Begomovirus, in the Family Geminiviridae. The begomoviruses all share the same whitefly vector, are highly recombinogenic and are distributed throughout tropical and subtropical regions where they seriously threaten the food security of the world's poorest people.The rapidly growing availability of diverse full genome sequences from across the world is increasing the feasibility of studying the large-scale population processes that underly observable pattern of virus diversity. In particular, characterizing the genetic structure of virus populations could potentially reveal much about how factors such as geographical distributions, host ranges and gene flow between populations combine to produce the discontinuous patterns of genetic diversity that we perceive as distinct virus species. Among the richest and most diverse full genome datasets that are available is that for the dicotyledonous plant infecting genus, We focus here on using a model-based population genetic approach to identify the genetically distinct sub-populations within the global begomovirus meta-population. We demonstrate the existence of at least seven major sub-populations that can further be sub-divided into as many as thirty four significantly differentiated and genetically cohesive minor sub-populations. Using the population structure framework revealed in the present study, we further explored the extent of gene flow and recombination between genetic populations.Begomovirus population structure revealed here could facilitate population genetics studies into how central parameters of population genetics namely selection, recombination, mutation, gene flow, and genetic drift shape the global begomovirus diversity.Although geographical barriers are apparently the most significant underlying cause of the seven major population sub-divisions, within the framework of these sub-divisions, we explore patterns of gene flow to reveal that both host range differences and genetic barriers to recombination have probably been major contributors to the minor population sub-divisions that we have identified. We believe that the global  The study of genome-wide patterns of sequence variation within and between closely related virus species can be used to efficiently infer the fine-scale genetic structures of virus populations. Information on population structures - particularly that pertaining to stratification and admixture (i.e. gene flow) - is valuable in a variety of situations. These include the establishment of sensible species/subspecies/strain classification criteria, the detection of geographical or biological barriers to gene flow, and the identification of demographic, epidemiological or evolutionary processes responsible for virus differentiation -3. More Begomovirus that are responsible for economically devastating diseases of many leguminaceous, solanaceous, curcurbitaceous and malvaceous crop species throughout the tropical and subtropical regions of the world [Bemasia tabaci, and have circular single stranded one (i.e. monopartite) or two (i.e. bipartite) component genomes ranging in size from ~2.7 Kb (for monopartite species) to ~5.4 Kb (for bipartite species) [It is therefore surprising that there have been no studies specifically aimed at identifying global-scale population genetic structures within agriculturally significant groups of plant pathogenic viruses such as the geminiviruses, potyviruses, tospoviruses, cucumoviruses and sobemoviruses. For example, virtually nothing is known about population stratification amongst the various geminivirus species within the genus he world -15. Begospecies) .Relationships amongst DNA-A and DNA-A-like sequences are widely used in formalized begomovirus species, strain and variant demarcation schemes -19. BaseBegomovirus taxonomic classification system is based almost entirely on traditional phylogenetic reconstruction and pairwise genetic distance estimators (such as Hamming or p-distances) [The current stances) -20,22. THowever, frequent inter-species genetic recombination is a prominent feature of begomovirus evolution -27 that Here we use such a population-genetics model-based clustering approach both to verify the existence of defined sub-populations within the global begomovirus meta-population and to track the movement of genetic material between these populations. Besides identifying hitherto unappreciated genetically discreet begomovirus sub-populations, our study provides interesting insights into how constraints on genetic recombination imposed by geographical distance and/or host range differences may contribute to taxonomically relevant patterns of begomovirus diversity.SA). Monte Carlo simulations indicated that although pairs of sites within the begomovirus genome did indeed display evidence of significant linkage disequilibrium , the corresponding ISA was 0.0367 - a low value providing evidence that many of the polymorphic loci considered are effectively in linkage equilibrium. ISA is expected to be zero when there is no linkage among pairs of polymorphisms. The estimated ISA value for our global begomovirus dataset was, for example considerably lower than that approximated for Helicobacter pylori (0.0607) [The admixture model implemented in STRUCTURE assigns individual genomes to populations under the assumption that all polymorphic sites within the genomes are in linkage equilibrium. We therefore tested the degree of linkage equilibrium that is evident within begomovirus genomes using LIAN 3.4 to calculate a standardized index of association between genome sites (I(0.0607)  and slig(0.0607) . In both2, against the physical distance separating pairs of sites  against physical distance. This analysis indicated that there was no systematic LD bias in our begomovirus dataset that might seriously impact its use in the inference of gross population structure.To investigate further the degrees of LD displayed by pairs of polymorphic sites we plotted two standard measures of LD, |D'| and rtes Fig. . There wOur initial analysis of population structure within the full begomovirus dataset aimed at discriminating between two to twelve sub-populations i.e. K = 2 to12) failed to yield an estimate of the true optimal sub-population number in that the value of Ln P(D) increased consistently with increasing K. However, the second-order rate of change of the likelihood function (ΔK) showed a clear peak at K = 8, reflecting the existence of at least eight genetically cohesive begomovirus sub-populations each displaying distinctive nucleotide distribution patterns. Although according to ΔK, the optimal number of sub-populations for the complete begomovirus dataset was eight, we chose the more conservative K = 7 for further analysis because this number of sub-populations yielded reasonably consistent clustering in repeated analysis runs. With K = 8, either the sweet potato-infecting viruses within the larger swepovirus-Asian legumovirus sub-population , the New Delhi tomato-Asian Cucurbit-infecting viruses (NDT-ACU), the Indo-Pak cotton-South Indian tomato viruses (IPC-SIT), and China-Japan-Southeast Asia viruses (Ch-J-SEA).For the sake of clarity, we named the seven sub-populations identified in the K = 7 analysis based on both the geographical location and hosts of the viruses assigned with the sub-populations. Schematic representations of the population structures revealed by our analysis are summarised in Fig The sequential increase in population stratification noted in the analysis series with K values ranging from two through seven Fig.  providesSince inconsistent sub-population splits were obtained with K > 7, we attempted to identify further population structures within the seven consistently defined sub-populations obtained with K = 7. Each one of these sub-populations was treated as a main population and each was analysed separately under the admixture model with uncorrelated allele frequencies.A second layer of population structure analysis was performed on each of the seven major sub-populations in isolation Fig . STRUCTUThe major IPC-SIT and Af-Med sub-populations apparently each contained four genetically cohesive minor sub-populations . The hierarchical AMOVA of the seven major sub-populations and the 34 minor sub-populations indicated that most of the observable genetic diversity is collectively attributable to fixed genetic differences between the 34 minor sub-populations (40.96% of the diversity) and seven major sub-populations  were applied to the various population partitions. The null hypothesis of no population structure was rejected with p-values < 0.001 by the Z-test [ST statistics (pairwise measures of population differentiation), were calculated for each of the 34 minor sub-populations [see additional file ST scores ranged from 0.09 to 0.92. Whereas an FST value of 0 between two populations would indicates that they were completely undifferentiated, a score of 1 would indicate that every observable genetic difference between individual members of the two populations could be used to distinguish between the populations. Overall, a very high degree of differentiation was noted between the Tomato chino La paz virus group and the African cassava mosaic virus minor sub-population. (FST = 0.92). The lowest degree of differentiation (FST = 0.09) was observed between the New World Tomato rugose and chloratic mottle and Tomato golden mottle virus groups. With the exceptions highlighted (the numbers in bold) in Table S1 [see additional file ST values.To further test whether the 34 identified minor sub-populations would be considered genetically distinct using alternative methodologies, two other statistical tests of population differentiation  consist of viruses that have collectively been sampled from six or more different host species, others contain viruses that have only ever been sampled from one species. Interestingly, the "broad host range" minor sub-populations are also apparently more admixed than the "narrow host range" minor sub-populations. Unfortunately we cannot tell from our analysis either whether recombination has facilitated the increased host-ranges that are apparent within these sub-populations or whether increased host ranges drive increased inter-sub-population recombination frequencies.It must, however, be stressed firstly that very little is known about the natural host ranges of any of these virus groups and, secondly, that there exist blatant sampling biases in favor of begomovirus species/strains that cause crop diseases. The fact remains however, that whereas certain of the minor sub-populations  of pairwise distances between groups of closely related sequences that apparently share a recent common ancestry , is computed and compared to the variance expected when all loci are in linkage equilibrium (VE). Only polymorphic sites were included for the analysis and a 5% critical value was obtained as described [2 [Testing for the presence and degree of linkage disequilibrium (LD) evident in a group of sequences is a significant aspect of population genetics. Moreover, the model-based approach we used to investigate the structure of begomovirus populations assumes that different polymorphic sites along the genomes being investigated display only limited degrees of LD. From the perspective of global begomovirus diversity it is very probable that, because of the extent of inter-species genetic exchange amongst begomoviruses, many sites will be effectively in linkage equilibrium. However it was essential that we test the degree of linkage equilibrium evident within our worldwide begomovirus population sample. A null hypothesis of linkage equilibrium was tested by Monte Carlo simulations using the program LIAN (version 3.4) . LIAN pe dataset . This prescribed . In addiescribed  and r2 [ribed [2 , were esribed [2 .Global begomovirus population structure was investigated using the program STRUCTURE (Version 2.0) . This prad hoc STRUCTURE runs were performed to determine the optimum number of iterations for the initial burn-in and estimation phases of the analysis so as to ensure the reliability of posterior probability estimates. Burn-in and parameter estimation iterations ranging from 20,000 to 40,000 did not yield significantly different results. From these preliminary analyses we determined that an initial burn-in of 40,000 iterations followed by 40,000 iterations for parameter estimation was sufficient. To estimate the number of populations (the K parameter), the begomovirus dataset was analyzed allowing the value of K to vary from 1 to 12. Five independent runs were carried out for each K value . As advised in the STRUCTURE user's manual, we set most of the parameters to their default values [opt) was identified as previously described [In the beginning, t values . Specifit values . This mot values . We co-et values . The lamescribed .opt, each individual was then assigned to one of the sub-populations, according to their respective estimated membership scores  for each of the different sub-populations. Individuals that could be assigned to two or more different sub-populations each with membership scores of 0.15 or higher were considered to be admixed. It is important to note that despite our expecting the admixture model to identify the correct number of sub-populations we also expected it to generally overestimate the proportion of admixed individuals by ignoring linkage between polymorphic nucleotide sites that were physically very close to one another within the begomovirus genomes. We also applied the linkage model for Kopt in order to account for potential physical linkage between loci when refining the sub-population assignment of difficult to assign individuals. For this model the burn-in and MCMC run lengths were set at 20000 and 40000 respectively, with a 10000 iteration admixture burn-in length.For Kad hoc parameter as described in [We used the first hierarchical sub-population cluster classification inferred by STRUCTURE to study finer-scale clustering within major begomovirus sub-populations. Each of the seven established major begomovirus sub-populations was considered as a major sub- population and analysed separately under the admixture model with uncorrelated allele frequencies . We used ΔK, an ribed in  to deterST statistics). The fixation indices estimated from the begomovirus sequence analyses were tested using a non-parametric permutation approach as described in [ST based estimates of population structure was also tested in ARLEQUIN using a permutation test (with 1000 randomised iterations) as in [The population stratifications inferred by STRUCTURE were tested by analysis of molecular variance (AMOVA) as implemented in ARLEQUIN (ver. 3.0) . AMOVA mribed in . Furthers) as in . Also, Ds) as in  was useds) as in . PermutaThe authors declare that they have no competing interests.HCP conceived, designed the study. HCP, DPS, AV, MS, BS and MR performed sequence alignments and population structure analysis. HCP and DPM interpreted data and wrote the manuscript. All authors have read and approved the final manuscript.Table S1. Differentiation between the 34 minor begomovirus sub-populations identified in this study. Data provided represent pairwise measures of population differentiation (FST). Non significant FST values based on permutation test are highlighted.Click here for file


In 32 healthy blood donors, 20 patients with histologically verified cancer and 18 patients with non-neoplastic diseases, the fluorescence polarization changes of fluorescein samples incorporated in mononuclear leucotyes were measured after incubation with PHA. The leucocytes of healthy persons, and 16/18 persons with non-neoplastic diseases, responded with a decrease in the degree of fluorescence polarization by about 20% from that in non-PHA-stimulated cells. In 19/20 patients with a variety of malignant tumours, the leucotyes did not respond to PHA stimulation with such a decrease. The exceptions among the patients with neoplastic and non-neoplastic diseases are considered, and may not be "false-negative" or "false-positive" respectively, but indicative of a particular situation in that disease. The biophysical mechanisms underlying the observed changes remain to be investigated.


Smear-negative tuberculosis occurs more frequently in human immunodeficiency virus (HIV)-infected patients than in non-HIV-infected patients. Besides, there are substantial numbers of patients who cannot produce sputum, making the diagnosis of pulmonary tuberculosis (PTB) difficult.To evaluate the relative yield of pre- and post-bronchoscopy sputum and bronchoalveolar lavage (BAL) in ‘sputum smear’-negative, HIV-positive patients.A tertiary care referral hospital in Addis Ababa.Mycobacterium tuberculosis (MTB) was done for all sputa and BAL.Acid-fast stain (AFS) using the concentration technique was done on 85 pre-bronchoscopy sputum and 120 BAL samples. Direct AFS was done on all BAL and 117 post-bronchoscopy sputum samples. Culture for P=0.002, P=0.028 respectively).MTB was isolated from 26 (21.7%), 23 (19.7%) and 13 (15.3%) of BAL, post- and pre-bronchoscopy sputum cultures respectively. AFS on pre-bronchoscopy sputum using concentration technique and direct AFS on BAL together detected 11 (41%) of the 27 culture-positive cases. In patients who could produce sputum, the sensitivity of pre-bronchoscopy sputum culture  was comparable to BAL  and post-bronchoscopy sputum . In patients who could not produce sputum, however, both BAL  and post-bronchoscopy sputum  detected significantly more patients than those who could produce sputum (In HIV-infected patients, AFS by concentration method on pre-bronchoscopy sputum and direct AFS on BAL in patients who cannot produce sputum are the preferred methods of making a rapid diagnosis. BAL culture seems to add little value in patients who can produce sputum; therefore, bronchoscopy should be deferred under such circumstances. M. tuberculosis in sputum AFB smear-negative, HIV-positive Ethiopian patients with atypical chest X-ray.The diagnosis of tuberculosis continues to be a challenge in developing countries. The human immunodeficiency syndrome (HIV) has worsened the situation by decreasing the sensitivity of sputum smear for acid-fast bacillus (AFB). As a result, the prevalence of smear-negative tuberculosis is on the rise.–3 Beside46Pneumocystis jirovecii (PJ). All patients who were ‘sputum smear’-negative for AFB underwent acid-fast stain after sputum liquefaction with sodium hypochlorite and concentration with a centrifuge and culture for M. tuberculosis. Those who tested negative for PJ in sputum sample and patients who could not produce sputum underwent bronchoscopy for bronchoalveolar lavage and post-bronchoscopy sputum examination for AFB and culture for M. tuberculosis.The study was conducted at Tikur Anbessa University Hospital, a tertiary care referral center in Addis Ababa, Ethiopia. Between March 2005 and July 2006, consecutive HIV-infected patients with atypical chest X-rays and whose sputum smear was negative for AFB or could not produce sputum at all were enrolled into the study. Patients were initially excluded from the study if their chest X-rays were judged as typical for tuberculosis or segmental/ lobar pneumonia and if they were offered therapeutic trial. Those who failed to respond to the treatment were later included into the study if their sputum was negative for AFB and if they were positive for HIV. Patients who could produce sputum were asked to give another set of sputum, after brushing their teeth and rinsing their mouth under tap water, for staining for Both the oral and nasal pharynges were anesthetized with 5 ml of 2% lidocaine solution using a sprayer before bronchoscopy. Transnasal fiber-optic bronchoscopy was performed with Olympus BF A-3R flexible bronchoscope after administering a local intranasal lidocaine jelly. The scope was then wedged to a segmental or sub-segmental bronchus of the abnormal area as determined by the chest X-ray and a lavage was performed by instilling between 80 and 120 ml of sterile normal saline and immediately sucking it with a suction machine. Bronchoalveolar lavage was continued until at least 50 ml of the instilled saline was recovered. As most of the patients were in respiratory distress, the procedure had to be done as quickly as possible. The tracheo-bronchial tree was also visualized for the presence of purulent secretion and localized lesions such as Kaposi sarcoma. Patients were monitored for their oxygen saturation and most required administration of intranasal oxygen. Post bronchoscopy, patients were asked to cough and produce sputum. The BAL fluid and post-bronchoscopy sputum were sent for acid-fast stain and culture.Initial smear-negative sputa, already pooled and kept at +4°C and bronchoalveolar lavage were transferred to a 15 ml screw-capped tube and mixed with an equal volume of sodium hypochlorite (5%). The tubes were incubated at room temperature for 15 min and shaken by hand at regular intervals. After addition of 8 ml of distilled water, the tubes were centrifuged at 3,000 rpm for 15 min. The supernatant of each tube was carefully discarded, the sediment was mixed with remaining fluid and direct smears were prepared by applying a drop of the resuspended sediment with a sterile pipette to a slide. The slides were dried in air, heat-fixed and stained by the Ziehl Nielsen technique.2 for 1 week, at 37°C in air for another 7 weeks and thereafter were checked once a week for mycobacterial growth. Growth of mycobacteria was confirmed by typical colony morphology and microscopy for AFB. In order to identify the LJ-grown isolates to species level, the nitrate reductase test, thiophene 2 carboxylic acid hydroxide (TCH) and the pyrazinamide test were performed.AFS using the Ziehl-Nielsen method was initially done on all BAL specimens and post-bronchoscopy sputum cultures and the results employed for the management of patients. Subsequently, mycobacterial culture on conventional Löwenstain-Jensen (LJ) egg medium and LJ medium containing pyruvate was performed for all pre- and post-bronchoscopy sputum and BAL fluid samples. Before culturing, the samples were digested and decontaminated from nonmycobacterial microorganisms by the sodium lauryl sulfate method. The tubeChest X-rays were read by two radiologists unaware of the diagnosis. Whenever there was discordance in the reading, they were asked to reach a consensus diagnosis. Chest X-rays were read as ‘highly likely’ , ‘likely’ or ‘less likely’ of the various pulmonary opportunistic infections including tuberculosis based on an already prepared diagnostic protocol format.The study was approved by the National Ethical Committee in Ethiopia and the Ethical Committee of the Karolinska Institute in Sweden. HIV testing and bronchoscopy were done after informed consent. Patients were referred for antiretroviral treatment, care and social support whenever applicable.After pre-coding of the variables, the raw data was entered into the computer using EPI-Info version 6.04D statistical package and exported to SPSS for windows version 11 statistical package for the purpose of analysis. Analysis was done using both the statistical packages. Frequencies, proportions and summary statistics were used to describe the study subjects in relation to relevant variables. For comparing the results of different test procedures, sensitivity, specificity and predictive values were calculated along with their 95% CI.mycobacterium tuberculosis. Bronchoscopy and bronchoalveolar lavage were done in 120 patients after excluding 11 patients in whom sputum examination was positive for P. jirovecii. Post-bronchoscopy sputum was obtained in 117 of the 120 patients. The relative yield of the various diagnostic methods and comparison of the prevalence among the different techniques are depicted in A total of 131 patients were enrolled into the study, of whom 85 (64.9%) were able to produce sputum and underwent AFS after sputum liquefaction with sodium hypochlorite and concentration with centrifuge and culture for Post-bronchoscopy sputum smear for AFB had the lowest yield, with only two specimens positive for AFB; and both were positive in BAL smear. However, post-bronchoscopy sputum culture was as good as BAL culture (sensitivity 88.5%). The sensitivity, specificity, positive and negative predictive values of the various diagnostic tests against their gold standard are shown in P= 0.002, P= 0.028 respectively). Besides, BAL smear was positive in only 3 (3.5%) cases amongst the 85 sputum producers, while it detected 6 (17%) amongst the sputum non-producers (P= 0.028).Amongst the 85 patients who could produce sputum, all BAL, post-bronchoscopy and pre-bronchoscopy sputum cultures detected similar number of patients. BAL and post-bronchoscopy sputum cultures were positive in 12 (14%) patients each, while pre-bronchoscopy sputum culture was positive in 13 (15.3%) patients. BAL and post-bronchoscopy sputum, on the other hand, detected MTB in 14 (40%) and 11 (31.4%) of the 35 patients who could not produce sputum for this difference was statistically significant in the two groups for both BAL and post-bronchoscopy sputum  patients were strongly suspected of having tuberculosis radiologically. The remaining patients were considered to have pneumocystis pneumonia and bacterial infection and were put on separate treatment until either the smear or culture results were available. The sensitivity and positive predictive value of chest X-ray in the diagnosis of tuberculosis amongst this group of patients were low.The results of our study have demonstrated that in HIV-infected patients presenting with atypical chest X-ray and negative sputum smear, culture from bronchoalveolar lavage is the most sensitive method for detection of tuberculosis. The overall sensitivity of sputum culture was relatively low compared to BAL (15.3% versus 21.7%) and this is comparable to findings from other studies.–11 Howev6BAL smear was positive in one-third of the patients positive by BAL culture and was complementary to sputum smear by concentration technique. However, all the 6 patients that complemented sputum smear were from among patients who could not produce sputum. Results of both smear and culture suggest, to a significant level, that the major benefit of bronchoscopic aspirate is in patients who cannot produce sputum. The two quick methods of direct BAL smear and sputum AFS by concentration method allowed the detection of nearly half of the culture-proven cases. This is welcome news to the treating physician who, otherwise, had to wait 8 weeks before the diagnosis was made by sputum culture.Post-bronchoscopy sputum culture is highly sensitive compared to BAL culture (sensitivity 88.5%). However, all cultures from post-bronchoscopy sputum were also positive in BAL culture and therefore did not add much to the overall diagnosis. It has been suggested in the past that the instillation of local anesthetic agents into the tracheo- bronchial tree during bronchoscopy will kill some of the mycobacteria and render the BAL less culture-positive compared to post-bronchoscopy sputum. Our finding, however, does not support this notion and BAL culture remains the most sensitive method for the diagnosis of tuberculosis.Induced sputum has been shown to have an excellent yield compared to BAL and therefore can be a practical solution for those who cannot expectorate adequate sputum.–14 UnforSeveral studies have demonstrated that transbronchial biopsy (TBBX) provides incremental diagnostic information not available from evaluation of sputum or bronchoalveolar lavage.–18 The pAs of recent, the polymerase chain reaction (PCR) has been shown to have a very high sensitivity and specificity in both sputum and BAL specimen and is increasingly being used for the diagnosis of tuberculosis.–21 The cIn conclusion, our study demonstrated that sputum AFS using the concentration method and BAL smear for AFB are rapid methods for the diagnosis of PTB and bronchoalveolar lavage is a biologic specimen with the highest yield and is the most useful. It has also demonstrated that the added value of BAL culture over sputum culture is in patients who cannot produce sputum. It is therefore reasonable to conclude, that in HIV-infected patients with negative sputum smear and atypical chest X ray, bronchoscopy as a diagnostic tool for PTB should be spared for patients who cannot produce sputum. Such a recommendation, however, requires a larger and more detailed study before it is implemented. We also recommend that sputum induction, being relatively simple and inexpensive, should be evaluated as an alternative for bronchoscopy in resource-constrained settings.


Changes in synaptic strength are believed to underlie learning and memory. We explore the idea that norepinephrine is an essential modulator of memory through its ability to regulate synaptic mechanisms. Emotional arousal leads to activation of the locus coeruleus with the subsequent release of norepineprine in the brain, resulting in the enhancement of memory. Norepinephrine activates both pre- and post-synaptic adrenergic receptors at central synapses with different functional outcomes, depending on the expression pattern of these receptors in specific neural circuitries underlying distinct behavioral processes. We review the evidence for noradrenergic modulation of synaptic plasticity with consideration of how this may contribute to the mechanisms of learning and memory. Specific cells and synapses within implicated neural circuits are recruited during learning, such that memory allocation is not random but rather is regulated by precise mechanisms which define where and how information is stored within the neural network . Nearly The results of numerous previous experiments implicate the amygdala in acquisition and retention of memory for emotionally charged events [reviewed in -12]. ThuRecent reviews have examined the role of the noradrenergic system in emotional memory , the infq-coupled α1receptors and Gi-coupled α2 receptors. Activation of three different subtypes of β-receptors , linked to Gs proteins, results in a rise in the intracellular cyclic AMP concentration and subsequent PKA activation.Norepinephrine, also called noradrenaline, is a catecholamine produced by dopamine β-hydroxylase  which isThe locus coeruleus is a nucleus composed of mostly medium-size neurons located within the dorsal wall of the rostral pons in the lateral floor of the fourth ventricle that serves as the principal site for synthesis of norepinephrine in the brain . The proThe mechanisms of memory could be best understood with the systematic application of associative learning paradigms. Short-term memory is normally defined and measured by retrieval tests that occur within the first few hours following the acquisition event, while long-term memory is typically measured using retrieval tests that occur at later times, following many hours or even days after the memory was acquired . NumerouA receptor antagonist picrotoxin, used in the memory-enhancing doses, showed a substantial increase in levels of NE in the amygdala, while systemic injections of the memory impairing doses of the GABAA receptor agonist muscimol resulted in decreased levels of NE  norepinephrine preaccumulated in slices from the hippocampus in both the dentate gyrus and the CA1 region [While the amygdala presents us with one of the clearest examples of how synaptic plasticity in a defined neural circuitry could control fear-related behavioral responses, the hippocampus is another important locus of the NE actions linked to the mechanisms of learning and memory. Using labeled norepinephrine, it was directly demonstrated that NE could be released in the hippocampus in an activity-dependent fashion. Thus, NMDA application was shown to induce the release of [1 region . A recen1 region . The res1-adrenoreceptor agonist and were blocked by the α1-adrenoreceptor antagonist, leading to the conclusion that the actions of norepinephrine may shift during development. Activation of noradrenergic systems during emotional arousal may enhance memory formation by inhibiting protein phosphatases that normally oppose the induction of LTP, because protein phosphatase inhibitors mimicked the effects of β-adrenoreceptor activation that enabled the induction of LTP during long trains of stimulation at CA1 synapses [This recent study builds on an existing literature supporting a role of norepinephrine in the functioning of hippocampal circuitry. Thus, it was reported previously that norepinephrine could induce long lasting modifications of synaptic responses in the dentate gyrus in rats, associated with differential actions on distinct projections  arising in the entorhinal cortex . The finsynapses . In the synapses . This plsynapses ,82-84. Ti-coupled α2 receptors and Gs-coupled β1, β2, and β3 receptors) or phospholipase C (through Gq-coupled α1receptors). This knowledge would result in understanding of how second messengers may interact with the mechanisms of synaptic plasticity in the brain in the context of specific forms of learning and memory.The better understanding of the mechanisms by which emotional arousal could enhance the brain's ability to store, retain, and subsequently recall information, would require a detailed characterization of the signaling pathways lying downstream of norepinephrine binding to adrenergic receptors. Norepinephrine activates various subtypes of adrenergic receptors, so it is not surprising that evidence is accumulating for the differential roles of signaling pathways tied to distinct receptor classes in synaptic plasticity. Thus, the extracellular signal-regulated MAP kinase (ERK) is activated by β-adrenoreceptors in somas and dendrites of CA1 pyramidal neurons in a PKA-dependent fashion . It play2 adrenoreceptors by NE in Purkinje cells could control short-term and long-term associative plasticity at the parallel fiber synapses [2-receptors interfered selectively with the induction of associative synaptic plasticity. Thus, noradrenergic modulation in this system could provide a mechanism for context-dependent modulation of associative plasticity and memory. In slices of the rat visual cortex, paired-pulse stimulation in the presence of NE resulted in a form of homosynaptic LTD [1 receptor antagonist and mimicked by the α1 agonist. In the developing visual cortex in rats, LTP at inhibitory synapses could be induced by the high frequency stimulation when excitatory neurotransmission was blocked [As the noradrenergic bundle sends projections throughout the central nervous system, it is expected that norepinephrine would modulate synaptic plasticity in other neural circuits besides the hippocampus and amygdala. Consistent with this prediction, it has been demonstrated recently that activation of αsynapses . In the ptic LTD . This no blocked . The ind2-adrenoreceptor-dependent inhibition of excitatory glutamatergic signaling was observed. Norepinephrine, acting at presynaptic α2 receptors, inhibited single fiber glutamatergic inputs from the nociceptive pontine parabrachial nucleus to the central amygdala [2-adrenoreceptors on inputs to sympathetic preganglionic neurons in slices of neonatal rat spinal cord also decreased glutamate release [2-dependent inhibition over both its own and glutamate release [2-dependent inhibition of calcium influx [In several brain circuits, αamygdala . This ef release . In thes release . At the m influx .Taken together, these findings indicate, that while NE can produce a variety of the effects on synaptic and neuronal mechanisms in different regions of the brain, the expression patterns of different NE receptor subtypes in specific neural circuits could determine the resulting functional outcome.s-linked β-adrenoreceptors activating the cAMP-dependent pathway [1-adrenoreceptors and decrease it through activation of α2-adrenoreceptors on GABAergic terminals [Presently, there is substantial evidence that activation of adrenoreceptors by norepinephrine can modulate GABAergic inhibitory systems in the brain. Thus, activation of the locus coeruleus resulted in suppression of feedforward interneurons in rat dentate gyrus, thereby promoting conditions for the induction of synaptic plasticity . In the  pathway . In the erminals . As the It is well established that emotional arousal modulates the formation of memory, and a substantial literature, of which only a fraction is cited in this review, points to a critical role for the release of norepinephrine in such modulation. The electrophysiological studies have begun to elucidate how norepinephrine could modulate both synaptic transmission and plasticity in specific neural circuits. Storing memories in the brain likely requires changes in the number, structure, and function of synapses . ConsideAMPA: α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate; cAMP: Cyclic adenosine monophosphate; ERK: extracellular signal-regulated kinase;GABA: γ-aminobutyric acid; GluR1: AMPA receptor subunit; LTP: long-term potentiation; LTD: long-term depression; MAPK: mitogen-activated protein kinase; NMDA: N-methyl-D-aspartic acid; PKA: cAMP-dependent protein kinaseThe authors declare that they have no competing interests.K.T. and V.Y.B. wrote, read and approved the final manuscript


We have purified epithelial cells from human colonic tumours by velocity sedimentation in an isokinetic density gradient of Ficoll in tissue culture medium. In frozen sections of colonic carcinoma, histochemically demonstrable N-acetyl-beta-D-glucosaminidase (HDAG) was observed primarily in epithelial cells. We used this enzyme as a histochemical marker of epithelial cells. Initial suspensions of cells from colonic tumours suspended with 0-25% trypsin contained an average of 24% of the nucleated cells with HDAG. In the purest fraction obtained from gradient centrifugations, an average of 74% of the nucleated cells contained HDAG. After centrifugation, the quarter of the density gradient which contained the most rapidly sedimenting cells was purified 2-4-fold over that in the initial suspension. Cells in this zone of the gradient also gave rise to colonies in soft agar. Cells from initial suspension resulted in 15-25% as many colonies of 7 or more cells in cultures inoculated with the same number of nucleated cells. For the most part, cells obtained from the other zones of the gradient did not give rise to colonies in soft agar.


To evaluate a new approach of recanalisation of nasolacrimal duct obstruction (RC-NLDO) in the treatment of the nasolacrimal duct obstruction (NLDO) and chronic dacryocystitis.583 patients with 641 eyes suffering from NLDO and chronic dacryocystitis were enrolled in this study. The RC-NLDO was performed in 506 eyes, with 135 eyes undergoing external dacryocystorhinostomy (EX-DCR) as controls. Patient follow-up for 54 months was evaluated by symptoms, dye disappearance test, lacrimal irrigation and digital subtraction dacryocystogram. The RC-NLDO was also performed in 12 rhesus monkeys for histopathological examination.The clinical success rates were 93.1% in 506 cases of RC-NLDO and 91.11% in 135 cases of EX-DCR. The success rates for second surgery were achieved in 85.19% on RC-NLDO and 40.0% on EX-DCR. No major intra- or postoperative complications were observed in the RC-NLDO group. The mean operative duration was 12.5 min for RC-NLDO and 40.3 min for EX-DCR (p<0.001). A pathological study in rhesus monkeys demonstrated that the RC-NLDO wounded epithelium in nasolacrimal duct healed completely within 1 month without granulation tissue formation.The findings demonstrate that the RC-NLDO is a simple and effective approach proven to recanalise the obstructed nasolacrimal duct with a comparable success rate to EX-DCR. Nasolacrimal duct obstruction (NLDO) and chronic dacryocystitis are common ophthalmic diseases. The external dacryocystorhinostomy (EX-DCR) has been the most effective and standard surgery in treating these conditions since 1904 when it was reported by Toti.Recanalisation of nasolacrimal duct obstruction (RC-NLDO) was an evolutionally developed surgical approach for treating these conditions to restore the native nasolacrimal duct, using a simple instrument, the lacrimal canaliser, which we created in 1994.This study adhered to the tenets of the Declaration of Helsinki and was approved by the Institutional Review Board (IRB)/Ethics Committee of Zhongshan Ophthalmic Center, Sun Yat-sen University. All cases were chosen from outpatients who were diagnosed as having NLDO and/or chronic dacryocystitis. Every patient underwent preoperatively comprehensive ophthalmic and intranasal examination. Dacryocystogram or digital subtraction dacryocystogram was performed in some cases.A total of 641 eyes of 583 consecutive patients were recruited from July 2003 to June 2006 with their signed informed consent forms, including 135 eyes of 126 patients undergoing the EX-DCR and 506 eyes of 457 patients undergoing the RC-NLDO. There were no statistical differences in patient demographics between these two groups. The male-to-female ratio was approximately 1:3, and the average age was 50 years. The duration of symptoms ranged from 6 months to 26 years (mean 5.1 years) in the RC-NLDO group and from 6 months to 17 years (mean 4.7 years) in the EX-DCR group.The instrument used for recanalisation of nasolacrimal duct obstruction was the lacrimal canaliser consisting of a console and its accessories . The conEX-DCR was performed under local anaesthesia in a standardised fashion.All patients were prescribed topical antibiotic eye-drops and a nasal mucosa astringent four times a day for 10 days. The lacrimal passage was irrigated with antibiotic solution weekly in the first half month after surgery. Postoperative evaluation and long-term follow-up were performed by the same doctors.Clinical success was defined by the results of the dye disappearance test, lacrimal irrigation and symptoms. “Full success” was noted if the fluorescent stain was positive in 5 min, indicating the free nasolacrimal passage, and the symptoms were completely resolved. The lacrimal irrigation was performed if no fluorescent stain was found, or the stain was found after more than 10 min. “Partial success” was noted if the pledget was stained with fluorescein after lacrimal irrigation and there was no reflux. “Partial success” was also scored for the patient who had some symptoms but no reflux in lacrimal irrigation. “Failure” was defined as no improvement or recurrence in tearing with severe reflux in irrigation at the last follow-up.Twelve rhesus monkeys (1.5–2 years old and weighing 4–6 kg) were purchased from Guangdong Medical Laboratory Animal Center, Guangdong, China. Experimental procedures were performed adhering to the ARVO statement for the Use of Animals in Ophthalmic and Vision Research. One eye randomly chosen from each monkey was used in RC-NLDO, and the other eye from each animal was used as an untreated normal control. The surgery and postoperative care were performed in the same manner as for the patients described above. The animals were killed by an overdose of barbiturates 7 days, 1 month, 2 months and 3 months after surgery. Specimens including canaliculi, lacrimal sac and nasolacrimal duct were carefully collected for histopathological examination.The patient preoperative conditions are summarised in The surgical outcomes are summarised in In the EX-DCR group, one patient (0.74%) suffered postoperative bleeding immediately after surgery; eight patients (5.93%) reported transient pain in the upper segment of the maxillary bone, but it was tolerable; and 30 patients (22.22%) complained about their visible scars. No infection or uncontrollable bleeding occurred. In the RC-NLDO group, four patients (0.79%) had postoperative periocular subcutaneous haematoma. No other complications were observed in this group.In normal rhesus monkey, the lacrimal sac is lined by stratified columnar epithelium containing scattered goblet cells on a broad basement membrane while the nasolacrimal duct is lined by a double layer of epithelium, a superficial layer of columnar cells and a basal layer of non-keratinised squamous cells . It diffThe healing process of the epithelium in the nasolacrimal duct after RC-NLDO surgery in the rhesus monkey was evaluated by histopathological examinations. Cross-sections of the specimens obtained immediately after RC-NLDO displayed the intact epithelium of the lacrimal sac and an almost total loss of epithelium in the nasolacrimal duct with a few residual cells appearing as small islands on the basal membrane . SectionThe ideal treatment for NLDO is to recanalise the obstructed duct and restore normal anatomical structure and physiological function of the lacrimal drainage system. The EX-DCR is a successful operation with a success rate of 80–95%, but it is a relatively complex procedure17In order to overcome these disadvantages, a number of therapeutic developments and promising advances in DCR have been reported recently, such as endonasal (endoscopic) DCR and endocanalicular laser DCR.Recanalisation of the nasolacrimal duct obstruction (RC-NLDO) was a simple and evolutional approach for treating NLDO and chronic dacryocystitis. In the last 5 years, we have been conducting this study with up to 54-month long-term follow-up to evaluate the RC-NLDO approach for comparison with EX-DCR. Our findings demonstrated that the RC-NLDO was a highly successful approach with an overall success rate at 93.18% for primary surgery and 85.19% for second repeated surgery. The pathological study in rhesus monkeys further confirmed that the surgically wounded epithelium in the nasolacrimal duct was starting to heal in a week and completely recovered within 1 month, creating a wide recanalised cavity.2,” the reopened area (S = 1.13 mm2) of the duct cavity in cross-section by RC-NLDO was 4–9-fold larger than the laser-created cavity (0.13–0.28 mm2). According to the Poiseuille law, the flow resistance is the fourth power inversely proportional to the radius of the duct, the tear flow resistance through the cavity created by RC-NLDO would be 16–81-fold lower than that created by laser treatment. Second is the lower incidence of false passage formation. No false passage formation is essential for success. In normal conditions, the soft tissue of the membranaceous nasolacrimal duct adheres tightly to its surrounding osseous nasolacrimal duct. Therefore, when there is no false passage formation, the direction of surgical scar contraction is acentric, which pulls the soft tissue to the wall of the osseous nasolacrimal duct, so the gently acentric contraction of the surgical scar would not obstruct the cavity and would not reduce the success rate. If the false passage was formed, the direction of the surgical scar contract would pull itself towards the centre of the reconstructed nasolacrimal duct, which would narrow or block the cavity. The false passage could be avoided in most cases if the electrocauterisation was performed simultaneously while slowly withdrawing the HFL probe after its tip was inserted into the nasal cavity during the RC-NLDO procedure.RC-NLDO has achieved a high success rate that was comparable with EX-DCR. This may be due to the following factors. First a larger lumen was created . In laseThe RC-NLDO technique has several other advantages, such as (1) minimal trauma and no facial cutaneous scar due to the surgery being performed without cutaneous incision and bone excision; (2) less disruption of the lacrimal pump function due to the surgery restoring a physiological tear passage without making a bypass draining system; (3) a simpler, easier and faster (average 12.5 min) procedure similar to conventional lacrimal probing.Usually, the lower canaliculus is ascendant in tear drainage (about 75%). Thus, the superior canaliculus was chosen for the HFL probe to pass through, which protected the function of tear drainage in case of any unexpected damage. The most obstructed points could be penetrated by rotational manipulation along with a slight force during electrocauterisation. In the procedure, the obstructed tissue was cauterised to an eschar crust tube tightly adhering to the wall of the reopened cavity, and became a transitorily sustaining membrane to the wall of the reconstructed nasolacrimal duct. Additionally, while the HFL probe is withdrawn, the rotational manipulation could keep the eschar crust tube intact and minimise the risk of postoperative bleeding, inflammation and synechia.The RC-NLDO is also a choice of treatment for patients who suffer from a lacrimal sac mucocoele, obstructed lacrimal duct with atrophic rhinitis or small lacrimal sac. The RC-NLDO is suitable for patients who failed to respond to a previous EX-DCR.RC-NLDO is not suitable for treating an obstructed osseous nasolacrimal duct which could be treated with EX-DCR. Contraindications for RC-NLDO also include acute dacryocystitis, suspicion of malignancy and patients suffering from severe hypertension or severe cardiac disease .In conclusion, a simple and evolutional approach RC-NLDO has been evaluated by long-term follow-up in a large patient population and animal pathological examination, and the findings demonstrated that this new approach has been proven to recanalise the nasolacrimal duct obstructions. When compared with EX-DCR, RC-NLDO is a new option for treating NLDO and chronic dacryocystitis with a similar or better clinical success rate. The advantages include its efficacy, minimal invasion, safety and simplicity. RC-NLDO is also an optimal choice for recurrent patients who failed to respond to EX-DCR and for patients with small lacrimal sacs or atrophic rhinitis who are not suitable for EX-DCR.


The Centers for Medicare and Medicaid Services (CMS) has implemented the CMS-Hierarchical Condition Category (CMS-HCC) model to risk adjust Medicare capitation payments. This study intends to assess the performance of the CMS-HCC risk adjustment method and to compare it to the Charlson and Elixhauser comorbidity measures in predicting in-hospital and six-month mortality in Medicare beneficiaries.st, 2006 and June 30th, 2006. Additionally, four disease-specific samples consisting of subgroups of patients with principal diagnoses of congestive heart failure (CHF), stroke, diabetes mellitus (DM), and acute myocardial infarction (AMI) were also selected. Four analytic files were generated for each sample by extracting inpatient and/or outpatient claims for each patient. Logistic regressions were used to compare the methods. Model performance was assessed using the c-statistic, the Akaike's information criterion (AIC), the Bayesian information criterion (BIC) and their 95% confidence intervals estimated using bootstrapping.The study used the 2005-2006 Chronic Condition Data Warehouse (CCW) 5% Medicare files. The primary study sample included all community-dwelling fee-for-service Medicare beneficiaries with a hospital admission between January 1The CMS-HCC had statistically significant higher c-statistic and lower AIC and BIC values than the Charlson and Elixhauser methods in predicting in-hospital and six-month mortality across all samples in analytic files that included claims from the index hospitalization. Exclusion of claims for the index hospitalization generally led to drops in model performance across all methods with the highest drops for the CMS-HCC method. However, the CMS-HCC still performed as well or better than the other two methods.The CMS-HCC method demonstrated better performance relative to the Charlson and Elixhauser methods in predicting in-hospital and six-month mortality. The CMS-HCC model is preferred over the Charlson and Elixhauser methods if information about the patient's diagnoses prior to the index hospitalization is available and used to code the risk adjusters. However, caution should be exercised in studies evaluating inpatient processes of care and where data on pre-index admission diagnoses are unavailable. Randomized controlled trials are not practically feasible in all situations given time, costs and/or ethical considerations. Observational studies using administrative claims data are being increasingly used in an attempt to address gaps in evidence where randomized trials are unavailable. However, the validity of the results from such secondary database analyses often face the threat of potential confounding associated with the differences in the baseline health status between patients. Risk adjustment methods are commonly used to address this threat with the most frequently used methods being the Charlson  and the The Centers for Medicare and Medicaid Services (CMS) has implemented a risk adjustment system, the CMS hierarchical condition categories (CMS-HCC) model to adjust capitation payments made to private plans in Medicare . It "useThis study attempts to assess the performance of the CMS-HCC as a risk adjustment method in predicting mortality and to compare it with the Charlson and Elixhauser methods. We compare these three risk adjustment methods among all Medicare beneficiaries with a hospital admission and disease-specific samples consisting of patients with principal diagnoses of congestive heart failure (CHF), stroke, diabetes mellitus (DM), and acute myocardial infarction (AMI).This study used data from the 2005 and 2006 Chronic Condition Data Warehouse (CCW) 5% Medicare files. Specifically, we used personal summary, inpatient, and outpatient files for a 5 percent random sample of Medicare beneficiaries. The inpatient file included one record per hospitalization summarizing all services rendered to a beneficiary from the time of hospital admission through discharge. The outpatient files included claims submitted by both institutional outpatient providers  and carrier claims submitted by non-institutional outpatient providers (e.g. physicians). We also used the personal summary file which captures demographic, enrollment, and administrative information on beneficiaries.st, 2006 and June 30th, 2006. Additionally, four disease-specific samples consisting of subgroups of patients who had a hospitalization with a primary diagnosis of (1) stroke ; (2) DM (ICD-9-CM: '250'); (3) AMI (ICD-9-CM: '410'); or (4) CHF  during the six-month observation period between January 1st , 2006 and June 30th, 2006 were also selected. We selected the four conditions because they provide a mix of acute and chronic conditions that are common reasons for hospital admissions. Approximately 47% of all Medicare patients in the 5% CCW impatient file were discharged from hospitals during 2006 with at least one diagnosis of the four conditions. The first hospitalization during the six-month observation period was identified as the index hospitalization. The admission date of the index hospitalization was identified as the index date. Additional inclusion criteria for the study samples were fee-for-service Medicare Part A and B coverage for at least 12-month prior to the index admission and Medicare enrollment up to six months after the index date or death, whichever came first.We evaluated the three risk adjustment methods in five samples. The primary study sample included all community-dwelling fee-for-service Medicare beneficiaries with a hospital admission between January 1The outcomes of interest were in-hospital all-cause mortality and six-month all-cause mortality. In-hospital mortality was defined as death during the index hospitalization stay. Six-month mortality was defined as death within 180 days of the index hospital admission. In-hospital mortality was also counted as part of the six-month mortality outcome. The vital status and date of death for each beneficiary was identified using the CCW personal summary file.The Charlson comorbidity index was originally derived to classify comorbidities that might alter the risk of mortality in individuals in longitudinal studies . NineteeThe Elixhauser comorbidities method derives a comprehensive set of 30 comorbidities which were associated with increases in hospital length of stay, hospital charges, and in-hospital mortality . The EliThe CMS-HCC model aggregates all ICD-9-CM codes into 189 condition categories which describe a broad set of similar diseases organized into organ systems . ConditiLogistic regressions estimating mortality were used to compare the three risk adjustment methods, namely the Charlson, Elixhauser, and CMS-HCC method. A summary score version and clinical condition indicators version was created for each of these risk adjustment methods, thus resulting in a comparison of six risk-adjustment approaches. Programs to generate the CMS-HCC, Elixhauser, and Charlson risk scores were downloaded from the websites of CMS , Agency Traditionally, only inpatient files were used to code Charlson and Elixhauser comorbidities ,2,5. HowClinical diagnoses (e.g. comorbidities) under the three risk adjustment methods were identified using the ICD-9-CM diagnosis codes on the claims included in each of the four analytic files for the five samples. Summary scores and binary indicators of the clinical diagnoses were created to allow comparison of the six risk adjustment approaches in predicting the two outcomes, in-hospital mortality and six-month mortality. A total of 240 logistic regression models were estimated [5 samples*4 analytic files*6 risk adjustments methods (3 summary score and 3 separate indicators) *2 outcomes]. Each model included variables on the patient's age (as a linear and quadratic term) and gender.Model performance was assessed using the c-statistic, the Akaike information criterion (AIC) , and thest 2006 and June 30th 2006. The mean age of the sample was 78 years and 40% were males. The inpatient mortality rate was 3% and six-month mortality rate was 14%. Across the four disease-specific samples a total of 2,339 DM, 9,828 stroke, 5,749 AMI, and 11,287 CHF patients were included in our study. The mean age of the patients ranged from 73.3 (DM) to 80.1 years (CHF) and the percentage of males ranged from 40.2% (stroke) to 49.6% (DM). Inpatient mortality was the highest for AMI (8.5%), followed by stroke (5.5%), CHF (3.6%), and DM (1.7%). Six-month mortality rates were similar and highest in CHF (23.3%) and AMI (23.1%) patients followed by stroke (18.1%) and DM (14.4%) patients.The primary sample included a total of 170,342 patients with a hospital admission between January 1Table Table The results for the models predicting risk of in-patient and six-month mortality showed that the CMS-HCC method had substantially higher discrimination than the other two risk adjustment methods when we included the index admission claim in coding the risk adjusters. This was done in the first (IPindex), second (IPpre12mo+index) and third analytic (IP+OPpre12mo+index) files which contained the index admission. When we only used the pre-index inpatient and outpatient claims  to code risk adjusters, the CMS-HCC generally still had better discrimination than the other two methods overall. However, the difference was much smaller. In models including the individual diagnosis indicators of each risk adjustment method, the Elixhauser method generally outperformed the Charlson method in both in-patient mortality and six-month mortality models with higher c-statistic values. Because our objective was to compare the CMS-HCC method to the Elixhauser and Charlson method, we did not conduct statistical tests to compare the Elixhauser and the Charlson methods against each other.Table The results of the AIC and BIC were consistent with those based on the c-statistic values (Data not shown. Results are available from the authors upon request). Overall, the CMS-HCC models performed better  than the Charlson and Elixhauser models in the analytic files across the five samples. However, some differences were not statistically significant. After excluding claims on the index date from all inpatient and outpatient claims within 12 months prior to the index admission, there were generally increases in the AICs and BICs.Several studies have been conducted to compare the performance of the two most common risk adjustment methods, the Charlson and the Elixhauser methods ,15,18,19There are several possible explanations for the superior performance of the CMS-HCC method. First, the CMS-HCC risk adjustment method captures more conditions than the Charlson and Elixhauser methods. The CMS-HCC aggregates 189 condition categories into 70 categories , while tInpatient mortality rates are often used to measure quality of care inside hospitals . Risk adIn addition, the predictive power of the CMS-HCC models predicting in-hospital mortality decreased when additional diagnostic information from inpatient and outpatient claims in the 12-month prior to admission was included. This is in contrast to the Charlson and Elixhauser models whose performance in predicting in-hospital mortality increased with the inclusion of additional information. This difference may occur due to the fact that the clinical complications during the index hospitalization included only in the CMS-HCC model play an important role in its predictive performance for in-hospital mortality and adding prior diagnostic information dilutes its prediction power Table .The Charlson and Elixhauser methods were originally designed to serve as risk adjusters using only inpatient or hospital discharge data. Health services researchers have been increasingly using both inpatient and outpatient data for coding these two risk adjusters given the wide availability of longitudinal administrative claims datasets. However, these data also make it possible to exclude diagnoses codes related to complications due to inpatient processes of care by only including diagnoses from inpatient and outpatient claims identified before the index hospitalization date . In this case, our results indicate that the CMS-HCC is more favorable than the other two methods because it captures more comprehensive diagnosis information than the Charlson and Elixhauser methods and complications due to inpatient processes of care are not an issue. However, one should be careful when using CMS-HCC without removing diagnoses from index hospitalization particularly in studies evaluating inpatient processes of care.There are several limitations of our study that should be addressed. First, some of our models using individual diagnosis indicators failed to converge. This may be due to our small sample sizes for some conditions which limited our ability to conduct a full comparison across all conditions and analytic files for the individual diagnosis indicator models across the three methods. However, all models converged for our primary sample of all patients with hospital admissions and the results were consistent with those from the models that converged in the disease-specific samples. Second, we limited our assessment of the outcomes to in-hospital and six-month mortality. Model performance may be different when examining a longer time horizon for mortality as well as for other health outcomes. Finally, our evaluation of the three risk-adjustment methods was conducted using samples of Medicare beneficiaries. Since the CMS-HCC risk score was originally developed and calibrated using data on Medicare beneficiaries whereas Charlson and Elixhauser methods were not, it is possible that our study results were more favorable for the CMS-HCC. However, one might argue that the CMS-HCC method was developed to predict Medicare expenditures and not mortality unlike the other two methods. Nevertheless, future evaluations of these risk adjustment methods in other patient populations are needed.The CMS-HCC risk adjustment models demonstrated better performance relative to the Charlson and Elixhauser models in predicting in-hospital and six-month mortality. The CMS-HCC model is preferred over the Charlson and Elixhauser methods if patient diagnoses prior to the index hospitalization are available and can be used to code these risk adjusters. However, it should be used cautiously in studies focused on evaluating inpatient processes of care when complications due to process of care cannot be identified and excluded based on available data.The data used for the study is not openly available. This study is approved by University of Pennsylvania Institutional Review Board.The authors declare that they have no competing interests.PL participated in study design, data analysis and manuscript writing. MK and JD participated in study design and manuscript writing. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/10/245/prepub


The effect of adriamycin (ADM) infusion-rate on cellular ADM concentrations and on clonogenicity of human haematopoietic cells was studied in vivo and in vitro. In patients an ADM dose of 30 mg m-2 was administered as a bolus injection, or as a 4 h or a 24 h infusion. In vitro the effect of ADM on clonogenic cell growth was determined after exposure during 5 min, 2 h and 24 h of human bone marrow cells to increasing ADM concentrations. ADM showed rapid intracellular accumulation, to levels 100-fold the plasma concentration in vivo or the incubation medium concentration in the in vitro experiments. After a bolus injection or 5 min exposure only approximately 10% of the cellular peak ADM was retained after elimination of the drug from the plasma or the incubation medium. Ninety percent of the ADM was apparently 'loosely' bound. After 4 h and 24 h constant-rate infusions and also after 2 h and 24 h incubations in vitro, the cells accumulated ADM gradually, and the subsequent washing-out of the cellular ADM was substantially less, most of the ADM being 'tightly' bound. Despite these different patterns of uptake and retention after in vivo short- and long-lasting infusion of the same total dose, the 'tightly-bound' cellular ADM concentrations were the same. Moreover, comparable cellular ADM concentrations, retained after efflux of the 'loosely-bound' cellular ADM fraction were equally cytotoxic to normal human clonogenic cells. Short-lasting cellular peak ADM concentrations which occur after a bolus injection or after short exposure to high ADM concentrations are not essential for the cytotoxic effect, in contrast to the retained, 'tightly-bound' cellular ADM levels.


In insect classical conditioning, octopamine  or dopamine has been suggested to mediate reinforcing properties of appetitive or aversive unconditioned stimulus, respectively. However, the roles of octopaminergic and dopaminergic neurons in memory recall have remained unclear.We studied the roles of octopaminergic and dopaminergic neurons in appetitive and aversive memory recall in olfactory and visual conditioning in crickets. We found that pharmacological blockade of octopamine and dopamine receptors impaired aversive memory recall and appetitive memory recall, respectively, thereby suggesting that activation of octopaminergic and dopaminergic neurons and the resulting release of octopamine and dopamine are needed for appetitive and aversive memory recall, respectively. On the basis of this finding, we propose a new model in which it is assumed that two types of synaptic connections are formed by conditioning and are activated during memory recall, one type being connections from neurons representing conditioned stimulus to neurons inducing conditioned response and the other being connections from neurons representing conditioned stimulus to octopaminergic or dopaminergic neurons representing appetitive or aversive unconditioned stimulus, respectively. The former is called 'stimulus-response connection' and the latter is called 'stimulus-stimulus connection' by theorists studying classical conditioning in higher vertebrates. Our model predicts that pharmacological blockade of octopamine or dopamine receptors during the first stage of second-order conditioning does not impair second-order conditioning, because it impairs the formation of the stimulus-response connection but not the stimulus-stimulus connection. The results of our study with a cross-modal second-order conditioning were in full accordance with this prediction.We suggest that insect classical conditioning involves the formation of two kinds of memory traces, which match to stimulus-stimulus connection and stimulus-response connection. This is the first study to suggest that classical conditioning in insects involves, as does classical conditioning in higher vertebrates, the formation of stimulus-stimulus connection and its activation for memory recall, which are often called cognitive processes. Gryllus bimaculatus, we have shown that pharmacological blockade of octopamine (OA) receptors impairs conditioning of olfactory, visual pattern or color stimuli with water reward, whereas blockade of dopamine (DA) receptors specifically impairs conditioning of these stimuli with sodium chloride punishment [Insects are useful models for the study of cellular and molecular mechanisms of learning -4. Therenishment -15.et al. [et al. [et al. [The roles of OA and DA for memory recall in insects, however, have remained controversial. In fruit-flies, disruption of DA-ergic synaptic transmission had no effects on memory recall after aversive olfactory conditioning . In honeet al.  reportedet al.  that the [et al.  argued t [et al. , howeverHere we show that OA and DA receptor antagonists impair appetitive and aversive memory recall, respectively, in olfactory and visual pattern conditioning in crickets. In order to account for this finding, we propose a new model of insect classical conditioning that assumes the involvement of two memory traces, one characterized as 'stimulus-stimulus connection' (S-S connection) and the other characterized as 'stimulus-response connection' (S-R connection) following the terminology of theorists studying classical conditioning in higher vertebrates -23, in cFirst, the effects of epinastine and mianserin, antagonists of insect OA receptors, on the recall of appetitive olfactory memory were studied. The specificity of these drugs for insect neural OA receptors has been demonstrated in biochemical studies ,25. ThreNext, the effects of OA receptor antagonists on the recall of aversive olfactory memory were studied. Three groups of animals were subjected to aversive conditioning trials to associate an odor with 20% sodium chloride solution. One day later, the groups were each injected with saline or saline containing epinastine or mianserin and were subjected to retention tests at 30 min after injection. The groups injected with epinastine and mianserin exhibited significantly increased preferences for the control odor (and thus significantly decreased preference for the punished odor), the levels of preferences being as high as that of the saline-injected control group Figure , right. We also found that fluphenazine, chlorpromazine and spiperone, antagonists of insect DA receptors, impair aversive olfactory memory recall. The specificity of these drugs for insect neural OA receptors has been examined in biochemical studies ,28. FourDA receptor antagonists had no effects on appetitive olfactory memory recall. Four groups of animals were subjected to appetitive conditioning trials. One day later, they were each injected with saline or saline containing fluphenazine, chlorpromazine or spiperone and tested at 30 min after injection. The groups injected with fluphenazine, chlorpromazine and spiperone exhibited no impairment of appetitive memory recall Figure , left. TBecause the effects of DA and OA receptor antagonists on memory recall were studied after two-trial appetitive conditioning and six-trial aversive conditioning, it could be argued that different effects of OA and DA receptor antagonists are ascribed to the difference in the number of trials. However, this is unlikely because we have observed that memory recall after six-trial appetitive conditioning is fully impaired by epinastine (data not shown).The impairment of appetitive memory recall and aversive memory recall by injection of OA and DA receptor antagonists, respectively, indicates that intact synaptic transmission from OA- and DA-ergic neurons is necessary for appetitive memory recall and aversive memory recall, respectively. Impairment of the recall of appetitive memory and aversive memory by blockade of OA and DA receptors, respectively, was not specific to 1-day (long-term) memory but was also found for 1-h (mid-term) memory [see Additional file We next investigated whether the finding that OA and DA receptor antagonists specifically impair the recall of appetitive memory and aversive memory, respectively, is applicable to the recall of visual memory. First, the effects of an OA receptor antagonist on the recall of appetitive visual memory were studied. Three groups of animals were subjected to appetitive conditioning of a visual pattern, using the procedure described previously . One dayNext, the effects of an OA receptor antagonist on the recall of aversive visual memory were studied. Another three groups of animals were subjected to aversive conditioning trials. One day later, the groups were each injected with saline or saline containing epinastine or mianserin and tested at 30 min after injection. The groups injected with epinastine and mianserin exhibited significant levels of memory recall, indicating that an OA receptor antagonist does not impair aversive memory recall Figure , right. Similarly, injection of DA receptor antagonists impaired aversive visual memory recall Figure , right. Injection of DA receptor antagonists had no effect on appetitive visual memory recall. Four groups of animals were subjected to appetitive visual conditioning. One day later, the groups were each injected with saline or saline containing fluphenazine, chlorpromazine or spiperone and tested at 30 min after injection. The groups injected with fluphenazine, chlorpromazine and spiperone exhibited significant levels of memory recall, the levels of which were as high as the level in the saline-injected group Figure , left. TWe previously concluded that OA/DA receptor antagonists impaired olfactory and visual pattern conditioning on the basis of observations that groups of crickets injected with these drugs 30 min before conditioning exhibited no conditioning effects, that is, no significantly changed preferences for the conditioned stimuli, in retention tests performed 1 day (for olfactory conditioning) or 30 min  after completing the conditioning ,14. The It is obvious that our conclusion that an OA antagonist and a DA antagonist impair appetitive and aversive olfactory conditioning is valid, because the effects of epinastine (OA receptor antagonist) and fluphenazine (DA receptor antagonist) do not last for 1 day [see Additional files In the case of visual pattern conditioning, however, our conclusion can be justified only for aversive conditioning. We observed that fluphenazine impaired aversive olfactory conditioning when it was injected 30 min before conditioning but not when it was injected 60 min before conditioning . The retWe performed, therefore, new experiments to re-examine the effect of epinastine on appetitive visual pattern conditioning. A group of animals was injected with epinastine 30 min before appetitive visual pattern conditioning and subjected to retention tests at 1 day after conditioning. The group exhibited no significantly increased preference for the rewarded pattern  is formed during conditioning. We evaluated this assumption by using a second-order conditioning procedure. Second-order conditioning is a technique for testing whether a stimulus can acquire the reinforcing property of a US by conditioning with the US and the stimulus can support new conditioning thereafter. In actual experiment, a stimulus (CS1) is paired with a US and then a new stimulus (CS2) is paired with the CS1 Figure . In our Second-order conditioning has been reported in honey bees ,30 and fWe then proceeded to the evaluation of our model by using the second-order conditioning procedure. First, we studied the effects of injection of an OA receptor antagonist before the initial stage of appetitive second-order conditioning. One group of animals was injected with saline containing epinastine, and 30 min later the group was subjected to the first appetitive conditioning stage in which CS1 was paired with an appetitive US Figure . The secIn contrast, epinastine impaired appetitive second-order conditioning when it was injected before the second conditioning stage. Two groups of animals were subjected to the first conditioning stage and each group was injected with saline or saline containing epinastine 30 min later. They were subjected to the second conditioning stage 30 min after the injection. The test was performed 1 day after completion of the second conditioning stage, assuring that epinastine had been fully metabolized at that time. Here, the time schedule was slightly changed from that shown in Figures Epinastine also impaired the recall of memory formed by appetitive second-order conditioning. Two groups of animals were subjected to appetitive second-order conditioning and each group was injected with saline or saline containing epinastine 30 min later. The final test was performed 30 min after the injection. The control group injected with saline before the test exhibited a significantly increased preference for the CS2. In contrast, the group injected with epinastine before the final test exhibited no significantly increased preference for the CS2 Figure . The impNext, we studied the effects of injection of a DA receptor antagonist before the initial stage of aversive second-order conditioning. A group of animals was injected with fluphenazine, and 30 min later the group was subjected to the first aversive conditioning stage to associate CS1 with aversive US. One day later, the group was subjected to the second CS2-CS1 pairing stage, and the final test was performed 1 h after the second conditioning stage. The group injected with fluphenazine before the first conditioning stage exhibited a significantly increased preference for the control pattern (and thus significantly decreased preference for the CS2) Figure , thus inIn contrast, fluphenazine impaired aversive second-order conditioning when it was injected before the second conditioning stage. Two groups were subjected to the first conditioning stage and each group was injected with saline or saline containing fluphenazine 30 min later. At 30 min after injection, they were subjected to the second conditioning stage. The final test was performed 1 day after completion of the second conditioning stage. We showed in another group of animals that aversive second-order conditioning could be achieved with this time schedule Figure , intact.P = 0.098), probably because aversive second-order conditioning trials yielded only a relatively low level of conditioning effect. On account of the imperfectness of data, the results are in good accordance with our model. It should be noted that alternative models in which different sets of OA/DA neurons participate in reinforcement and memory recall do not account for our finding, since these models predict an impairment of second-order conditioning when OA/DA receptors are blocked at any stage of conditioning.Fluphenazine also impaired the recall of memory formed by aversive second-order conditioning. Two groups were subjected to aversive second-order conditioning trials. The groups were injected with saline or saline containing fluphenazine at 30 min after completion of the conditioning trials and tested at 30 min after injection. The control group injected with saline before the test exhibited a significantly increased preference for the control pattern Figure . In contWe found, at first, that epinastine and mianserin, OA receptor antagonists, impair appetitive memory recall without affecting aversive memory recall, whereas fluphenazine, chlorpromazine, and spiperone, DA receptor antagonists, impair aversive memory recall without affecting appetitive memory recall, in olfactory and visual pattern learning in crickets. Although the specificity of some of antagonists used in this study is not perfect, as we have discussed previously , we obseIn order to account for this finding, we proposed a model with the assumptions that (1) two synaptic connections, that is, synaptic connection from neurons representing CS to OA/DA neurons representing US  and et al. [et al. [Our finding that OA-ergic signaling and DA-ergic signaling are needed for appetitive and aversive memory recall, respectively, is in accordance with some previous findings but not with others. In honey bees, Hammer  reportedet al.  suggeste [et al.  argued t [et al. , but it Results of other studies, however, are inconsistent with our findings. A study using transgenic fruit-flies has suggested that intact DA-ergic synaptic transmission is not needed for memory recall after pairing olfactory CS with electric shock US . In honeBased on our findings in crickets and those reported in other species of insects, we propose that activations of OA/DA neurons are needed for memory recall in some forms of classical conditioning in insects but not in other forms of classical conditioning. Such diversity is noted in classical conditioning in higher vertebrates as is discussed below. The critical factors for the requirement of OA/DA-ergic signaling in appetitive/aversive memory recall in insect classical conditioning should be determined.Second-order conditioning has been reported in honey bees ,30 and fAcheta domesticus, the mushroom body has been suggested to participate in olfactory learning [An important future subject is to determine the sites of convergence of CS and US for olfactory and visual pattern learning in crickets. In fruit-flies ,32 and hlearning . In fruilearning . Thus, tlearning  and studIn higher vertebrates, two theories to account for classical conditioning have been proposed. In one theory, classical conditioning is viewed as the formation of a new reflex pathway for the CS to evoke a CR, as a result of pairing of the CS with a US (S-R theory -23). AccWe conclude that activation of OA- and DA-ergic neurons is necessary for appetitive and aversive memory recall, respectively, in crickets. We also suggest that the S-S or cognitive account of classical conditioning proposed for higher vertebrates is applicable to insects. Because the brain of insects, which we refers to as 'microbrain' ,39, consG. bimaculatus, at 1 week after the imaginal molt were used. Three days before the start of the experiment, animals were deprived of drinking water to enhance their motivation to search for water.Adult male crickets, We used classical conditioning and operant testing procedures described previously ,14,40,41The methods used for the test were described previously . All groThe procedure for visual conditioning was described previously . A blackThe procedure for the visual pattern preference test was described previously . All groFor achieving second-order conditioning, groups of animals were subjected to CS1-US pairing trials and then CS2-CS1 pairing trials  conditioning were each injected with 3 μl of physiological saline  or salintr/(tr+tnr) × 100, where tr was the time spent exploring the odor or pattern associated with reward and tnr was the time spent exploring the odor or pattern not associated with reward. In aversive conditioning, relative preference was determined using the PI for unpunished odor or pattern, defined as tnp/(tnp+tp) × 100, where tnp was the time spent exploring the odor or pattern not associated with punishment and tp was the time spent exploring the odor or pattern associated with punishment. Wilcoxon's test (WCX test) was used to compare preferences before and after training, and the Mann-Whitney test (M-W test) was used to compare preferences of different groups. We found no significant differences in pre-conditioning odor or visual pattern preferences among different groups of animals .An odor or a pattern was considered to have been visited when the cricket probed it with its mouth or pulpi. The time spent visiting each odor or pattern was measured cumulatively. In appetitive conditioning, relative preference of each animal was determined using the preference index (PI) for rewarded odor or pattern, defined as CS: conditioned stimulus; CR: conditioned response; DA: dopamine; ITI: inter-trial interval; M-U model: Mizunami-Unoki model; M-W test: Mann-Whitney test; OA: octopamine; PI: preference index; P/UP: paired/unpaired; S-R: stimulus-response; S-S: stimulus-stimulus; US: unconditioned stimulus; UR: unconditioned response; WCX test: Wilcoxon's test.MM conceived, designed and supervised the study and wrote the manuscript. MM and SU conceived the model. SU, YM, DH and AH carried out the experiments. YM supervised the experiments. All authors participated in the discussion and approved the final manuscript.An octopamine or dopamine receptor antagonist does not impair maintenance of appetitive or aversive 1-day olfactory memory, respectively. Four groups of animals were subjected to two-trial appetitive or six-trial aversive olfactory conditioning (A) or eight-trial appetitive or twelve-trial aversive visual conditioning (B). At 1 day after conditioning, they were each injected with 3 μl of saline containing 1 μM epinastine or 500 μM fluphenazine at 1 day before the final test. Preference indexes for rewarded odor (in the case of appetitive conditioning) or unpunished control odor (in the case of aversive conditioning) before (white bars) and after conditioning (black bars) are shown with means + SEM. The number of animals is shown at each data point. The results of statistical comparison before and after conditioning (Wilcoxon's test) are shown as asterisks (** P < 0.01). All groups exhibited significant levels of conditioning effects at 1 day after drug injection, at which time the drug had been fully metabolized, thus indicating that injection of epinastine or fluphenazine does not impair maintenance of appetitive or aversive memory, respectively.Click here for fileAn octopamine or dopamine receptor antagonist impairs recall, but not maintenance, of appetitive or aversive 1-hour olfactory memory, respectively. (A) Six groups of animals were each subjected to two-trial appetitive or six-trial aversive olfactory conditioning. At 30 min after conditioning, they were each injected with 3 μl of saline or saline containing 1 μM epinastine or 500 μM fluphenazine. Their odor preferences were tested at 30 min after injection. (B) Four groups of animals were each subjected to two-trial appetitive or six-trial aversive olfactory conditioning. At 30 min after conditioning, they were each injected with 3 μl of saline or saline containing 1 μM epinastine or 500 μM fluphenazine. Their odor preferences were tested at 1 day after injection. Preference indexes for rewarded odor (in the case of appetitive conditioning) or unpunished control odor (in the case of aversive conditioning) before (white bars) and after conditioning (black bars) are shown with means + SEM. The results of statistical comparison before and after conditioning (Wilcoxon's test) are shown as asterisks . Epinastine fully impaired appetitive 1-h memory recall, but it had no effect on aversive 1-h memory recall. In contrast, fluphenazine fully impaired aversive 1-h memory recall, but it had no effect on appetitive 1-h memory recall. Injection of these drugs did not impair maintenance of memory, because no impairment of memory was observed when the final test was performed at 1 day after injection, at which time the drug had been fully metabolized.Click here for fileAn octopamine or dopamine receptor antagonist does not impair maintenance of memory after second-order conditioning. One group of animals was subjected to appetitive second-order conditioning with four first-stage trials and six second-stage trials, and 30 min later the group was injected with 3 μl of saline containing 1 μM epinastine. The final test was performed at 1 day after injection. The other group of animals was subjected to aversive second-order conditioning with six first-stage trials and eight second-stage trials, and 30 min later the group was injected with 3 μl of saline containing 500 μM fluphenazine. The final test was performed at 1 day after injection. Preference indexes for rewarded pattern (in the case of appetitive conditioning) or unpunished control pattern (in the case of aversive conditioning) before (white bars) and after conditioning (black bars) are shown with means + SEM. The results of statistical comparison before and after conditioning (Wilcoxon's test) are shown as asterisks . Both groups exhibited significant levels of second-order conditioning effects, indicating that injection of these drugs did not impair memory maintenance.Click here for file