family that involved in Drosophila leg developmentBombyx mori sister of odd and bowl (Bmsob). Therefore, we next focused on the Bmsob gene that is the unique gene having sequence information within the mapped region.To map the ap locus . Within caffolds . Both ofBmsob gene in the wild type and the ap mutant using rapid amplification of cDNA ends (RACE) technology, 1923 bp and 1924 bp, respectively (2H2-type zinc finger structures. The 5′-untranslated region (5′-UTR) of both was 85 bp long, and the 3′-UTR was 527 bp and 528 bp long, respectively, and both had two exons. There were 15 single base mutations in Bmsob open reading frames (ORFs) between Dazao and the ap mutant, of which 13 were synonymous mutations and two were missense mutations . In addition, there were eight single base mutations and one base insertion in the Bmsob UTR in the ap mutant. In order to study the relationship between the candidate gene and the ap mutant further, we undertook a more detailed analysis of the Bmsob gene.Subsequently, according to the two predicted genes and ESTs, we obtained the full-length cDNA of ectively . The lenap mutant suggest its candidate gene is expressed in particular spatial temporal patterns. Therefore, we undertook a detailed analysis of the temporal expression pattern of the Bmsob gene. Given that the embryonic period is crucial for the development of limbs, we examined the temporal expression pattern of Bmsob mRNA at nine different developmental stages of the embryo and newly hatched larvae. The semi-quantitative RT-PCR analysis showed that expression of the Bmsob gene was detected first after oviposition and then increased gradually; it reached a peak level at 4 days after egg laying and decreased gradually thereafter  showed that the expression level of Bmsob was decreased markedly in the ap mutant compared to the wild type at both stages  experiments to validate interactions between the Bmsob protein and the BmAntp, BmUbx and BmAbd-B genes. The results showed that the Bmsob protein could bind directly to the BmUbx gene but not to the BmAntp or BmAbd-B genes  screening showed that the sob gene might be involved in the development of legs and wings22Drosophila sob gene participates in the wing developmentBmsob as the only predicted gene on the scaffolds within our mapping region should be probably responsible for the multiple mutation phenotypes of the ap mutant, suggesting that Bmsob has important roles in the development of legs, wings and the reproductive system in the silkworm. Furthermore, the expression patterns of the Bmsob gene are consistent with the development of the embryo and wings , and eight single base mutations and one base insertion into the UTR  were obtained from the Silkworm Gene Bank at Southwest University, Chongqing, China. Silkworms were reared with fresh mulberry leaves at 25 °C under a photoperiod of 12h light/12h dark. The B. mori ovary cell line BmN-SWU1The wild-type strain Dazao and the Eggs from non-mated females were dissected and rinsed with double-distilled water. For scanning electron microscopic observation, the eggs were processed as previously describedap mutant, we used a single-pair cross between a Dazao female and an ap male to produce F1 offspring. Because there is no recombination in female silkworms, 22 progeny from a single-pair backcross between an F1 female and an ap male were used for the linkage analysis and 384 F2 progeny with the ap phenotype from selfing F1 (Dazao × ap) × (Dazao × ap) were used for the recombination analysis. Genomic DNA was extracted from parent moths, F1 moths and each F2 individual of third instar larvae as previously describedB. mori genome sequenceap were used for linkage analysis  from Dazao wild-type embryos of different developmental stages (from newly laid eggs to newly hatched larvae), the whole body of several developmental stages (from day 4 of the 4th instar larvae to day 5 of the pupae) and the wing discs of larvae and the wings of pupae and moths (from day 1 of the 5th instar larvae to the adult moth). The cDNA was synthesized using the PrimeScript reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer’s protocol. The gene for Actin3 of B. mori was used as an internal control. For qRT-PCR, total RNA was isolated from 4 days old Dazao and ap embryos and reverse transcription was carried out as described above. The ap/ap embryos were from ap/+ females × ap/ap males and we distinguished ap/ap embryos from ap/+ by the apodal phenotype. The eukaryotic translation initiation factor 4A (silkworm microarray probe ID: sw22934) was used as an internal control. The qRT-PCR experiments were performed using the CFX96 Real-Time System  with an iTaq Universal SYBR Green Supermix (Bio-Rad), according to the manufacturer’s recommended procedure. The specific primers for RT-PCR and qRT-PCR are listed in To analyze gene expression patterns, the semi-quantitative RT-PCR experiments were performed. Total RNAs were isolated with TRIzolhttp://www.ncbi.nlm.nih.gov/protein), SilkDBhttp://silkworm.genomics.org.cn/), Flybasehttp://flybase.org/), BeetleBasehttp://beetlebase.org/), VectorBasehttps://www.vectorbase.org/) and Heliconius Genomehttp://www.butterflygenome.org/). Accession numbers are listed in Protein sequences were downloaded from the following websites: National Center for Biotechnology Information (NCBI) Protein Database  were seeded in 24-well culture plates . After 24 h, cells were co-transfected with 400 ng pfsLSV40-Bmsob-Dazao plasmid or pfsLSV40-Bmsob-ap plasmid and 100 ng pIZ-Rluc control plasmid. At 48 h after transfection, a GloMax-Multi Detection System and a Dual-Glo Luciferase Assay Kit  were used to quantify luciferase activity. All assays were performed in triplicate.We constructed recombinant plasmids from a pfsLSV40 vector  that contained the ap mutant embryos as described above. For the hybridization experiment, we mixed equal amounts of total RNA from at least seven biological replicates to create one sample. Microarray hybridization and raw data normalization were carried out by Capital Bio Corp.  as describedap mutant compared to the wild type if it displayed at least a twofold change.Total RNA was extracted from the wild type and Bmsob and BmDsp were cloned into the pGEX-4T-1 vector with a glutathione S-transferase (GST) tag and pET-28a (+) vector with a His tag, respectively. Primers are listed in Escherichia coli strain BL21 to express GST-tagged Bmsob (GST-sob) and His-tagged BmDsp (His-Dsp) proteins. The GST-sob protein and His-Dsp protein were purified using a GSTrap FF column and a HisTrap HP column , respectively, according to the manufacturers’ protocol. Purified proteins were verified through western blotting as described previouslyThe ORF of Bmsob and BmDsp binding sites, we used two different transcription factor binding site prediction programs, Tfsitescan (http://www.ifti.org/cgi-bin/ifti/Tfsitescan.pl) and PROMO . The features related to the gene binding sites are G+C-rich and A+T-rich sequences for Bmsob and BmDsp, respectively, in accordance with their structural domains50For the prediction of How to cite this article: Chen, P. et al. Molecular mapping and characterization of the silkworm apodal mutant. Sci. Rep.6, 18956; doi: 10.1038/srep18956 (2016).


Transthyretin (TTR) is an amyloidogenic tetrameric protein, present in human plasma, associated with several familial amyloidoses. Variability of TTR is not only due to point mutations in the encoding gene but also to post-translational modifications (PTMs) at Cys10, being the most common PTMs the S-sulfonation, S-glycinylcysteinylation, S-cysteinylation and S-glutathionylation. It is thought that PTMs at Cys10 may play an important biological role in the onset and pathological process of the amyloidosis. Recently we reported the development of a methodology for quantification of PTMs in serum samples, as well as for the determination of serum TTR levels, from healthy (wt-TTR) and ATTR V30M individuals which involves an enrichment step by immunoprecipitation followed by mass spectrometry analysis of (i) the intact TTR protein and (ii) targeted LC-MS analysis of peptides carrying the PTMs of interest  in press). Analysis of serum samples by the combination of the two methods affords complementary information on the relative and absolute amounts of the selected TTR PTM forms.We aimed at describing the applicability of our mass spectrometry methodology among healthy controls and V30M-TTR patients (cases) at different disease stages followed at our institution and other three spanish health facilities. Inclusion criteriae for cases consisted of a positive genetic testing for V30M-TTR status and a signed ethics’ committee approved informed consent. Mass spectrometric analysis was performed as detailed in our previouis work.A total of 50 healthy controls and 29 patients were included after signing the informed consent. Demographic and clinical characteristics were gathered and correlated to the proteomic analysis profile according to our validated methodology. Significant differences were found for total TTR as well as for specific TTR Cys-10 PTMs between the different V30M ATTR stages as well as between controls and symptomatic patients.Quantification of wt:V30M TTR ratio and quantification of Cys-10 PTM.Isoforms is a feasible, reproducible and robust method by intact TTR and targeted LC-MS in the TTR V30M population. Significant differences for wt:V30M TTR ratio as well as for some specific PTMs have been found in a small cohort of V30M-TTR patients at different ATTR stages. These results need to be confirmed in a bigger cohort of patients with ATTR.


Multi-drug resistant efflux transporters found in Blood-Brain Barrier (BBB) acts as a functional barrier, by pumping out most of the drugs into the blood. Previous studies showed focused ultrasound (FUS) induced microbubble oscillation can disrupt the BBB by loosening the tight junctions in the brain endothelial cells; however, no study was performed to investigate its impact on the functional barrier of the BBB. In this study, the BBB in rat brains were disrupted using the MRI guided FUS and microbubbles. The immunofluorescence study evaluated the expression of the P-glycoprotein (P-gp), the most dominant multi-drug resistant protein found in the BBB. Intensity of the P-gp expression at the BBB disruption (BBBD) regions was significantly reduced (63.2 ± 18.4%) compared to the control area. The magnitude of the BBBD and the level of the P-gp down-regulation were significantly correlated. Both the immunofluorescence and histologic analysis at the BBBD regions revealed no apparent damage in the brain endothelial cells. The results demonstrate that the FUS and microbubbles can induce a localized down-regulation of P-gp expression in rat brain. The study suggests a clinically translation of this method to treat neural diseases through targeted delivery of the wide ranges of brain disorder related drugs. Many effective therapeutic drugs have been developed to treat central nervous system (CNS) related diseases. However, the effectiveness of most of those drugs had been hindered by the presence of a barrier between the CNS and the circulating blood; the blood-brain barrier (BBB). The BBB is a physical and metabolic barrier, which serves to regulate the flow of essential components into and out of the central nervous system (CNS) and to prevent the influence of toxic compounds and pathogens to the CNS12Different strategies have been used to overcome the BBB, including direct drug injection/infusion68Recently, focused ultrasound (FUS) combined with microbubbles has emerged as a promising method to temporarily permeabilize the BBB in a targeted region enabling a localized drug delivery for brain tumors and other disorders of the CNS11215Even with the disruption of the physical barrier of the BBB, there is still another barrier which impedes the effective delivery of the drugs into the brain. ATP-binding cassette (ABC) efflux transporters such as P-glycoprotein (P-gp) in the brain the endothelial cells serve as a functional barrier, preventing the brain uptake of harmful compounds171921in-vivo experiment on rat brains. In this work, we limited ourselves to focus on the expression of P-gp, the most known multi-drug resistance transporter that is expressed at the brain endothelial cellsThe purpose of this work was to investigate the impact of FUS and microbubbles on the functional barrier of the BBB through an From a pilot study, successful BBB disruptions were confirmed through enhanced the intensity of a MR contrast agent in T1-weighted MR images. The range of feasible acoustic pressure for BBB disruption was found at range of 0.6 MPa–0.65 MPa peak negative pressure see .The FUS and microbubbles successfully disrupted BBB of a rat brain as shown in This localized down-regulation of P-gp expression resulted in all three rats. Areas were selected over the BBB disruption region from contrast enhanced T1-weighted MR images. Areas at corresponding opposite side of the brain were used as controls. Fluorescence images at the selected areas were analyzed for the Evans Blue and the P-gp expression. For a detailed analysis, the contrast (MR) and the fluorescence (Evans Blue and P-gp) intensities between the sonicated and the control areas at a total of 31 selected areas in all three rats were compared statistically . In totaThe gradient of the acoustic pressure field around the focal region induced different magnitude of BBB openings at the sonication region See . In ordeH&E histologic analysis of the acute specimens revealed that sonications resulted in either no apparent change in the tissue or in some extravasated red blood cells as shown in In addition, any presence of adverse impacts of FUS and microbubbles on brain endothelial cells functions was observed by comparing RECA-1 expression at the BBB disruption region and the control region. The RECA-1 expression was not reduced at the sonication region compared to the control region see , demonstmdr1a knockout miceThe overall results of our study demonstrate the inhibition of the P-gp by the FUS and microbubbles. Also, there was a significant relationship between the level of the down-regulation of the P-gp expression and the magnitude of the BBB opening. The BBB disruptions and the down-regulations of the P-gp expression involved no damage to the tissue and the functions of endothelial cells of the brain. These results suggest direct translation of the technique to medicine. P-gp substrates include recognized anticancer drugs such as Doxorubicine, daunorubicine, vinblastine, vincristine, etoposide, teniposide117in-vitro cell studies3233in-vivo in the BBB.In brain disease such as epilepsy, depression, or schizophrenia patients are resistant to current medications despite adequate choice of therapeutic drugs at maximum tolerated doses, mainly due to the low retention rate of the drug by the multi-drug transporters at the BBB2425282717Past studies have shown that the predictable oscillation of microbubbles, also known as ‘stable’ cavitation, generates mechanical stress and shear stress to the blood vessel wall353613Recent applications of FUS combined with microbubbles as a therapeutic method have shown several promising biological effects, including BBB disruption accompanied by reversible changes in neuron responses4445There are few limitations in this study. First, no actual P-gp substrate drug was administered in this study. Our past work showed FUS and microbubbles not only enhanced the delivery of the chemotherapy agent doxorubicin to the rat brain tumors, but also retained drug concentrations in sonicated tumors for at least 24 hoursIn this work, we only observed a down-regulation of P-gp expression at 24 hours after the BBB disruption. In the correlation analysis, the contrast enhanced MR image was taken immediately after the FUS treatment with microbubbles, while the P-gp and the Evans Blue fluorescence intensities were measured on brains that were perfused and fixed 24 hours after the FUS treatment. In Lastly, even though the P-gp is a most known active efflux transporter found in the BBB, there are other multi-drug efflux transporters such as breast cancer resistance protein (BCRP) and members of the multi-drug resistance protein (MRP) family, and their substrates vary. Our study focused in P-gp, but future work needs to investigate the other transporters found in BBB.in-vivo experiment for the P-gp evaluation. Remaining four rats were used for RECA-1 fluorescence analysis and Hematoxylin and Eosin (H&E) histology for assessment of any damage in rat brain endothelial cell.All experiments were done in accordance with procedures approved by the Daegu-Gyeongbuk Medical Innovation Foundation (DGMIF) Institutional Animal Care and Use Committee (IACUC). All procedures and animals handlings were performed following the ethical guidelines for animal studies. The animals were anesthetized during all procedures and were constantly monitored throughout the experiment. No pain or suffering was evident as a result of the procedure. A total of 25 male Sprague-Dawley rats  were used for this study. First 18 rats were used in pilot study to define experimental settings for stable BBB disruption (BBBD). Three rats were used for the in situ pressure accounting for the attenuation due to the skull.  system  was used to sonicate rat brains for BBB disruption. The system schematic is shown in in-vivo experiment of P-gp expression analysis.In prior to experiment on P-gp, a pilot study was performed with 18 rats. The pilot study aimed to refine the experimental settings, ultrasound parameters and microbubble conditions for safe and repeatable BBB disruptions. During sonications, the acoustic emission signal from FUS induced microbubble oscillation was observed through a PZT hydrophone located at the center of the transducer. A range of sufficient and adequate ultrasound intensity was found by monitoring the magnitude of subharmonic signal intensity see . This me48in-vivo experiment, rats were anesthetized with mixture of Zoletil 25 mg/kg  and Rumpun 4.6 mg/kg  intramuscularly. The hair on their heads was removed using a shaving razor and hair removal cream. The animals were placed supine on a MR-compatible animal bed as described in For in vivo , respectively. 0.5 ml Evans Blue  was injected intravenously in order to determine the BBB disruption regions through both histologic and immunofluorescence analysisin-vivo experiment for P-gp evaluation. Following perfusion, all brains were extracted and two of them were prepared for RECA-1 fluorescence imaging and the remaining two were prepared for a histologic analysis. The details are described in the following sections.In order to assess any presence of damage in brain tissue and endothelial cells due to BBB disruptions, additional 4 rats sonicated. The procedure and protocol of the sonication was identical to the Imaging was performed using the 9.4T preclinical MRI system described above. An 86-mm inner diameter volume coil was used for RF transmission and signal reception. T2-weighted imaging was used to select the sonication targets and detection of edema. 2D RARE pulse sequence was used for acquisition of T1-weighted images for the evaluation of BBB disruption  images were recorded on a confocal fluorescence microscope  equipped with a 10 X objective. Two channels were acquired sequentially with the following excitation and emission parameters: 488 nm, 500 to 540 nm for P-gp and RECA-1; 550 nm, 590 to 620 nm for Evans Blue, respectively. To remove background from the image, we performed background subtraction on Metamorph software .th section (250 μm apart) was stained with H&E. The author who evaluated the histology was blind to the FUS exposure parameters. The images were recorded on an upright microscope  using 10 X and 20X objectives.To evaluate the histological effects of the ultrasound protocol used in this study, the extracted brains were embedded in paraffin, and serially sectioned at 5 μm sections in the axial plane (perpendicular to the direction of ultrasound beam propagation). Every 50The MR contrast intensity, Evans Blue and P-gp fluorescence intensities for the sonicated and contralateral (control) regions were compared using a two-tailed paired student’s t test. The correlation analysis between P-gp down-regulation, MR contrast enhancement and Evans Blue fluorescence intensity were evaluated using two-tailed Pearson correlation. For all statistical analysis, values of p < 0.05 were considered statistically significant.How to cite this article: Cho, H. et al. Localized Down-regulation of P-glycoprotein by Focused Ultrasound and Microbubbles induced Blood-Brain Barrier Disruption in Rat Brain. Sci. Rep.6, 31201; doi: 10.1038/srep31201 (2016).