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


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


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


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


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


Coccidioides in Southwest US and Central America. Primary pulmonary infection is initiated by inhalation of air-borne arthroconidia. Since, lung is the first organ that encounters arthroconidia, different components of the pulmonary innate immune system may be involved in the regulation of host defense. Pulmonary surfactant proteins (SP)-A and SP-D have been recognized to play an important role in binding and phagocytosis of various microorganisms, but their roles in Coccidioides infection are not known.Coccidioidomycosis or Valley Fever is caused by Coccidioides posadasii infection, and binding of SP-A and SP-D to Coccidioidal antigens. Mice were challenged intranasally with a lethal dose of C. posadasii (n = 30 arthroconidia) and bronchoalveolar lavage fluid (BALF) samples were collected on day 10, post infection. In another group of animals, mice were immunized with protective formalin killed spherule (FKS) vaccine prior to infection. The concentrations of BALF SP-A, SP-D, total phospholipid were measured using enzyme linked immunosorbent assay and biochemical assays.In this study, we studied the changes in amounts of pulmonary SP-A, SP-D and phospholipid in murine model of C. posadasii infected mice, the concentrations of total phospholipid, SP-A and SP-D were 17 % , 38 %  and 4 %  of those in lavage fluid samples of non-infected control mice, respectively. However, the concentrations of SP-A and SP-D remained unchanged in BALF samples of C. posadasii protected mice after immunization with FKS vaccine. Also, we found that both SP-A and SP-D bind to Coccidiodal antigens.We found that in lavage fluid samples of C. posadasii infection perturbs the pulmonary SP-A, SP-D, and phospholipids, potentially enabling the disease progression and promoting fungal dissemination.Our results suggest that the  Coccidioides immitis or posadasii [C. posadasii or C. immitis, are the most virulent fungal pathogens enlisted in Select Agent list and pose a risk for bioterrorism [Coccidioides [Coccidioides.Coccidioidomycosis or Valley Fever is a fungal disease caused by the biphasic, highly virulent, soil-fungus osadasii . It is eosadasii . C. posaerrorism . The prierrorism -4. Previidioides ,6. HowevStreptococci [Pseudomonas aeruginosa [At alveolar level in the lung, the innate immune system is composed of many cell types and chemical mediators, including surfactant. The pulmonary surfactant is a complex mixture of lipids 88–90%) and proteins 10–12%), synthesized by type II epithelial cells and Clara cells. It lines the alveoli, and helps in maintaining normal lung function . Among f8–90% and–12%, synptococci , Pseudomruginosa . Also, iruginosa . Both SPruginosa . Recent ruginosa . HoweverC. posadasii and C. posadasii protected mice after immunization with protective formalin killed spherule (FKS) vaccine, and binding of pulmonary collectins to Coccidioidal antigens.This study focuses on analyzing the changes in amounts of the SP-A and SP-D in the bronchoalveolar lavage fluid (BALF) samples from mice infected with lethal dose of C. posadasii infection. The BALB/c mice were used to study the changes in pulmonary surfactant after intranasal challenge with C. posadasii. And, the C57BL6 mice were used to study the changes in pulmonary surfactant after vaccination with protective FKS vaccine. Mice were housed in Biosafety Level-3 animal facility at UTHSCSA and provided with food and water ad libitum. All experimental animal care and treatment protocols were reviewed and approved by Institutional Animal Care and Use Committee.BALB/c and C57BL6 mice  from Jackson Laboratory  were used in this study. Both mouse strains are susceptible to C. immitis (now posadasii) Silveira strain, cultured on 1 % glucose-0.5 % yeast extract agar (GYE), was used for infecting the mice [C. posadasii were carried out in Biosafety Level-3 facility at UTHSCSA.C. posadasii arthroconidia) suspended in endotoxin-free 0.15 M NaCl  using sterile pyrogen-free microtip. Mice were held in an upright position for 1–2 min to resume normal breathing after injection. Control mice were challenged with equal volume of endotoxin-free 0.15 M NaCl.Mice were anaesthetized after intramuscular injection of ketamine- xylazine (75 μg/g body weight ketamine and 10 μg/g body weight xylazine) and were then challenged intranasally with a lethal dose of arthroconidia arthroconidia were used to prepare FKS as described earlier [2 incubator. The spherules were collected from the harvested culture, washed in endotoxin-free water and killed with 1 % formalin. FKS preparation was checked for sterility and lyophilized. C57BL6 female mice (age 6 weeks old) were immunized intramuscularly twice and subcutaneously once at one week interval with FKS (0.7 mg/dose each time). The mice in FKS immunized, infected group were then challenged intranasally with 30 C. posadasii arthroconidia, 15 days after last immunization, as described above. earlier . BrieflyMice were anaesthetized as mentioned above, prior to sacrifice on day 10, post intranasal infection. This standard procedure was used for intranasal injection since it does not cause respiratory depression during anaesthesia. The lung and spleen tissues were collected in sterile 0.15 M NaCl for studying fungal load.The fungal burden was studied by plating ten fold dilutions of lung and spleen homogenates in 0.15 M saline on Mycosel agar plates  and incubating for 72 h at 30°C. The cfu counts were recorded and normalized with organ weight.At the time of necropsy, we collected BALF by injecting 1 ml endotoxin-free 0.15 M NaCl solution  three times, via an angiocatheter  placed in the trachea. The volume of the input solution was kept constant  and approximately, 90–95 % of the solution was recovered consistently. The BALF was centrifuged at 500 rpm for 10 min at 4°C to remove cells. The cell free BALF supernatant was filtered through 0.2 μm syringe filters  and stored at -80°C for further analysis.The total protein concentration was measured in BALF specimens using micro bicinchonic acid protein assay kit  against bovine serum albumin (BSA) standard protein. The total phospholipid content in lipid extracts of BALF specimens was determined using the method of Stewart, against Dipalmitoyl-phosphatidylcholine  standard solutions ,18. BrieThe BALF and lung tissue homogenate samples  were run on 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) running gel and transferred on nitrocellulose membranes  overnight at 15 mA current. The nonspecific sites on the membrane with transferred proteins were blocked by 15% nonfat milk in Tris-buffered saline containing 0.05% tween 20 (TBST). The membrane was washed and incubated for 1 h with diluted (1:500) primary anti-human SP-A polyclonal antibody raised in rabbit  or anti-mouse SP-D antibody . After washing the membrane with TBST, the membrane was incubated for 1 h with 1:10,000 diluted alkaline phosphatase conjugated anti-rabbit IgG raised in goat . The immunoreactive bands were observed by alkaline phosphatase conjugate system . Purified human SP-A  and recombinant human SP-D  were run with the samples.C. posadasii mycelia , prepared as a toluene-induced lysate of young nio, TX, ) were al3, pH 9.6. The wells were washed three times with deionized water, and nonspecific sites were blocked with a buffer containing 0.25% BSA, 0.05% tween 20, 0.17 M boric acid and 0.12 M NaCl, pH 8.5. The wells were washed and incubated for 2 h with rabbit anti-human SP-A or rabbit anti-mouse SP-D antibody. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody  was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent . The reaction was stopped by adding 50 μl of 2 N H2SO4 and read at 450 nm spectrophotometrically. The regression coefficient for a least-square linear fit to the standard curve of SP-A and SP-D was 0.99. The limits of detection for SP-A and SP-D were 2 ng/ml.The concentrations of SP-A and SP-D were measured in BALF samples as described earlier . The ant3 buffer, pH 9.6) or CDN-filtrate  or BSA  at room temperature. The nonspecific binding was blocked with phosphate buffered saline (pH 7.4) containing 0.1% triton-X 100 and 3% nonfat milk (buffer A). The purified human SP-A and recombinant human SP-D diluted in 20 mM Tris (pH 7.4) containing 0.15 M NaCl, 5 mM CaCl2 and 1 mg/ml BSA were then added to the wells and incubated for 3 h at 37°C. The wells were then washed with buffer A and incubated for 1 h at room temperature with diluted (1:1000 in buffer A) anti-SP-A and anti-SP-D antibodies. After washing the wells, the horseradish peroxidase conjugated anti-rabbit IgG antibody  was added. After incubation for 2 h, the wells were washed again and incubated with tetramethylbenzidine substrate reagent . The reaction was stopped by adding 2 N H2SO4 and read at 405 nm spectrophotometrically.A microtiter well based method  was usedThe coating of Coccidioidal antigens (CDN-lysate and CDN-filtrate) to the plates was confirmed using a positive control antibody that recognizes Coccidioidal antigens as described earlier . The alkStatistical analyses of the data (t-test or ANOVA) were done using Prism Software . The p value <0.05 was considered significant.C. posadasii infected mice survived till the day of sacrifice (day 10 post infection). However, the mice were lethargic and lost body weight (Table C. posadasii infected mice.All of the C. posadasii infected mice (58 μg) when compared to non-infected saline injected controls .The mean protein content of BALF samples from infected mice was 788 μg versus 326 μg protein in BALF samples from non-infected saline injected control mice, after 10 days of intranasal infection  .The anti-human-SP-A and anti-mouse SP-D antibodies recognized 34 kDa and 43 kDa monomer bands of SP-A and SP-D in BALF and lung tissue samples Fig . The upp001) Fig . No signC. posadasii were recovered from both lung and spleens of infected animals indicating the presence of active infection  C57BL6 mice when compared to FKS immunized, non-infected mice  versus FKS immunized, non-infected mice (538.4 μg).No significant changes were seen in SP-A or SP-D levels in BALF samples of protected  coated onto microtiter wells Fig . Both SPC. posadasii infected mice, but were intact in lungs of C. posadasii protected mice after immunization with protective FKS vaccine. Furthermore, our results suggest that both SP-A and SP-D bind to Coccidioidal antigens. This is the first study where the amounts of SP-A and SP-D were measured in BALF samples from mice infected with lethal dose of C. posadasii and the binding of pulmonary collectins to Coccidioidal antigens was assessed. Since lung is the first organ of the body that comes into contact with air-borne C. posadasii arthroconidia, we hypothesized that the pulmonary surfactant may play an important role in regulating the immune response against C. posadasii. Among four surfactant proteins, SP-A and SP-D, interact with most of the clinically important fungal pathogens including Pneumocystis carinii [Cryptococcus neoformans [Aspergillus fumigatus [Candida albicans [C. albicans and directly inhibit growth by aggregation of the organism without involvement of macrophage dependent phagocytosis [A. fumigatus conidia and C. neoformans by alveolar macrophages and neutrophils [A. fumigatus [C. posadasii infection versus the unaltered amounts in the lungs of protected mice and binding of collectins to Coccidioidal antigens indicate that pulmonary collectins may be involved in uptake/phagocytosis of C. posadasii by antigen presenting cells and downstream immune regulation.In the present study we found that the levels of pulmonary surfactant collectins were altered in the lungs of  carinii Cryptococoformans , Aspergiumigatus  and Candalbicans . SP-D haocytosis . On the trophils ,29. In sumigatus . The decC. posadasii infected mice  and growth factors are released during infection and inflammation. These cytokines and growth factors affect the synthesis and secretion of pulmonary surfactant by pulmonary epithelial cells [C. posadasii protected mice, but not of SP-A (Fig Besides changes in amounts of SP-A and SP-D, a decrease in the amount of BALF phospholipids was also observed in ce Table . Earlier carinii ,30. To d carinii . Likewisal cells ,33. In tSP-A Fig . We specC. posadasii infected patients with disseminated disease is not very effective [Coccidioides infection in murine model of coccidioidomycosis, but failed in humans [C. posadasii and C. immitis are highly virulent organisms, cause endemic infection, and pose a risk for bioterrorism, there is an urgent need for discovery of improved therapeutic drugs and regimens or preventive vaccines [At present, the treatment of ffective . Treatmen humans . More prvaccines ,35.in vivo administration of artificial or natural surfactant in C. posadasii infected mice may be useful in determining their therapeutic usefulness. We speculate that the findings from our present study would initiate similar studies to understand the role of pulmonary innate immune components in infectious diseases caused by C. posadasii or other virulent respiratory pathogens.In future, clearance experiments after SA designed and co-ordinated the study, performed assays and statistical analysis and drafted the manuscript. JJC assessed lung pathology. DMM prepared and immunized mice with FKS. All authors read and approved the final manuscript.


People are unpredictable. One night you may crave Italian food, but another only Thai will do. One day you might finish a crossword puzzle in record time, and the next not a single clue prompts an answer. Such behavioral variation has been found in laboratory studies, too: a person's ability to find a faint image on a screen varies widely from one viewing to the next. Similarly, when an animal repeatedly receives the same stimulus—for example, a faint image—a neuron in a region of the animal's visual brain might be very active upon one presentation and relatively quiet the next.Across the cerebral cortex—the brain region that integrates the senses and controls voluntary movement—neurons are notorious for their unpredictable behavior. The neurons themselves don't create this noise; when directly stimulated with an electrode multiple times, neurons will give the same response every time. Most neurons, however, receive signals from a host of other neurons. These various signals combine to form a seemingly noisy electrical input, which shows up as fluctuations in the recipient neuron's membrane potential—a difference in electrical charge between the inside and outside of the cell's membrane. Neuron function is intimately tied to the membrane potential, which is usually maintained within a narrow range, called the resting potential. But incoming signals can push the resting potential higher or lower. If the membrane potential rises above a certain threshold, the neuron fires, sending an electrical signal down its length. In this way, the brain relays and processes information.PLoS Biology, Matteo Carandini addresses this longstanding mystery of neuron variability and comes up with a different answer. Carandini simultaneously measured the membrane potentials and firing patterns of individual neurons in the cat visual cortex. He found, surprisingly, that the membrane potentials varied much less than the firing patterns, ruling out noisy inputs as the cause of neurons' noisy outputs. Instead, the neurons amplified noise in the signals they received.Since the 1960s, neuroscientists trying to account for the cortex's variable responses have pointed to noisy inputs from other parts of the brain as the prime suspect. In this issue of Carandini then used a simple model of neuron behavior to explain why this would occur. He started with a tried-and-true approximation of neuron behavior, called the rectification model: a neuron doesn't fire until its membrane potential rises above a threshold, but once it crosses this threshold, its firing rate is correlated with the strength of incoming signals. Then he added the assumption that the neurons receive signals with some randomness. Given these minimal assumptions, Carandini showed that neurons fed a noisy signal will tend to amplify the noise in the signal. Importantly, his model reproduced a well-known phenomenon: as cortical neurons' average firing rate goes up, their firing rate also becomes more variable—that is, they get noisier.Carandini's model also predicted something new: as the firing rate continues to increase, the firing rate should become more consistent and less noisy—which he calls saturation of variability. Carandini's measurements in cats showed neurons actually behave this way, a key validation of his model.It's not clear whether this amplification of variability is something that helps or hampers the brain. Despite being a nuisance to neuroscientists, such fluctuations could be crucial to how the brain functions, Carandini speculates. Without some variability in their cortex, animals would act like cameras or other simple machines that respond the same way each time to a stimulus. It's advantageous for behavior, and hence brains, to be adaptable. But amplifying noise in a signal seems to run counter to relaying and processing the information in the signal. Carandini suggests that what appears as noise in the experiments are signals from other parts of the cortex—that is, noise is in the eye of the beholder. Now that the source of the variability is clear, neuroscientists can study whether it serves a function in the brain.


Lamptey and Wilson discuss the implications of a new study showing that combining treatment with prevention is the best approach to tackling the HIV pandemic. There has been a recent and dramatic rise in global funding for HIV/AIDS, from US$2.1 billion in 2001 to US$6.1 billion in 2004 [The WHO “3 by 5” Initiative: This initiative aims to place 3 million people in low- and middle-income countries on ART by the end of 2005.The US President's Emergency Plan for HIV/AIDS Relief: This initiative aims to treat 2 million HIV-infected people with ART, to prevent 7 million new infections, and to care for 10 million HIV-infected individuals and AIDS orphans in five years (2004–2009).The Global Fund to Fight AIDS, Tuberculosis, and Malaria: In the five years following its inception (2002–2007), the Global Fund aims to provide 1.6 million people with ART and 52 million people with VCT, and to support more than 1 million orphans with medical services, education, and community care.The scale of the proposed increase in the number of patients receiving ART raises numerous questions about the treatment itself. Which drugs will be used? How much will it cost? How will their quality be monitored and assured? How will they be distributed? Who will be eligible? How will the desired level of treatment be sustained? Is there adequate infrastructure and human resources to support the expanded services?The commitment of substantial funding to treatment in resource-poor countries also has implications for the prevention efforts in those same countries. In many Western countries and Brazil (the sources of the majority of the available data on the subject), the impressive drop in mortality due to HIV following increased access to ART is coupled with a disheartening rise in the number of new cases of HIV, as emphasis and funding are shifted from prevention to treatment . CountriOf course, prevention and treatment are not mutually exclusive. Successful prevention efforts mean fewer patients will need the costly drug treatment programs, helping extend the sustainability of ART. In turn, the success of ART in prolonging healthy living helps prevention efforts by reducing the stigma associated with self-education and responsible behaviors.PLoS Medicine, “Integrating HIV Prevention and Treatment: From Slogans to Impact,” Salomon and colleagues use mathematical modeling to assess the epidemiologic impact of treatment and prevention efforts, and to quantify the opportunities and potential risks of large-scale treatment roll-out. Using a variety of different scenarios, they propose methods for establishing the most effective balance between spending on prevention and spending on treatment.In their study in the January 2005 issue of Modeling is a technique used by many scientists, including epidemiologists and statisticians, to create a mathematical equation that can be used to determine which variables affect an outcome of interest, and to what extent. Once the influential variables are determined, a baseline model is established that includes those variables and reflects their relative importance to the outcome. The effect of changing the value of any of these variables, or several of them, can then be tested, and new outcomes projected. HIV modeling is inexact and requires far better data but can nevertheless provide important insights.Salomon and colleagues used mathematical modeling to assess the effect of changing aspects of the HIV/AIDS “equation” on the future course of the HIV/AIDS epidemic. First, a baseline model was created to fit expected HIV/AIDS projections for the year 2020 if there were to be no change in the current epidemiologic trends—no ART scale-up, and no changes in prevention efforts or behavior. Heterosexual contact is the predominant mode of HIV transmission across Africa, and Salomon and colleagues' study modeled the disease only within the heterosexual population. The model was also tailored to take into account epidemiologic, demographic, and sociologic patterns in the eastern, central/western and southern regions of Africa. Using the baseline models tailored to each region, the effects of prevention and treatment efforts were then measured.Two treatment-centered scenarios were tested in which the World Health Organization's “3 by 5” initiative see  was achiBaseline projections in Salomon and colleagues' study showed that without any behavioral change or ART scale-up, the HIV/AIDS prevalence rate would remain relatively stable, but the number of new infections would increase by 52.3 million by 2020. Treatment-centered scenarios reduced the total number of new infections through 2020 by a maximum of 3 million, or 6%, while indicating that the number of AIDS deaths through 2020 would decline by 13%, to 32.4 million. A prevention-centered strategy would provide greater reductions in incidence (36%) and similar mortality reductions by 2020, but more modest mortality benefits over the next five to ten years.The scenarios in which all of these statistics were most improved, however, were those that combined both prevention and treatment efforts. In the scenario in which treatment enhanced prevention, Salomon and colleagues projected 29 million averted infections (55%) and 10 million averted deaths (27%) through the year 2020. However, if a narrow focus on treatment scale-up leads to reduced effectiveness of prevention efforts, the benefits of a combined response would be considerably smaller—9 million averted infections (17%) and 6 million averted deaths (16%) .Combining treatment with effective prevention efforts could reduce the resource needs for treatment dramatically in the long term. In the various scenarios the numbers of people being treated in 2020 ranges from 9.2 million in a treatment-only scenario with mixed effects, to 4.2 million in a combined response with positive treatment–prevention synergies.The authors have demonstrated through mathematical modeling that the integration of treatment and prevention is epidemiologically sound. However, an integrated and comprehensive program  is not oEffective prevention makes treatment more affordable and sustainable. Effective prevention can lead to a substantial reduction in the number of new infections and therefore ultimately will lead to a reduction in the number of people who will need treatment. The reduction of adult HIV/AIDS prevalence in Uganda from 18.5% to 6% over the last several years has reduced the number of those eventually needing treatment by nearly 68% [arly 68% . Unless arly 68% . SalomonSuccessful treatment and care can make prevention more acceptable and effective. Widespread access to treatment could bring millions of people into health-care settings, providing new opportunities for health-care workers to deliver and reinforce HIV prevention messages and interventions [ventions . Improveventions .Prevention can make treatment more accessible. The early establishment of community-based prevention services in rural Ghana was instrumental in reducing the stigma of AIDS and improving the knowledge and attitude of the community prior to the development of ART and VCT services . This process also made it easier for community and implementing agencies to identify and refer patients needing treatment services.Expanded care and prevention activities have synergistic effects. Continued effective treatment, care, and prevention programs will reduce the number of orphans and vulnerable children, reduce mother to child transmission of the virus, and improve the lives of families and the strength of communities.Integration ensures that prevention activities are not neglected. The world has a unique opportunity, as ART services are launched and expanded, to simultaneously bolster prevention efforts [ efforts . Experie efforts . In addiIntegration can provide opportunities to address vulnerable groups more effectively. A commitment to providing large-scale treatment helps to focus attention on communities at greatest risk, particularly in lower prevalence contexts. This provides an opportunity to address the prevention and treatment needs of vulnerable groups more effectively.Treatment resources can help improve infrastructure for prevention and other health services. The training of health providers and improvements in laboratory services, pharmacy, logistics, commodity management, and health information systems can benefit both treatment and prevention services. Further, in many countries, a large number of health-care workers are themselves infected. Treatment can help to preserve the lives and productivity of these critically needed AIDS prevention and treatment workers, as well as those of other health professionals.A long-term decline in AIDS deaths may be preventing new infections. The short-term decline in AIDS deaths is driven by effective care and treatment programs, but a long-term decline may be driven by the prevention of new infections. Integrated and comprehensive strategies are more likely to lead to affordable, sustainable programs.Success requires dramatic expansion of both ART and prevention. Globally, fewer than one in five people at high risk of infection have access to proven HIV prevention interventions [ventions  and lessventions . Unless The results of Salomon and colleagues' model need to be validated. Further operational research is needed to validate the findings of this study.


Imbalance in the health workforce is a major concern in both developed and developing countries. It is a complex issue that encompasses a wide range of possible situations. This paper aims to contribute not only to a better understanding of the issues related to imbalance through a critical review of its definition and nature, but also to the development of an analytical framework. The framework emphasizes the number and types of factors affecting health workforce imbalances, and facilitates the development of policy tools and their assessment. Moreover, to facilitate comparisons between health workforce imbalances, a typology of imbalances is proposed that differentiates between profession/specialty imbalances, geographical imbalances, institutional and services imbalances and gender imbalances. Imbalance in the health workforce is a major challenge for health policy-makers, since human resources – the different kinds of clinical and non-clinical staff who make each individual and public health intervention happen – are the most important of the health system's inputs . ImbalanAlthough imbalance in the health workforce is an important issue for policy-makers, various elements contribute to obscuring policy development. First, many reports of shortages are not borne out by the evidence. Rosenfeld and Moses  show thaThe objective of this paper is to contribute to a better understanding of the issues related to imbalance through a critical review of its definition and nature and the development of an analytical framework.There are various approaches to defining imbalances . From anOne of the key questions regarding imbalances is how long these last: Is the imbalance temporary or permanent? In a competitive labour market, we should expect most imbalances to be resolved over time. Imbalances will tend to disappear faster the greater the reaction speed and also the greater the elasticity of supply (or demand) . This tyIn contrast, a static imbalance occurs because supply does not increase or decrease; market equilibrium is therefore not achieved. For instance, wage adjustments may respond slowly to shifts in demand or supply as a result of institutional and regulatory arrangements, imperfect market competition  and wage-control policies. Another example is physicians' education: because of the length of time required to educate physicians, changes in available supply take a long time to react significantly. Lack of information on the state of the various labour markets can also be a factor in the speed of market adjustment. To make proper labour market decisions, households and firms must be informed of the existing market conditions across markets. They must therefore know what wages are paid and the nature and location of job openings and available workers.Moreover, we should also differentiate between qualitative and quantitative imbalance. In a tight labour market, employers might not find the ideal candidate, but will still recruit someone. Under these conditions, the issue is the quality of job candidates rather than the quantity of people willing and able to do the job . From thTo better understand the role of factors affecting health workforce imbalances and to facilitate the development of policy tools, a conceptual framework is presented in this section.Factors affecting health workforce imbalances are numerous and complex, but focusing on crucial elements should permit insight into the issue of health workforce imbalances. The framework is depicted in Fig. Central to this framework are the demand for and supply of health labour. Also included in the framework is the health care system, and in particular, some of its features that are likely to have an impact on health workforce imbalances. Policies constitute another crucial element of the framework. In effect, health policies but also non-health-oriented policies can have an impact on health workforce imbalances. The framework also incorporates financial, physical and knowledge resources that contribute to model the health workforce.Finally, "global" factors such as economic, sociodemographic, political, geographical and cultural factors are included. These elements contribute directly or indirectly to shaping and transforming the entire society and hence the health workforce.The first element of the framework to be examined is the demand for health personnel. The demand for health personnel can be considered as a derived demand for health services. Accordingly, we should consider factors determining the demand for health services. Personal characteristics – such as health needs, cultural and sociodemographic characteristics – and economic factors play an important role.It has often been proposed that the planning of human resources for health be based solely on estimates of health needs in the population . HoweverSeveral studies have attempted to estimate the impact of economic factors on the demand for health care. In particular in the United States, studies have attempted to estimate price and income elasticities of demand for medical services -13. MeasAnother element influencing the demand for health care is the value of a patient's time, such as travel time and waiting time. Acton  found thOther factors affect the demand for health labour. In particular, some specific features of the health care system and its features, policies, resources and environmental factors do have an impact on the demand for health labour. Their respective role will further discussed later.After reviewing factors affecting the demand for health labour, we shall now turn to those affecting the supply of the health workforce. In particular, we shall consider the following elements: factors affecting the choice for a health professional training/education, participation in the health labour market and migration.The availability of a renewed health workforce, as well as the type of profession and specialty chosen by individuals, is a major concern for health decision-makers. These issues are of particular relevance, especially since the number of younger people, predominantly women, choosing a nursing career is declining in some countries and since in professional training/education, individuals' choices do not always match the absorptive capacity of the market.From an economic perspective, the decision to undertake professional training/education is considered an investment decision. To emphasize the essential similarities of these investments to other kinds of investments, economists refer to them as investment in human capital . Since iBased on the human capital approach, rate of return on education can be estimated. An average rate of return that is high and rising for a given profession will attract more individuals to that profession. On the other hand, a lower and decreasing average rate of return will discourage individuals from choosing that profession. Nowak and Preston , using tBesides the human capital approach, the choice of a profession can also be explained by sociopsychological factors. For instance, individuals may choose a profession because it is highly valued by the society or for family tradition. In the health sector, the satisfaction afforded by caring for people and assisting them to improve their health is an important element used by nursing schools to attract new enrollees. In the light of this approach, the decline in the number of individuals choosing nursing as a career might also be explained by the fact that this profession is now less socially valued than before ,20.The economic theory of the decision to work views the decision as a choice concerning how people spend their time. Individuals face a trade-off between labour and leisure. They decide how much of their time to spend working for pay or participating in leisure activities, the latter being activities that are not work-related.An issue that has drawn a lot of attention recently is the impact of wage increases on labour participation, in particular for nurses. In the short term, higher wages can have at least two effects on the labour supply of current qualified nurses: first, qualified nurses who are working in other occupations may return to nursing activities; second, nurses now in practice may respond by working more hours. In the long run, higher wages in nursing relative to other occupations make nursing an attractive profession and will draw more people into nurse training programmes.In their literature review of wage elasticity of nursing labour supply, Antonazzo et al.  and ChihMoreover, hospitals are also using a variety of strategies to recruit new staff. A survey of hospitals in the United States shows that richer benefits, such as health insurance and vacation time, are the most common incentives used. In addition, hospitals may offer other recruitment and retention benefits, such as tuition reimbursement, flexible hours and signing bonuses based on experience or length of commitment . Many coEconomic factors also play a role in physician's participation in the labour market, as demonstrated by the impact of cost-containment policies in Canada, where most provincial governments have implemented an assortment of controls of health care expenses. Threshold reductions were introduced, so that fees payable to individual physicians were reduced as billing exceeded an agreed threshold. As a consequence, physicians who had billed at the threshold level chose to take leaves of absence rather than receive a level of reimbursement they considered inadequate .When health personnel choose an alternative or additional occupation, this is likely to have consequences on health labour supply. In developing countries, and particularly in Africa, attempts to reform the health care sector have frequently failed to respond to the aspirations of staff concerning remuneration and working conditions. Salaries are often inadequate and may be paid late, and health workers try to solve their financial problems in a variety of ways . PrivateParker and Rickam  examinedThe relative importance of wage is also emphasized by studies investigating job satisfaction. There is support in the empirical literature for the existence of job dissatisfaction among nurses, and the link between job dissatisfaction and job exit ,31. In tMigration of health personnel can have a serious impact on the supply of human resources in health, because it may exacerbate health personnel imbalances in "sending" countries. It is suggested that migration is an "individual, spontaneous and voluntary act" that is motivated by the perceived net gain of migrating – that is, the gain will offset the tangible and intangible costs of moving . DecisioThe reality for many health workers in developing countries is to be underpaid, poorly motivated and increasingly dissatisfied and sceptical . The relAs the health workforce is part of the health care system, we shall also consider features of the health care system that are likely to have an impact on the demand and supply of health labour. In particular, we shall examine market failures, the diversity of stakeholders, the supply-demand adjustment time lag and hospitals' potential monopsony power.From an economic perspective, the health care market is characterized by market failures – that is, the assumptions for perfect competition are violated. From a societal perspective, in the presence of market failures such as externalities – imperfect knowledge, asymmetry of information and uncertainty – market mechanisms lead to a non-optimal demand and/or supply in health services. In other words, shortages and surpluses are likely to result from the health care market.Most markets are characterized by market failures, but what is unique to the health services market is the extent of these market failures . GovernmThe health care system is characterized by a wide range of institutional stakeholders involved in shaping human resources for health, all of whom may have different objectives ,42. The In the case of Mozambique, whereas the policy of employing national professionals by cooperation agencies has met with warm support from national cadres, its effect on the health sector is problematic . The proMoreover, adjustments between the demand and supply for health personnel may take a long time. In the health care field; the time lag between education and practising may be quite substantial. To obtain licensure to practise medicine requires lengthy education and training, and the long lag time between a changed student intake and a change in supply has been noted : supply monopsony. One example is the potential monopsony power of hospitals in hiring nurses or the ministry of health in hiring the health workforce. The amount of labour demanded will influence the price the monopsonist must pay for it. In contrast to the situation in a competitive market, the monopsony is a price maker, not a price taker. Monopsony results in lower wages and lower employment of nurses compared to a competitive market.A single entity that is the sole purchaser of labour is a A number of studies have tested whether or not hospitals possess monopsony power with respect to nurses, and the results are contradictory. Sullivan  and StaiIn contrast, providers' power may enable the latter to restrict the supply of human resources for health. Seldon, Jung and Cavazos  suggest The type off regulation associated with a profession plays an important role regarding the supply of members of a profession. Regulation has, by tradition, been achieved through a combination of direct government regulation and, to a large extent, through rules adopted by professional associations, whose self-regulatory powers enable them to establish both entry requirements and rules regarding professional conduct .Such barriers to entry exist in particular for doctors, but also in other health professions, such as dentistry. Some argue that these barriers constitute a means to limit entry into the profession, and hence maintain high incomes. Muzondo and Pazderka  establisHealth and non-health policies contribute to shaping the health care system and have an influence on the demand and supply of health labour.Health policy can be defined as a formal statement or procedure within institutions (notably government) that defines priorities and the parameters for action in response to health needs, available resources and other political pressures. Health policy is often enacted through legislation or other forms of rule-making that create regulations and incentives for providing health services and programmes and access to them. For instance, the decision to introduce or expand health insurance coverage is likely to have an impact on the demand for health services. This is illustrated by the RAND Health Insurance Experiment, a controlled experiment that increased knowledge about the effect of different insurance copayments on use of medical services. Insurance copayments ranged from zero to 95%. The RAND study concluded that as the co-insurance rose, overall use and expenditure fell for adults and children combined .Non-health policies reflect state interventions in areas such as employment, education and regional development that contribute to shaping the health workforce. These policies do not directly address health issues, but have an indirect impact on such issues. In France, a controversial new regulation was introduced that reduced the workweek to a maximum of 35 hours in an attempt both to create hundreds of thousands of new jobs and to achieve greater flexibility in the labour force. Unions responded by demanding the creation of more posts in public hospitals.Financial, physical and knowledge resources are crucial to any type of health care workforce. The level of resources attributed to the health care system, and how these resources are used, will have a significant impact on health workforce issues.In terms of financial resources, human resources account for a high proportion of national budgets assigned to the health sector . Health Physical resources include human resources within the health sector and other sectors; buildings and engineering services such as sanitation, water and heating systems for community use and for the use of medical care institutions; and equipment and supplies. Finally, the health workforce is also constrained by its human capital. This human capital can be associated with the qualification and education of the health workforce. Education of the health workforce is the systematic instruction, schooling or training given in preparation for work.Economic, sociodemographic, cultural, and geographical factors contribute to shaping and transforming society and hence have a direct or indirect impact on health workforce issues.From an economic perspective, for instance, there is evidence of a correlation between the level of economic development of a country and its level of human resources for health. Countries with higher GDP per capita are said to spend more on health care than countries with lower income, as demonstrated by cross-sectional studies,  and hencMoreover, both the demand and supply are likely to be affected by sociodemographic elements such as the age distribution of the population. On the demand side, the ageing of the population is giving rise to an increase in the demand for health services and health personnel, especially nurses for home care.On the supply side, the ageing of the health workforce, and in particular of nurses, has serious implications for the future of the nursing labour market. For example, the Institute of Medicine noted that older registered nurses have a reduced capacity to perform certain tasks . It was Geographical and cultural factors also play a role in determining the demand and supply of human resources. Geographical characteristics affect the organization of health services delivery. For instance, a country with many islands or with isolated population groups will face particular challenges in terms of health workforce issues. Similarly, significant climatic changes are likely to give rise to changes in health needs, which in turn will call for changes in health services and in the health workforce. Finally, both cultural and political values also affect the demand for and supply of human resources for health.This section considers a typology of imbalances, and differentiates between the following:• Profession/specialty imbalances: Under this category, we consider imbalance in the various health professions, such as doctors or nurses, as well as shortages within a profession, e.g. shortage of one type of specialists.• Geographical imbalances: These are disparities between urban and rural regions and poor and rich regions.• Institutional and services imbalances: These are differences in health workforce supply between health care facilities, as well as between services.• Gender imbalances: These are disparities in female/male representation in the health workforce.Imbalances have been reported for almost all health professions, and in particular for nurses. The United States General Accounting Office  reports Major variations occur in the number of health care workers per capita population and in the skill mix employed across countries, as depicted in Fig. Virtually all countries suffer from a geographical maldistribution of human resources for health, and the primary area of concern is usually the physician workforce . In bothUnderstandably, most health care professionals prefer to settle in urban areas, which offer opportunities for professional development as well as education and other amenities for themselves and their families. But it is in the rural and remote areas, especially in the developing countries, that most severe public health problems are found.The geographical maldistribution of doctors has been the object of particular attention. In general there is a higher concentration of general practitioners in the inner suburbs of the metropolitan areas. According to the Australian Medical Workforce Advisory Committee , the rea• historical• lifestyle-related: access to amenities• spouse/husband-related: greater employment opportunities• child-related: better access to secondary and tertiary education services• professional, family and social ties and professional ambitions.The geographical distribution of health care personnel is an important issue in many countries. Managua, the capital of Nicaragua, contains one-fifth of the country's population but around half of the available health personnel . In BangIn Indonesia the geographical distribution of physicians is a particular concern, since Indonesia's vast size and difficult geography present a tremendous challenge to health service delivery . It is dTo improve the geographical distribution of physicians, governments often have used combinations of compulsory service and incentives. So far, there is virtually no country in the world that has solved the problem of a rural/urban imbalance of the physician workforce . This doInstitutional imbalances occur when some health care facilities have too many staff because of prestige, working conditions, ability to generate additional income, or other situation-specific factors, while others are understaffed . InstituImbalance between the types of health services provided may also arise. In particular, we can consider the issue of curative versus preventive care. In effect, it has been estimated that most diseases (80%) and accidents are preventable through known methodologies, yet at present there is an imbalance in the funding of medical research, with only 1%-2% going to prevention and 98%-99% spent on curative approaches . This imIn many countries, women still tend to concentrate in the lower-status health occupations and to be a minority among more highly trained professionals and managers. In Bangladesh, the distribution by gender of the health workforce shows that the total proportion of women accounts for little more than one-fifth in health services . The disFemale general practitioners have been shown to practise differently from males, managing different types of medical conditions, with some differences due to patient mix and patient selectivity, and others inherent in the sex of physician. In some more traditional areas, some women will not seek care for themselves or even for their children because they do not have access to a female provider .This framework can be used to assess policy reforms and their impact on health workforce imbalances; it also provides a common framework for cross-country comparisons. This framework emphasizes the number and type of factors affecting health workforce imbalances, illustrating the complexity of this issue. From a policy perspective, it is particularly interesting to identify factors that policy-makers can influence in order to remedy imbalance problems.Various monetary and non-monetary incentives are used to influence the supply and/or demand for the health workforce. For example, subsidies, grants and scholarships are examples of incentives that can be used to attract more nursing students, whereas wage increases, additional benefits and working hours flexibility are examples of commonly used incentives to attract or retain the health workforce.The numerous factors and actors involved in the health workforce imbalance issue call for a coherent health workforce vision and policy. In that context, health planning plays an important role since it contributes to shaping the health care system. Moreover, since from a societal perspective market mechanisms alone do not allow an adequate demand/supply of health personnel to be reached, public interventions such as human resources planning are a means to correct for market failures. Health planning involves a time horizon. Forecasting the future number of health personnel needed and developing policies to meet such figures are common to any health care system. Physicians represent the profession for which more planning effort has been expended to achieve a workforce of appropriate size than for any other health profession. Countries' desire to meet population health needs and to avoid social welfare losses resulting from a shortage or an oversupply are factors explaining, to a large extent, the importance attributed to planning in the context of public health policies.The policy implications of forecasting either a shortage or a surplus of health care personnel are different, and hence attempts at projections must be rigorous. For instance, referring to previous studies predicting significant surpluses, Cooper  notes thAs a result of forecasted physician surpluses, various policy recommendations have been formulated. The United States Institute of Medicine  publisheThis framework also apprehends the different types of imbalances. This is important since the choice of a policy will also depend on the type of imbalance. Significant disparities in human resources for health between health occupation, regions, gender or health services are recognized as classic problems of imbalance. However, the question of a public/private imbalance is more debatable. One the one hand, we can argue that for equity and access, a health care system should have a strong public component. On the other hand, we can imagine a private-sector oriented health care system with mechanisms to ensure access to the poor.In an attempt to contribute to a better understanding of imbalances in the health workforce, this paper has discussed a framework for human resources for health and proposed a typology of imbalances. Although the term "imbalance" is commonly used with respect to the health workforce, it is clear that imbalance in the health workforce encompasses a wide range of possible situations and is a complex issue. The use of a framework should facilitate the development of policy tools and their assessment.None declared.All authors participated in writing the original text and read and approved the final manuscript.