The use of complementary and alternative medicine (CAM) among cancer patients is widespread and mostly self-administrated. Today, one of the most relevant topics is the nondisclosure of CAM use to doctors. This general lack of communication exposes patients to dangerous behaviors and to less reliable information channels, such as the Web. The Italian context scarcely differs from this trend. Today, we are able to mine and analyze systematically the unstructured information available in the Web, to get an insight of people’s opinions, beliefs, and rumors concerning health topics.Our aim was to analyze Italian Web conversations about CAM, identifying the most relevant Web sources, therapies, and diseases and measure the related sentiment.Data have been collected using the Web Intelligence tool ifMONITOR. The workflow consisted of 6 phases: (1) eligibility criteria definition for the ifMONITOR search profile; (2) creation of a CAM terminology database; (3) generic Web search and automatic filtering, the results have been manually revised to refine the search profile, and stored in the ifMONITOR database; (4) automatic classification using the CAM database terms; (5) selection of the final sample and manual sentiment analysis using a 1-5 score range; (6) manual indexing of the Web sources and CAM therapies type retrieved. Descriptive univariate statistics were computed for each item: absolute frequency, percentage, central tendency (mean sentiment score [MSS]), and variability (standard variation σ).Overall, 212 Web sources, 423 Web documents, and 868 opinions have been retrieved. The overall sentiment measured tends to a good score (3.6 of 5). Quite a high polarization in the opinions of the conversation partaking emerged from standard variation analysis (σ≥1). In total, 126 of 212 (59.4%) Web sources retrieved were nonhealth-related. Facebook  and Yahoo Answers  were the most relevant. In total, 94 CAM therapies have been retrieved. Most belong to the “biologically based therapies or nutrition” category: 339 of 868 opinions (39.1%), showing an MSS of 3.9 (σ=0.83). Within nutrition, “diets” collected 154 opinions (18.4%) with an MSS of 3.8 (σ=0.87); “food as CAM” overall collected 112 opinions (12.8%) with a MSS of 4 (σ=0.68). Excluding diets and food, the most discussed CAM therapy is the controversial Italian “Di Bella multitherapy” with 102 opinions (11.8%) with an MSS of 3.4 (σ=1.21). Breast cancer was the most mentioned disease: 81 opinions of 868.Conversations about CAM and cancer are ubiquitous. There is a great concern about the biologically based therapies, perceived as harmless and useful, under-rating all risks related to dangerous interactions or malnutrition. Our results can be useful to doctors to be aware of the implications of these beliefs for the clinical practice. Web conversation exploitation could be a strategy to gain insights of people’s perspective for other controversial topics. Complementary and alternative medicine (CAM) is an umbrella definition for a wide range of medical practices that are no part of the standard medical care ,2. TheseA survey of the National Institutes of Health reports an increasing trend in CAM usage among American people from 1999-2012 . This trThe Italian context scarcely differs from these trends. A national survey performed by Italian Center for Social Studies and Investments (CENSIS) reports that 23.4% of Italian people resorted to unconventional medicine such as homeopathy and herbal remedies at least once in 2008 . D’ArenaCurrent literature reports that patients would like to talk with their doctors about CAM, but they do not ,6,9,16. On one hand, usually doctors do not ask patients about their CAM consumption habits . Maybe tThis general lack of communication between doctors and patients about CAM has relevant consequences.First, it exposes patients to potentially dangerous behaviors ,15,23,24This trend is amplified through the virtual communities that crowd the Web ,40. TodaWe collected data about CAM information requests to the SPL of the Centro di Riferimento Oncologico (CRO) Aviano, National Cancer Institute. From 2008 to 2013, 218 of 2313 overall questions requested to SPL (9.4%) were CAM-related. But the trend is constantly increasing. In 2008, we recorded 24 CAM-related questions of 387 (6.2%). In 2012, the CAM-related questions were 38 of 198 (19.2%), and in 2013, 46 of 282 (16.3%). Overall, 183 requests of 218 (84%) regarded biologically based therapies such as diet, food, or natural remedies effectiveness; interactions among remedies and drugs; and remedies availability. We have to point out that these numbers reflect only the questions recorded through the SPL access form. Due to our policy , this foBased on the literature and the preliminary data, we aim to analyze Italian Web conversation about CAM. In particular, we aim to identify the most relevant Italian Web conversation sources; the cancer CAM therapies most discussed by Italian Web users and the corresponding sentiment; and what is the most discussed disease.Our data collection methodology could be assimilated to the theoretical sampling  because Within our search, we followed a systematic approach, which was organized into 6 phases, with a strictly functional approach. We have used ifMONITOR for services “a” and “b” in phase 3 and for service “c” in phase 4. Websites, forums, blogs, communities, and social networks of any kind and topic addressed to the general public and containing conversations about CAM topics have been included. In particular, Facebook profiles, pages, and groups of users and YouTube channels were also included (depending on the privacy settings). Otherwise, we decided to leave out Twitter because the length of the messages is too short to express a complex opinion about the topic taken into account. The Web search was also limited to Web documents published between January 1, 2013, and May 31, 2014. We also limited the Web search only to Italian language Web documents, to meet the perspective of Italian people with low literacy skills.This phase is manual and has been executed by CRO librarians. They built up a CAM database containing relevant terms to be used as a reference in subsequent phases. For each CAM therapy identified, a structured template was filled up with related synonymies and references to specific philosophy or religion; names of people, organizations, and places; book titles; anatomy and physiology terms; substances or drugs; principles or processes; methods; instruments or tools; effects or side effects; diseases. Because the final goal was to analyze Web-based people conversations, it was important to capture the terminology that is actually known by the people themselves. For these reasons, 3 main kinds of sources of information have been considered: 1) scientific and medical resources  scientif,49, (2) This phase is aimed at collecting all the materials to be analyzed later exploiting ifMONITOR services “a” and “b.”The final result of phase 3 has been a set of 3708 documents, largely referring to people’s conversations, which were processed in phase 4.This fully automated phase is aimed at classifying the 3708 documents retrieved in which CAM therapy is mentioned in the document. The starting point has been the terms included in the CAM therapies database.This phase was mainly manual, and it was aimed at identifying a sample of documents to be analyzed in detail. The result constituted 838 Web documents.The manual analysis of the documents, starting from the documents of the most successful Web-based sources . These criteria enabled us to consider the most popular sources and Web conversations first.This process continued up to the accumulation of 423 Web documents, when we reached the saturation of the sample . We consFor each of the 423 documents, manual tagging was added, concerning the sentiment about the perceived effectiveness or ineffectiveness of the CAM therapy mentioned through a numerical score ranging from 1 to 5 . The score assignment was performed by a team of 4 trained Web intelligence analysts. They performed individually the sentiment analysis on different sets of documents, later they compared together the score assignment, thus limiting information bias due to interoperator discretionary.We did not perform any assessment of the medical and scientific reliability of these statements. According to the literature ,5,24,49,Furthermore, librarians indexed the Web sources according to the criteria of health and nonhealth thematic areas. Web sources are websites, blogs, forums, and social networks . Then they gathered Web sources according to their specific subject matters, as shown in The content classification allowed us to identify the most discussed CAM therapies; the most relevant Web sources; and the most discussed cancer type. Furthermore, the semantic analysis allowed us to measure the popularity and sentiment about the identified CAM and identify the pros and cons issues in the discussions about the most discussed CAM therapies.Statistical AnalysisDescriptive statistical analysis was performed using Microsoft Excel. Absolute (n) and relative frequencies (%) with reference to total Web sources, total Web documents, and/or total opinions included in the final sample were computed. For each item expressed using a numerical score  mean scores (mean sentiment score [MSS]) as a central tendency index and standard deviations from the mean (σ) as an indicator of variability were also calculated.The CAM Therapy Database included a set of 224 CAM therapies: 19 cancer treatments ; 205 general remedies used to manage diseases symptoms, treatments side effects, or to cope with the stress . The number of terms collected referring to the 224 CAM therapies is 3405.Phase 3 to 6: From Classification to Sentiment AnalysisThe final sample included 423 Web documents extracted from 212 Web sources, showing 868 opinions.Ten Web sources including 39 Web documents tagged automatically by ifMONITOR have become inaccessible before Phase 6. Nevertheless, all of them were automatically indexed by URL, title, author, date, CAM therapy mentioned, and opinion. But they lack information about Web sources or Web documents subjects (for instance topic of the single Web document). The 868 opinions retrieved stated an overall MSS of 3.6 (σ=1.0).We found 244 of 868 (28.1%) opinions related to a specific disease: 81 of 244 (33.2%) were breast cancer related; 64 (26.2%) were pancreatic cancer related; 29 (11.9%) were melanoma related; 27 (11.1%) were lung cancer related; 21 (8.6%) were bone marrow cancer related; 14 (5.7%) were ovarian cancer related; 6 (2.5%) were peritoneum cancer related; 1 (0.4%) were neck cancer; and 1 (0.4%) were mandible cancer related.We found 95 of 212 websites (44.8%), 45 social networks (21.3%), 41 blog (19.3%), and 28 forums (13.2%).Within social networks, 22 of 45 (49%) were public personal profiles, 12 (27%) were groups of users, and 11 (24%) were company or personal pages.We found 83 of 212 (39.2%) “health-related” Web sources producing 176 of 423 (41.6%) Web documents. Among the “health-related” group, we found 46 of 212 (21.7%) Web sources focused on CAM topics producing 103 of 423 (24.3%) Web documents. The 377 opinions of 868 (43.4%) of this group, expressed a MSS of 3.7 (σ=0.8). The Web sources focused on “oncology” topics were 8 of 212 (3.8%), producing 37 of 423 (8.7%) Web documents.On the other hand, 126 of 212 (59.4%) were “nonhealth-related” Web sources, producing 237 of 423 (56%) Web documents. Among “nonhealth-related” group, we found 66 of 212 (31.1%) Web sources focused on a wide range of different topics that produced 142 of 423 Web documents (33.6%). The 476 opinions of 868 (54.8%) retrieved into this group, expressed a MSS of 3.6 (σ=1.1).The highest MSS (3.7) was observed among “other issues,” “oncology,” “health and wellness,” and “CAM” categories. The lower (2.7) within the “debunking” category. Complete results are displayed in Yahoo Answers is the most retrieved Web source: 41 of 423 (9.7%). The Facebook group “Quelli che il cancro (e non solo) lo curo a modo mio” , counted 30 Web documents of 423 (7.1%). This group changed its privacy settings: today the access is limited to the registered members only. But if we sum all Facebook profiles, groups, and pages together, we count 89 of 423 (21%) Web documents, nearly double that of Yahoo Answers. The third Web source per number, 14 of 423 (3.3%), was Greenstyle.it, a Web magazine fostering ecologic lifestyles owned by HTML.it, an Italian network of publishing and advertising companies.We found 94 therapies or remedies mentioned in the conversations retrieved. Among these, 68 (72%) were included in the CAM keyword set. Twenty-six (28%) are therapies that emerged during the manual reviews of ifMONITOR’s search output in phase 3. Some of these are typical Italian alternative cancer remedies, such as “Giuseppe Nacci’s” diet, an Italian variation of “Gerson Therapy,” “Gianfranco Pantellini’s” therapy, based on potassium ascorbate consumption, “Alessiani’s Water,” enriched with minerals extracted from Romans catacombs.The most discussed CAM therapies belong to the “biologically based therapies” group, with 702 of 868 (80.9%) opinions retrieved. The MSS observed was 3.6 (σ=1.0). Among “biologically based therapies” the “nutrition” category had the higher rate of opinions, 339 of 868 (39.1%), expressing an MSS of 3.9 (σ=0.8). “Chemical-based medicines,” counted 186 opinions of 868 (21.4%) showing an MSS of 3.3 (σ=1.3). “Plant-based medicines” counted 144 opinions of 868 (16.6%), and an MSS of 3.6 (σ=1.0). The complete results are displayed in We identified different approaches concerning the “nutrition” issues. On one hand, most of the time, Web users talked about the effectiveness of a certain diet or alimentary regimen in cancer treatment or prevention. For instance, “vegetarianism” or “veganism,” “raw foodism,” “alkaline diet,” “Gerson therapy.” On the other hand, they often discussed about the healing properties of a specific food or spice. For instance, grapefruit, garlic, turmeric, mushrooms, lemon, or green tea.Finally, the discussions on dietary regimen included also the use of supplements, for instance, vitamins. “Diets” overall collected 154 opinions of 868 (18.4%) with an MSS of 3.8 (σ=0.9). “Food as CAM” overall collected 112 opinions of 868 (12.8%) with a MSS of 4 (σ=0.7). “Supplements” collected 73 opinions of 868 (7.8%) with an MSS of 3.8 (σ=0.9). The main results are summarized in Excluding diets and food, the most discussed CAM therapy is the “Di Bella multitherapy.” We retrieved 102 opinions of 868 (11.8%) with an MSS of 3.4 (σ=1.2). “Di Bella multitherapy” and a reportage of “Le Iene,”(the Hyenas) Italian comedy and satirical TV show with reports on politics and consumers issues, on “alkaline diet” and “veganism” were also the most recurring topic: 16 of 423 (3.8%) Web documents retrieved.We found 29 of 212 (13.7%) Web sources containing 58 of 423 (13.7%) Web documents and 102 of 868 (11.7%) opinions about “Di Bella multitherapy.” This is a controversial cancer treatment invented by the Italian physician Luigi Di Bella, based on a mix of somatostatin, melatonin, hormones, and vitamins. In the early 1990s, this treatment had a wide appeal among Italian cancer patients . The triThis therapy was mostly discussed among “Nonhealth-related” web sources, with 65 out of 102 (63.7%) opinions retrieved showing an MSS of 3.4 (σ=1.1). Most of the conversation were retrieved among the “other issues” category, with 35 of 102 (34.4%) opinions showing an MSS of 3.1 (σ=1.1). No opinions were retrieved into the “debunking” and “health and wellness” category.Conversely, we counted 37 of 102 (36.3%) opinions into “health-related” Web sources showing an overall MSS of 3.3 (σ=0.8). We observed that it was discussed almost only in the “oncology” category, with 29 of 102 (28.4%) opinions retrieved showing an MSS of 3.6 (σ=1.1).We also found that 23 of 423 (5.4%) Web documents have the “Di Bella multitherapy” as main topic of the conversation, distributed among: 6 in “oncology,” 1 in “CAM,” 2 in “mainstream newspaper on the Web,” 2 in “pseudoscience and conspiracy theories,” and 12 in “other issues” categories.The conversations retrieved were mainly focused on the discussion about effectiveness or ineffectiveness of “Di Bella therapy,” with 60 of 102 (58.8%) opinions showing an MSS of 3.8 (σ=1.3). The lowest MSS observed is 2.5 (σ=1.1) achieved by the topic “plot against therapy’s trial” means that the 5 users believe to the plot theory against the “Di Bella multitherapy.”The arguments in support of the Di Bella multitherapy expressed by users in the Web conversations were the absence of side effects, the effectiveness in treating neoplasm otherwise terminal, and its effectiveness while conventional therapies are suspended. There is a widespread belief that its real effectiveness cannot be proven because the trial was intentionally compromised. To prove this last statement, users often report that the well-known oncologist Umberto Veronesi during an interview confirmed the effectiveness of the somatostatin in breast cancer treatment. The points against the Di Bella multitherapy were mainly two: the therapy has not been recognized as a standard therapy by the Italian National Health Service because of the lack of scientific evidence and the therapy has high initial costs.Our survey confirms the existence of Web-based conversations about cancer CAM therapies among Italian Web users. The overall sentiment on CAM effectiveness tends to range from neutral to good, with a certain degree of variability in opinions (MSS±: 3.6±1.0).The conversations about CAM were retrieved mostly from “nonhealth-related” rather than “health-related” web sources. Facebook and Yahoo Answers together covered almost one third of the conversation retrieved. Beyond them, most of the CAM conversations are widespread within Web sources that deal with very different topics. For instance, political activism, economy, marketing, video games, sport, gastronomy, leisure, and weather forecasts. The overall sentiment observed in the “nonhealth-related” category is quite neutral with high level of polarization (MSS±: 3.3±1.3).A similar overall sentiment emerges in the “health-related” category, although with less variability (MSS±: 3.4±1.1). Despite the low number of Web sources retrieved, we found a high rate of opinions in the “oncology” category. This is because they are mostly conversations among patient-dedicated forums users. The sentiment observed in this category is quite good (MSS=3.8), with some variability among the conversations partaking (σ=1.1).The most discussed CAM therapies belong largely to the “biologically-based” category. In particular, the “nutrition” has a good score with low polarization (MSS±: 3.9±0.8). “Nutritional” CAMs are also considered very effective for cancer prevention, side-effects management, and as cancer cure also. Nutritional conversations concern the effectiveness of diets, such as “veganism.” But they also concern the healing properties of a single food. In this case, the consensus is high and with quite low variation (MSS±: 44±0.9). “Plant-based” medicines are also perceived as quite effective (MSS±: 3.6±1.0). Against the number of opinion retrieved, we observed an almost neutral sentiment score with a quite high polarization of opinions (MSS±: 3.3±1.3) among “chemical-based” medicines.Only a third (28.1%) of the opinions retrieved mentioned a specific neoplasm: mostly breast or ovarian cancer. In the other case, people referred only to cancer in general.Contrary to what we expected, we collected a low number of very popular CAM, such as homeopathy, Chinese traditional medicine, acupuncture, yoga, and reflexology. At the same time, almost a third (28%) of the CAMs that emerged during the manual reviews in phase 3 were unexpected or uncommon. They are all “biologically based.”Finally, particularly significant is the finding of a considerable amount of opinions about the “Di Bella multitherapy.” We observed that Di Bella multitherapy is mentioned mostly within the “nonhealth-related” Web sources. The corresponding MSS tends to a neutral value, with a very high rate of polarization, especially among the “nonhealth-related” Web sources (σ 1.3). It is interesting to observe that the higher MSS is recorded among the “oncology” and “pseudoscience and conspiracy theories” categories together.These findings on Di Bella Multitherapy are particularly relevant if we compare its popularity with other similar pseudoscientific cancer treatments, such as the “Simoncini Baking Soda” therapy. This latter is notably less discussed, with a very low MSS and a very high level of polarization (σ 1.4). This proves that Di Bella multitherapy is considered more than a pseudocure by those partaking in the Web conversations that were considered.Our findings in number and distributions of conversations confirm those reported in the Social Oncology Project for the United States : cancer The distribution of the conversations complies with the results of the CENSIS national report : only 13This complies with Quattrociocchi  and BessThe amount of conversations retrieved about nutritional and herbal remedies together comply with the trends in CAM consumption reported by current literature ,15,35,61Despite the wide consumption of some popular CAM therapies reported by the literature among the Italian cancer patients ,18,62, wContrary to the trends observed by the Social Oncology Project , the volOur study has some limitations. The sample is limited in number, and it is not representative of all the Italian Web conversations in the period taken into account. Furthermore, it was not possible to acquire demographic information about Web users. Although we have included a consistent number of Web users, this may not be representative of the entire Italian population. The use of mean and standard deviation for summarizing the opinions collected using a Likert-type ordinal scale is controversial; however, given the adequate sample size of each group and the nearly normal distribution of our data, the parametric approach is considered acceptable .Conversations on CAM and cancer are ubiquitous. Most of them flow through nonhealth-related Web sources. We need to know people’s information background to better understand their “implicit” knowledge about CAM issues and cancer. We found that unsubstantiated beliefs such as Di Bella multitherapy last for many years, despite scientific evidence. This particularly happens with the cancer patients’ Web-based communities that were retrieved. Understanding the reasons for this persistency against scientific evidence needs further research. There is a big concern about the biologically based therapies and remedies, in particular diet and nutrition and naturopathy issues. These are perceived as harmless and useful, under-rating all risks related to dangerous interactions or malnutrition.Based on our data, we suggest that doctors should be aware of the implications of these beliefs for the clinical practice. At the same time, it is necessary to keep themselves up to date about the ever-changing CAM issues, to foster patients’ information requests and avoid decisions based only on autonomous Web searches.We can exploit Web conversation analysis as a strategy to get insights of people perspective and improve new information strategies and resources even for other controversial topics such as clinical trials, sexuality and fertility, and HPV vaccination.


While the maintenance of “essential” genes is ensured by their lethal double null mutations, the expansion of “dangerous” gene families, implicated in cancer and other severe genetic diseases in human, remains puzzling. Could gene susceptibility to dominant deleterious mutations be somehow responsible for this striking evolutionary expansion of “dangerous” gene families?We proposed such an evolutionary model suggesting that this counterintuitive expansion of “dangerous” gene families is in fact a consequence of their susceptibility to deleterious mutations and purifying selection in polyploid species that arose from two rounds of whole genome duplication (WGD) events dating back from the onset of jawed vertebrates, some 500MY ago ,2. All Wversus 35%; 48% : 3,844/8,095; P=1.3×10−128, χ2). We also found that the retention of ohnologs is more strongly related to their “dangerousness” than their “essentiality” [Our data mining analyses, based on the 20,506 human protein coding genes, first revealed a strong correlation between the retention of ohnologs and their susceptibility to dominant deleterious mutations in humans . It appedirect and indirect effects of many genomic properties, such as essentiality, expression levels or divergence rates, on the retention of ohnologs.To go beyond mere correlations, we also performed mediation analyses, following the approach of Pearl , and quaversus 35%; 7.5% : 18/239; P=1.2×10−18, χ2). These mediation analyses also showed  the speciation event concomitant to WGD, and (ii) the dominance of deleterious mutations leading to purifying selection in post-WGD species.All in all, this supports a new evolutionary model relying on a non-adaptive mechanism that hinges on (


Canis familiaris). It is assumed that reproductive hormones influence tumorigenesis in this species, although the precise role of the endocrine milieu and reproductive state is subject to continuing discussion. In line with this, a recent systematic review of available data on the development of mammary neoplasms revealed weak evidence for risk reduction after neutering and an effect of age at neutering. Investigation of several hormone receptors has revealed decreased expression of estrogen receptor-alpha , progesterone (P4) receptor (PGR), prolactin (PRL) receptor (PRLR) and growth hormone receptor (GHR) associated with neoplastic differentiation of mammary tissues. In other studies, increased levels of estrogens, progesterone and prolactin were found in serum and/or tissue homogenates of dogs with malignant neoplasms. However, the association between these entities within one animal population was never previously examined. Therefore, this study investigated the association between circulating serum concentrations of estradiol-17β, progesterone and prolactin, and gene expression of ERα (ESR1), ERβ (ESR2), PGR, PRLR, PRL and GHR, with respect to reproductive state (spayed vs. intact) and cycle stage (anestrus vs. diestrus). Additionally, the expression of E-cadherin (CDH-1) was evaluated as a possible indicator of metastatic potential.Neoplasms of the mammary gland are among the most common diseases in female domestic dogs  contains supplementary material, which is available to authorized users. Estrogens , progesterone P4), prolactin (PRL) and growth hormone (GH) are essential for physiological mammary growth and development, which not only take place during pregnancy but also occur with every reproductive cycle. While estrogens induce ductal growth, P4 is essential together with PRL for lengthening and tertiary branching of the ductal system and stimulation of lobulo-alveolar development , prolact–8. In adin vivo and cell viability in vitro [Despite the presumed influence of reproductive hormones, studies on endocrine therapy for CMT are rare. Gene or protein expression of ERα (ESR1) and/or ERβ (ESR2), PGR, PRLR and GHR were detected in CMTs, and decreased receptor expression was associated with malignancy and/or a worse prognosis , 24–32. in vitro , 34. Thein vitro –37, is nin vitro , 39. Lowin vitro .Markers of tumor invasion are used in human medicine to estimate survival time and prognosis . E-cadheMastectomy is regarded as the most effective treatment for CMT so far. However, further therapeutic options should be sought, which could be used in combination with surgery or in cases of non-operable lesions and inflammatory mammary carcinomas. Knowledge of the interplay between hormones and their receptors during the reproductive cycle, after spaying or during tumorigenesis, could provide a basis for new and advanced therapeutic approaches for dogs with CMT.i.e., ERα (ESR1), ERβ (ESR2), PGR, PRLR, PRL, as well as GHR, in normal mammary tissues as well as in mammary neoplasms in female dogs, taking into account their reproductive state and cycle stage. Furthermore, gene expression of CDH-1 was analyzed as a marker for tumor invasiveness in mammary tissues showing different degrees of malignancy. In addition, the influence of reproductive state and cycle stages were assessed for all parameters.Up to now, there is no information on the effect of serum steroid hormone and PRL levels on their respective receptor gene expression in normal and neoplastic mammary tissues in dogs. Our goal was, therefore, to study the associations between circulating serum concentrations of E2, P4 and PRL, and the expression of genes encoding for their respective receptors, A prospective study was carried out with 32 privately owned female dogs that were presented with mammary neoplasms to the Clinic of Reproductive Medicine, Vetsuisse Faculty in Zurich between 2009 and 2013. Altogether, 56 mammary neoplasms and 28 normal mammary tissue samples were collected from these patients. Eleven dogs had only one mammary neoplasm and 21 bitches had two or more lesions. Mastectomy was performed in 28 dogs to remove the masses. The following surgery techniques were used: unilateral mastectomy (n = 12), unilateral mastectomy combined with regional mastectomy (n = 3), regional mastectomy (n = 11), excisional biopsy (n = 2). Three of four dogs with inoperable cancer were euthanized at initial presentation because of their poor general condition. The fourth dog was euthanized two months later because of severe dyspnea due to pulmonary metastasis.The phase of the estrous cycle at presentation was determined based on history, clinical examination and blood hormone assays (P4 and E2).All owners consented to their animal becoming a research participant and animal experimentation was approved by the Cantonal Veterinary Authority in Zurich, Switzerland (permission No. 136/2009 and 165/2012).In order to account for the possible pulsatile release and circadian rhythm of PRL secretion , 50, twoSerum PRL concentrations were measured with a previously validated heterologous radioimmunoassay (RIA) . The int125I-RIA  according to the manufacturer’s instructions, with slight modifications as described previously and validated for the dog [3H-RIA [E2 and P4 were determined from the first serum sample. E2 concentrations were determined by a solid-phase  the dog , 53. The [3H-RIA , 54. TheNeoplastic and normal mammary tissue samples from each dog were collected by surgical mastectomy, biopsy or immediately post mortem. Tissue samples were fixed in 10 % buffered formalin for 24 h and embedded in paraffin. Sections from each block were stained with hematoxylin and eosin (HE) for histopathological diagnosis according to the criteria of Goldschmidt , and theThe experimental procedure was based on our previously established protocol . Thus, oExcess paraffin was manually removed from each representative tissue section in a warm water bath (37 °C), and all sections were transferred into a 1.5 ml plastic Eppendorf tube . Total RNA was extracted using the RNeasy FFPE Kit  according to the manufacturer’s instructions. The RNA concentration was measured with a NanoDrop 2000 UV–Vis  Spectrophotometer . The RNA quality was assessed by checking the RNA integrity numbers (RIN) and samples with similar RIN were used for all groups. Extracted RNA was stored at −80 °C until analysis.™ Reverse Transcriptase (Qiagen) and random hexamers  as primers according to the manufacturer’s instructions. All reactions were carried out in an Eppendorf Mastercycler .In order to remove genomic DNA contamination, 50 ng RNA per reaction was DNAse treated  in accordance with the manufacturer’s instructions. Afterwards, RNA was reverse transcribed into complementary DNA (cDNA) using Sensiscript Semi-quantitative real-time (TaqMan) PCR reactions for ERα ESR1), ERβ (ESR2), PGR, PRL, PRLR, GHR and CDH-1 were performed as described previously , 57 usin, ERβ . A on 3.0.2  and the on 3.0.2  were useThe ages of dogs at the time of blood and tissue sampling ranged from 4.6 to 16.4 years . Bitches were of different breeds, the most frequent being mixed breed dogs (n = 9), Jack Russell terriers (n = 3), Boxers (n = 2) and dachshunds (n = 2).Twenty-four bitches were intact, of which 13 were in anestrus and 11 in diestrus. Eight dogs had been previously spayed before the time of mastectomy, six between the ages of 7 and 10 years, one before 18 months of age, and in one dog no information was available.The group of benign mammary neoplasms consisted of 19 tissue samples out of 12 dogs, the malignant neoplasia group of 37 samples from 24 dogs. Benign neoplasms included complex adenomas (n = 10), simple adenomas (n = 6), mixed benign mammary tumors (n = 2) and one ductal adenoma. The malignant group consisted of complex carcinomas (n = 14), simple carcinomas (n = 12), anaplastic carcinomas (n = 3), solid carcinomas (n = 3), ductal carcinomas (n = 3), one adeno-squamous carcinoma, and one carcinoma and malignant myoepithelioma. Normal tissue was available from 28 dogs.Serum levels of E2, P4 and PRL varied between 0.52 and 16.32 pg/ml, 0.05 and 91.4 ng/ml, and 1.32 and 8.91 ng/ml, respectively (Table Gene expression of ERα ESR1), PGR, PRLR, GHR and CDH-1 was detected in normal mammary tissues and in all mammary neoplasms, except in one carcinoma. The PRL and ERβ (ESR2) mRNA expression was below the detection limit in most of the samples in all groups, revealing more negative than positive results and, thereby, indicating their low expression levels. Corpus luteum tissue (mid-diestrus) used as a positive control tested positively for both factors  (p = 0.03), PGR (p = 0.008) and PRLR (p = 0.001). Thus, malignant tumors showed significantly lower ERα (ESR1) and PGR gene expression than normal mammary tissues . In contrast to these finding, the corresponding models for PGR and ERα (ESR1) did not show a relationship with their hormone levels .GHR gene expression was also predicted best by “tissue group” (p = 0.008). Consequently, GHR mRNA levels were significantly lower in malignant neoplasms compared to normal mammary tissue  for both “tissue group and reproductive state and/or cycle stage”. Among diestrous bitches, CDH-1 gene expression was highest in benign neoplasms compared to normal mammary tissue and malignant neoplasms , PGR, GHR and PRLR gene expression in the mammary tissue of dogs. Only PRLR gene expression was significantly decreased concurrently with the formation of benign tumors. Increased levels of serum PRL, but not of P4 and E2, were associated with a decrease in gene expression of the respective receptors only in malignant mammary neoplasms. No evidence was found for the presence of a paracrine/autocrine action of PRL in the canine mammary gland. CDH-1 mRNA expression was higher in benign compared to malignant neoplasms and normal mammary tissue and thus may serve as a prognostic marker.


Anas platyrhynchos), run the risk of genetic introgression by farmed conspecifics released for hunting purposes. We tested whether bill morphology of free-living birds has changed since large-scale releases of farmed mallards started. Three groups of mallards from Sweden, Norway and Finland were compared: historical wild , present-day wild, and present-day farmed. Higher density of bill lamellae was observed in historical wild mallards . Farmed mallards had wider bills than present-day and historical wild ones. Present-day wild and farmed mallards also had higher and shorter bills than historical wild mallards. Present-day mallards thus tend to have more “goose-like” bills  than their ancestors. Our study suggests that surviving released mallards affect morphological traits in wild population by introgression. We discuss how such anthropogenic impact may lead to a maladapted and genetically compromised wild mallard population. Our study system has bearing on other taxa where large-scale releases of conspecifics with ‘alien genes’ may cause a cryptic invasive process that nevertheless has fitness consequences for individual birds.Wild populations of the world’s most common dabbling duck, the mallard ( Norway and Finland), where releases are not practiced. While the study by Champagnon et al. To test the hypothesis that an influx of non-native genes leads to morphological change in the wild population we here compare bill morphology in present-day wild, present-day farmed, and ‘historical pre-release’ mallards from Sweden, Norway and Finland. An earlier test of this hypothesis was restricted to France, a country where massive releases are practiced No approval from the Swedish Animal Ethics Board is needed to trap and ring birds in Sweden. However, national ringing licenses were obtained by the Swedish Museum of National History in Stockholm (license number 632). Strict protocols were followed during all steps where live birds were handled in the study to ensure the safety of the animals. Handling time was kept to a minimum when live birds were measured and no animals were killed specifically for this study. No other specific permissions were needed, for locations or activities, to perform this study. Hunters who donated mallards had hunting permits issued by the Swedish Environmental Protection Agency. All hunts were carried out on private lands with permission from the land owners.Bill morphology was studied in 384 mallards originating from Sweden, Norway and Finland and belonging to three groups; ‘historical wild’ from museum collections, ‘present-day wild’ and ‘present-day hand-reared’ (farmed) see . The wilA photograph was taken of the underside of the bill of each specimen. A ruler was placed alongside the bill for scale reference . The proData on bill height, width and length were also obtained from each bird using a caliper measuring to the nearest 0.01 mm. Height and width were measured over the center of the nostrils . Length We used IBM SPSS Statistics version 20 and its univariate general linear models (GLM) to test whether bill measurements as well as lamellar density differed between sexes, and between sampling groups  for a given sex. To contrast groups for significant model outcomes, pairwise Tukey’s post-hoc tests were used. A significance level of 0.05 was used in all tests.2>0.068, all F1 values >15.065, all p<0.001) . Post-hoc tests revealed that historical wild males had significantly higher lamellar density than farmed mallards (p = 0.020), historical wild mallards also tended to have a higher lamellar density than present-day wild mallards for the same position (p = 0.051). No significant differences were found in any other position in either sex (p>0.116)  . When map>0.116) .2 = 0.225, F1 = 109.439, p<0.001; 2 = 0.084, F2 = 12.286, p<0.001; females: r2 = 0.041, F2 = 3.683, p = 0.028). Post-hoc tests revealed that historical wild mallards had flatter bills than farmed mallards in both sexes (p≤0.036 in both cases). In males, historical mallards also had flatter bills than present-day wild mallards (p<0.001) whereas in females there was a tendency for a difference between the two groups (p<0.068). No differences in bill height were found between farmed and present-day wild mallards in either sex (p>0.451 in both sexes)  , Fig. 4A2 = 0.116, F1 = 50.288, p<0.001; 2 = 0.346, F2 = 65.901, p<0.001; females: r2 = 0.600, F2 = 97.685, p<0.001). Post-hoc tests showed that farmed mallards had the widest bills and historical wild the narrowest   , Fig. 4B2 = 0.327, F1 = 187.495, p<0.001), and there was an effect of group within each sex . Post-hoc tests showed that historical mallards had longer bills than farmed (p≤0.023 in both sexes), while present-day wild were intermediate and could not be separated from any of the two other groups (p≥0.113)  , Fig. 4CThis study provides evidence that farmed mallards have more “goose-like” bill proportions than do historical and to some extent present-day wild ducks, i.e. they have less lamellae per centimeter, higher, wider, and shorter bills. Our results also show that today’s wild mallards have changed in the direction of farmed mallards since 1971.We found a lower lamellar density in the second centimeter of the bill in the Swedish farmed population, but only in males. Champagnon et al. Males consistently had a bigger bill  than females, which can be explained by simple allometry as males are larger in general. However, when comparing historical wild, present-day wild and farmed mallards, the pattern is more elusive. Historical mallards of both sexes had longer bills than farmed present-day mallards, a result similar to that reported by Pehrsson Anas cyanoptera). However, a correction term should be species-specific since bill proportions (soft parts as well as bone) may differ substantially between species and thus also the shrinkage patterns Historical wild mallards had flatter bills than present-day birds  and historical wild mallards also had the narrowest bills whereas farmed birds had the widest. This is consistent with the results reported by Pehrsson Pehrsson In short, this study shows that bills of farmed mallards have more “goose-like” proportions compared to historical wild mallards, and that wild mallards in northern Europe have changed in the direction of farmed birds with respect to bill morphology during the time large-scale release programs have been practiced. This change corroborates patterns described by Pehrsson Even though the survival of farmed mallards is low once released into nature A scientific implication of this study is that a deeper understanding is needed of the extent to which shipping of captive stock between countries and survival of released birds have altered natural genetic variation and geographic structuring in European mallards. If farmed stock have unique genetic markers it is valuable to know how abundant such individuals are in different geographic areas, and how well their spatial occurrence correlates with release activity. To increase our knowledge about survival, movements and breeding abilities of farmed mallards, comprehensive individual marking efforts are needed. The pairing process and breeding success of surviving farmed mallards in nature are also essential to study in order to assess the degree of introgression of ‘farmed genes’ into the wild population.Even though completely wild-like mallards may be nearly impossible to maintain long-term in captivity, there have been several suggestions on how to keep the differences to a minimum. Accordingly, mallard breeders can be encouraged to let the captive stock come in contact with wild birds by using open pens that allow the latter to enter the breeding facility. Secondly, breeders can be encouraged to use local or regional stock instead of importing birds or eggs from other countries. Thirdly, in order to limit drift in bill traits, ducks can be offered feeding conditions that more resemble the natural.Phragmites australis) In a larger context, this study exemplifies how releases of conspecifics with a non-native genome can affect wild populations. Cryptic invasions, whether it is by alien species like the common reed (S1 FileRaw Data.(XLSX)Click here for additional data file.


Shigella dysenteriae type 1 (Sd1) isolates. Because of substantial interest in Sd1, formerly a common cause of epidemics of severe dysentery, we went back to the specimens and repeated the microbiology. We have confirmed that all Shigella dysenteriae isolated in our study were non-type 1. We have made the following corrections to the text:In the original article, we reported detection of 6 Shigella species were isolated from 262 (24%) of 1,096 stool specimens.”In the “Results” section of the Abstract, the first sentence should read, “Shigella isolation and incidence rates” section of the Results should read, “For the period; 1 May, 2008 through 31 Dec, 2010, 242 (23%) Shigella bacteria were isolated from 1,096 stool specimens (data not shown).”The last sentence of the “Shigella isolates were S. flexneri (64%) followed by S. dysenteriae (11%) S. sonnei (9%), and S. boydii (5%). Species could not be determined for 12% of isolates. All 27 isolates of S. dysenteriae were non-type 1 (The “Species distribution” section of the Results should read, “Most n-type 1 .”Shigella incidence and monthly rainfall” should be a section heading, and the subsequent text in that paragraph should be beneath it.In the last paragraph of the Results, “We have also revised


The ventral prefrontal cortex is an integral part of the neural circuitry that is dysregulated in mood and anxiety disorders. However, the contribution of its distinct sub-regions to the regulation of negative emotion are poorly understood. Recently we implicated both the ventrolateral prefrontal cortex (vlPFC) and anterior orbitofrontal cortex (antOFC) in the regulation of conditioned fear and anxiety responses to a social stimulus, i.e., human intruder, in the marmoset monkey. In the present study we extend our investigations to determine the role of these two regions in regulating innate responses and coping strategies to a predator stimulus, i.e., a model snake. Both the vlPFC and antOFC lesioned groups exhibited enhanced anxiety-related responses to the snake in comparison to controls. Both groups also showed a reduction in active coping behavior. These results indicate that the vlPFC and antOFC contribute independently to the regulation of both innate fear and, as previously reported, conditioned fear, and highlight the importance of these regions in producing stimulus-appropriate coping responses. The finding that dysregulation in two distinct prefrontal regions produces the apparently similar behavioral phenotype of heightened negative emotion provides insight into the varied etiology that may underlie this symptom across a wide variety of neuropsychiatric conditions with implications for personalized treatment strategies. Ever since the 19th century case report of Phineas Gage, whose emotional character dramatically changed after considerable damage to his ventromedial prefrontal cortex, the PFC has been the focus of investigation for the regulation of emotions. Although negative emotions such as anxiety and fear are adaptive responses, the appropriate regulation of such negative emotions is crucial for a healthy mental life. When dysregulated, excessive fear and anxiety can become maladaptive and interfere with one's personal and social well-being. Recent studies using brain-imaging technologies have reported abnormal activities within the prefrontal areas of patients suffering from such disruptive anxiety disorders. When exposed to fear-inducing stimuli such as phobic objects , patients with posttraumatic stress disorder (PTSD), panic disorder and specific phobia exhibit reduced ventromedial PFC activity  and vlPFC (area 12) in a new world monkey, the common marmoset. Two distinct tests of negative emotion were studied, Pavlovian discriminative fear conditioning and a test of anxiety typically used in monkeys, the human intruder test. Lesions of both regions resulted in stronger, less adaptable conditioned fear responses and heightened anxiety  Act.Callithrix jacchus; 26 females, 23 males, average age 2.7 years ranging 1.8–4.2) were presented with a model snake in their home cage. The data from a subset of these animals (31) had been used in Shiba et al.  .49 naïve common marmosets  and dark brownish in color with black stripes. The model snake was contained in a triangular prism box made of opaque white Perspex (26 cm × 26 cm × 29.5 cm triangle sides × 30 cm high). By removing the sliding door of the box, the snake could be revealed to the subject. The box was designed to conceal the snake from all marmosets except the target subject. The animals had never seen the snake or the box before the experiment.Test procedures were identical to the ones previously described . For recording the vocalizations, the shotgun microphone was used to ensure that the target animal's calls could easily be distinguished from any other animals' calls in the room. The calls were analyzed with sound spectrogram . Since many of the behaviors were only displayed in the presence of the snake, only distance and locomotion could be scored across all phases. Inter-rater reliability was assessed by comparing the observers' scores on 15 randomly chosen animals (Table p < 0.0001) ensuring that the correlations between variables are overall significantly different from zero. The component axes are rotated to maximize the loadings of variables onto each component. The paradigm was designed to test the psychological constructs underlying the various behaviors expressed by animals in response to the model snake, which are not completely independent from each other , Friedman test was used to compare the means across the phases, and post-hoc comparison was performed with Wilcoxon signed-rank test. For correlational analyses, Pearson's r was used for the variables that were normally distributed or Spearman's ρ was used for those that violated the normality assumption.All analyses were performed using a statistical software SPSS (ver. 17–21). For the “Snake” phase, principal component analysis (PCA) was performed to reduce the separate but correlated measures into weighted composites that reflect underlying psychological dimensions Field, . Adequacr Field, . Thus, or Field,  and usedF = 2.46 p = 0.131].14 marmosets  that were not included in Experiment 1, were used. The housing condition and diet were the same as described in Section 2.1.1. All of the animals had experience of a discriminative fear conditioning paradigm and HIT as part of a previously reported behavioral study  or vlPFC (n = 3). The animals had at least a 2-week recovery period before behavioral testing.Surgical procedures have been described in an earlier report  were applied to the z-scores and the products were summed for each component  was used to carry out statistical analyses. To calculate the component scores of each animal, first, behavioral scores were standardized using the mean and standard deviation of all experimental groups, then, the component score coefficients obtained from the PCA with the larger sample  = 71.25, p < 0.0001, for locomotion, χ2(3) = 33.67, p < 0.0001] . A similar pattern of variable loadings on the emotionality component was reported in the human intruder paradigm  = 19.46, p < 0.001; post-hoc pairwise comparison of Phase: “snake” vs. “pre-snake” p < 0.001, “snake” vs. “post-snake” p = 0.008]  = 6.10, p = 0.002; post-hoc pairwise comparison of Phase: “snake” vs. “separated” p = 0.003, “snake” vs. “pre-snake” p < 0.001, “snake” vs. “post-snake” p = 0.012] , main effect of Phase: ] Figure . Locomot] Figure .F = 12.65, p = 0.001, post-hoc pairwise comparison for “emotionality” component: “antOFC” vs. “control” p = 0.007, “vlPFC” vs. “control” p = 0.030]  = 3.90, p = 0.052; post-hoc pairwise comparison for “control” vs. “antOFC” p = 0.018]  = 5.35, p = 0.024; post-hoc pairwise comparison for “control” vs. “antOFC” p = 0.016, “control” vs. “vlPFC” p = 0.018]  = 2.92, p = 0.096; post-hoc pairwise comparison for “control” vs. “vlPFC” p = 0.045]  = 3.45. p = 0.069; post-hoc pairwise comparison for “control” vs. “vlPFC” p = 0.028]  < 1] , Group × Component interaction ] Figure . There w] Figure , both an] Figure  and the ] Figure  and fewe] Figure . The gro] Figure .F = 12.65, p = 0.001, post-hoc pairwise comparison for “coping strategy” component: “antOFC” vs. “control” p = 0.039, “vlPFC” vs. “control” p = 0.005]  = 6.19, p = 0.045; post-hoc pairwise comparison Mann-Whitney Test, “antOFC” vs. “control” U = 2.00, p = 0.050, “vlPFC” vs. “control” U = 2.00, p = 0.027]  = 1.65, p = 0.237] , Group × Component interaction ] Figure . There w] Figure . The gro] Figure .Marmosets showed a relatively consistent pattern of behaviors in response to the presence of a predator threat, i.e., model snake, in their home cage (Experiment 1), although the extent to which individual animals displayed these behaviors differed quite considerably. Seven distinct behaviors and vocalizations were identified. PCA revealed two underlying components, which were labeled “emotionality” and “coping strategy,” based on the pattern of behaviors and vocalizations loading on each of the components. Compared to the sham-operated controls, marmosets with excitotoxic lesions of either vlPFC or antOFC had significantly higher “emotionality” scores, reflecting the animal's heightened anxiety/fear-related responses to the snake (Experiment 2). The lesioned animals also had a reduced “coping strategy” score. In particular, they emitted markedly fewer mobbing calls than controls. These results support the hypothesis that ventral PFC plays a role, not only in regulating learned fear and anxiety, as shown in our previous study (Agustín-Pavón et al., Fear of snakes has been widely exploited to induce anxiety/fear responses experimentally in primates. Compared to the periods before and after exposure to the snake, during the snake presentation, all marmosets displayed an avoidance response, spending more time at the back half of the cage and showing reduced locomotion. They also displayed varied levels of “attentional” responses directed at the snake, in the form of head cocks Menzel,  and starMarked individual variability between marmoset's behavioral emotionality responses to the snake has been reported previously and proposed to represent a spectrum of anxiety trait present within a population (Shiba et al., The finding that selective lesions of the antOFC and vlPFC in marmosets heightened the emotional responses to predator threat is consistent with the heightened emotionality they displayed to a human intruder (Agustín-Pavón et al., Given that the OFC, including areas 11 and 13, send projections to the GABAergic intercalated cells within the amygdala, which in turn issue inhibitory projections to the central nucleus (Ghashghaei and Barbas, Besides the contribution of antOFC to regulation of emotional responses to predator threat our current study also demonstrated that lesions of the vlPFC, independently from that of the antOFC, result in enhanced anxiety/fear-related responses. This is consistent with our previous finding that selective excitotoxic lesions of the vlPFC resulted in less adaptable conditioned fear responses, overall heightened behavioral and autonomic responses in fear discriminative conditioning and enhanced anxiety-related behaviors in response to a human intruder (Agustín-Pavón et al., reduction in response to predator threat should be contrasted with the marked increase in tsik and tsik-egg calls made by these same vlPFC lesioned animals, when compared to controls, in response to a human intruder (Agustín-Pavón et al., Besides a marked increase in emotional reactivity to predator threat, lesions of either the antOFC or vlPFC also significantly attenuated “coping strategy” responses. This effect was mainly driven by reduced numbers of tsik vocalizations, the mobbing and alarm call made to a threatening stimulus (Bezerra and Souto, One potential explanation for the opposing effects of lesions on vocalizations in response to predator threat and a human intruder may lie in the interaction between overall levels of emotionality and the coping strategy adopted. Emotional reactivity and coping response are not necessarily independent psychological dimensions. The strength of the emotional response may interact directly with the cognitive strategy adopted and may follow an inverted U-shaped function. Hence, when emotional reactivity to a human intruder, which is normally much less than that to a snake, is increased following PFC lesions, this may increase the likelihood that an animal adopts an active/aggressive strategy. In contrast, when emotional reactivity to a snake is increased following PFC lesions, the overall level of reactivity may be considerably greater, such that it acts to reduce the likelihood of an animal adopting an active/aggressive strategy and instead induces a withdrawal response, including an inhibition of vocalizations. In support of this, those animals in the colony that show the most extreme withdrawal response to the snake tend to also stay silent.per se. Given that marmosets display distinct patterns of behavior, including distinct vocalizations, in response to a human intruder and snake, then it is essential that animals recognize the different social and biologically relevant stimuli, and implement the appropriate coping behaviors. A snake commonly predates on marmoset monkeys in the wild (Ferrari and Ferrari, However, an alternative and equally plausible explanation is that the lesions disrupted the decision making process per se, remains to be determined. The finding that the pattern of emotion dysregulation appears similar following lesions of these two anatomically distinct regions leaves open the question as to their differential contributions. Given that activity in OFC neurons codes for upcoming appetitive and aversive motivational outcomes (Murray et al., In conclusion, the present study demonstrates that localized excitotoxic lesions of either the primate antOFC or vlPFC leads to enhanced fear-related responses to a predator threat, which implicates these ventral prefrontal sub-regions, not only in the regulation of conditioned fear and anxiety, as we had shown previously (Agustín-Pavón et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The stimulus feature that is most efficiently encoded by bursts, however, need not coincide with one of such classical features. We therefore searched for the optimal feature among all those that could be expressed as a linear transformation of the time-dependent input current. We found that bursting neurons transmitted 6 times more information about such more general features. The relevant events in the stimulus were located in a time window spanning ~100 ms before and ~20 ms after burst onset. Most importantly, the neural code employed by the simple and the biologically realistic models was largely the same, implying that the simple thalamic neuron model contains the essential ingredients that account for the computational properties of the thalamic burst code. Thus, our results suggest the n-spike burst code is a general property of thalamic neurons.Thalamic neurons have been long assumed to fire in tonic mode during perceptive states, and in burst mode during sleep and unconsciousness. However, recent evidence suggests that bursts may also be relevant in the encoding of sensory information. Here, we explore the neural code of such thalamic bursts. In order to assess whether the burst code is generic or whether it depends on the detailed properties of each bursting neuron, we analyzed two neuron models incorporating different levels of biological detail. One of the models contained no information of the biophysical processes entailed in spike generation, and described neuron activity at a phenomenological level. The second model represented the evolution of the individual ionic conductances involved in spiking and bursting, and required a large number of parameters. We analyzed the models' input selectivity using reverse correlation methods and information theory. We found that  Thalamic neurons can respond to input in either tonic or bursting modes  or duration, may provide downstream cells with information not conveyed by the temporal placement of bursts . The first model contains a simple integrate-and-fire mechanism with an added slow IT current to enable bursting  and the integrate and fire or burst (IFB) models. The two models contain different levels of biological detail. Figure IT) conductance that permits bursting. Unless stated explicitly, we used the original parameters defined by Smith et al.  where gL and EL are the conductance and reversal potential, respectively. The IT current is defined as IT = gTm∞h(Vm − ET) where the activation function m∞(Vm) = Θ(Vm − Vh) is modeled in terms of a Heaviside step function Θ(x) = 1 if x > 0, and Θ(x) = 0 if x ≤ 0. The inactivation variable h is governed by dh∕dt = −h∕τ−h when Vm>Vh, and by dh∕dt = (1−h)∕τ+h otherwise. The channel is hence open for Vm < Vh, and the inward Ca2+ current depolarizes the neuron. However, as Vm surpasses Vh, the inactivating variable h drops to zero with a time constant τ−h = 20 ms, thereby closing the channel. When the membrane potential decreases below Vh, h returns to unity with a slow time constant τ+h = 20 ms, re-opening the channel. We use the same parameters as Smith et al. .The leak conductance h et al. , namely,The more complex MC model was built using Hodgkin-Huxely conductances with further conductances simulating burst behavior , where gL = 0.1 mS and EL = −72 mV. The INa current is defined as NaI = gNa3m∞ (0.85−n)(mV − NaE), where gNa = 42 mS and ENa = 55 mV. The activation function m∞ follows the general equilibrium equation x∞ = αx∕(αx+βx), where x may be m, n or h. The gating variables αm and βm are defined as αm(Vm) = 0.1(Vm+29.7−σNa)∕(1−exp(−(Vm+29.7−σNa)∕10)) and βm(Vm) = 4(exp(−0.0556(Vm+54.7−σNa))), with σNa = 3. The variable n participates both in the equation for INa and IK (see below). In the equation for INa, it is preceded by a negative sign. Therefore, the term 0.85−h operates as an inactivating variable. Its evolution is governed by dn∕dt = 200(n∞−n)∕7(τn). Here, n∞ follows the general equilibrium expression, with αn(Vm) = 0.01(Vm+45.7−σK)∕(1−exp(−0.1(Vm+45.7−σK))), βn(Vm) = 0.125(exp(−(Vm+55.7−σK)∕80)), τn(Vm) = 1∕(αn+βn) and σK = 10. The IK current is defined as KI = 4gKn where the activation variable S∞ is S∞ = 1∕(exp(−(Vm+65)∕7.8)) and the inactivation variable h is governed by dh∕dt = 2(h∞−h)∕τh, with h∞(Vm) = 1∕(1+exp(−(Vm−θh)∕kh)) and τh = h∞exp((Vm+162.3)∕(17.8)+20). The parameters are gT = 0.3 mS, ET = 120 mV, θh = −81 mV and hK = 6.25 mV−1. The ISag current is defined as ISag = gSagH2(mV − Esag). Its gating variable H is governed by dH∕dt = (H∞−H)∕τH where the equilibrium variable is H∞ = 1∕(1+exp((Vm+69)∕7.1)) and τH = 1000∕(exp((Vm+66.4)∕9.3)+exp(−(Vm+81.6)∕13)). The persistent sodium current INaP is defined as INaP − gNaP3mP∞(mV − NaE), with mP∞, αPm and βPm follow the general expressions for m∞. The tuning constant σNaP = −5 and its conductance is gNaP = 9 mS. Like the IFB-T model, we also produced a tonic variant (MC-T) by removing all burst-associated conductances (gT = gSag = gNaP = 0 mS) and increasing gNa to 120 mS. For both tonic and bursting models, the membrane capacitance is 1 μFcm−2OU∕dt = ((μOU−OU)+uξ)∕τOU where μOU is the stimulus' mean, τOU is the correlation time, ξ is Gaussian distributed white noise with unit variance  colored noise signal  ratio where N:S = 5. The amplitude of the OU stimulus for each model were 0.8μA (MC), 0.5μA (IFB), 5μA (MC-T), and 3μA (IFB-T). These values produced physiologically plausible firing rates within the range of 5.8 ± 3.6 Hz and cross-trial variability of 0.43 ± 0.05 ms  or larger (2h) time steps.Both models were integrated with a backward-implicit implementation of the Euler-Murayama stochastic integration method  with respect to the mean M(t)  and V(t) were estimated as the mean and the variance across trials.When presented with multiple trials of the same stimulus, neurons do not produce identical responses. In particular, the number of spikes in a given time window may fluctuate. The Fano factor is a relative measure of the variance of the spike count I to quantify the information carried by the entire response. We also used a derived information measure ΔI . Under the ergodicity assumption  is therefore P(r) = 〈 P(r∣t)〉t, where 〈 …〉t represents a temporal average.The conditional probability of observing a particular response word We then calculated Shannon mutual information between the response and stimulus Shannon, T → ∞ of the ratio I∕T. In practice, we found that this ratio tends to a well-defined limit for T>20 ms. As T is increased further, a stable value is obtained inside the range T ∈  ms. For T > 60 ms, however, the estimates deteriorate rapidly due to limited sampling. For these reasons, here we set T = 40 ms (L = 8) bins to avoid sampling limitations. For shortness, information rates are reported just as information throughout the paper.The information rate is defined as the limit for I contains the contributions of independent and correlated spiking activity. Previous studies have reported information being carried by trains of independent spikes. For example, different stimuli may induce different spike counts within a set time window  it is possible to compute the independent distribution Pind(r∣t), where all correlations across time are ignored  = 〈 Pind(r∣t)〉t, the quantity ΔI is given byWe assessed the amount of information carried by stimulus-modulated correlations by using the measure ΔI and ΔI estimates .The stimulus amplitude at burst onset x(t)], where t is taken from a window spanning the stimulus prior to burst onset, starting at t+Tstart—with Tstart < 0—and ending at burst onset t = 0.The stimulus minimum in a time window preceding burst onset min∕ReHil[x(t)]}, where Im and Re indicate the imaginary and real part, respectively  are encoded by n-spike bursts, we estimated Shannon mutual information between F and burst size . The marginal distribution is P(f) = < P(f∣n)>n. The total entropy then reads,To quantify the extent up to which specific stimulus features , which represents the average stimulus deflection surrounding an n-spike event de Boer and Kuyper   of burst containing n spikes. Those features may or may not be the same, for different n-values. Therefore, although they are useful to predict whether a burst of specifically n spikes is expected, they are not necessarily useful to discriminate between different n-values. In order to identify the stimulus features that optimally discriminate between bursts of different duration, we turn to multi-discriminant analysis (MDA) , in decreasing order of effectiveness in discriminating clusters. The components of each vector represent the weights with which each time bin participates in the selected axis  Vj, obtaining a set of scalar quantities yij, where the index i runs over the set of stimulus windows. Using the descretized projected values (M = 32) we calculated the distributions P(yj∣n) and P(yj), and the associated entropies H(Yj) and H(Yj∣N) using Equations . The mutual information was then found by computing I = H(Yj)−H(Yj∣N)). We limited the analysis to the most significant discriminants, which in our data included j = 1 and j = 2.Once the vectors are obtained, in order to calculate the mutual information corresponding to the vector M(t) and variance V(t) of the spike count in 20 ms non-overlapping windows across 100 trials , as shown in Figures V(t)∕M(t) < 1) and with super-Poissonian (V(t)∕M(t)>1) behavior, see Figures V(t)∕M(t)>1) are indeed due to bursting rather than other causes like, for instance, fluctuations due to insufficient sampling.The variability of neuron responses to repeated presentations of a stimulus is usually quantified by means of the Fano factor, that is, by the ratio between the spike count variance and the spike count mean, both measured in a given time window. The Fano factor of a neuron governed by a Poisson firing mechanism is equal to one. Systematic deviations from unity indicate the presence of correlations between spikes  and 9.8% (IFB) of the total information I. The tonic firing MC-T and IFB-T models conveyed much less information in correlations, 1.1 and 2.0%, respectively. Responses where bursts were removed directly (BR) also conveyed significantly less information through stimulus-modulated correlations, yielding 2.3% (MC) and 1.1% (IFB). Hence, the spike correlations induced by bursting convey information about the stimulus, which is consistent with previous in-vivo results  for different burst sizes from the MC (A1) and IFB (B1) models . For both models, all n-ETAs contain a pre-onset hyperpolarization followed by a post-onset depolarization (Time = 0 ms indicates burst onset). The double-peak structure observed for positive times in both models closely resembles the results obtained by Samengo et al.  amplitude.To determine the stimulus features encoded by bursts of different length, we used reverse correlation methods Rieke, . We stimo et al.  for quadn.In other brain areas, bursts were shown to encode instantaneous stimulus parameters, like slope  and negative stimulus integral driving the IFB model (B2), single-spike events (n = 1) slightly departed from the common trend. Interestingly, the case of n = 1 is special. While for the purposes of the analysis we take all n = 1 events as single spikes, the ensemble of all such events can be separated into two distinct classes. Some of the single spikes are fired without activation of the IT current; these are purely tonic spikes. There are other events with n = 1, however, that are fired with a significant activation of the IT current; in a strictly dynamical sense, these are 1-spike bursts. A more detailed discussion of this distinction is given as supplementary information.Almost all parameters varied monotonically with increasing In summary, both models encode similar ranges of instantaneous stimulus parameters with the same overall increasing or decreasing trends. The similarity survives despite the different levels of biological complexity contained in the models.n-spike bursts that have increased or decreased variance, as compared to a random selection of stimuli. The features described by those directions are the uncorrelated components of the stimulus that are associated with an n-spike burst. The ETA is a specific linear combination of such features . The idea is to search for stimulus directions associated with n, the two most relevant directions corresponded to decreased variances. The corresponding eigenvectors are displayed in Figure n-dependent structure.For the two models studied here, and for all values of Notably, in both models the most relevant eigenvector only contained pre-onset structures. The second eigenvector, instead, contained both pre- and post-onset features. Hence, some of the features triggering bursts (the ones in the most relevant eigenvector) are statistically uncorrelated with the features terminating bursts , 1-spike bursts (blue curve) require an initial membrane depolarization at −50 ms followed by the priming hyperpolarization/trigger. Single IFB model spikes are evoked by a hyperpolarization followed by a sharp depolarization at onset. In both models, larger events (n>1) display hyperpolarization triggers whose shape is largely unchanged for different n, indicating little discrimination.Qualitatively, the lowest-variance STC vector (panels A1 and B1) represents the pre-onset priming of the V479 evoking MC model responses (A2) displays n-dependent structure both pre- and post-onset. Pre-onset n-dependent regions range from −90 ms to onset, the majority of this dependence stems from the difference between vectors associated with single spikes and multi-spike bursts. Post-onset, these vectors show n-dependent structure extending up to 38 ms after burst onset.The second lowest variance vector V380 (B1) shows a difference in stimulus preference between single spikes and bursts beginning −200 ms before onset. The second lowest variance vectors V379 (B2) showed a graded dependence on n. As n increases, the pre-onset hyperpolarization decreases in depth but increases in duration.For the IFB model, the lowest variance ETC vector n varies are encoded in the presence or absence of bursts, but not in the distinction between bursts of different lengths. Distinctions between bursts can only encode stimulus features that vary significantly with n. In order to make these statements quantitative, in the following sections we calculate the amount of information encoded by bursts.The stimulus features that remain unchanged as n and stimulus feature F . The results are displayed in Figures In Figures IT-mediated bursting neurons tested here, slope coding was negligible. Bursts from both models could discriminate Nint, that is, the amount of negative charge entering the neuron in the 250 ms prior to burst onset. Interestingly, the encoding of Nint was a time-demanding process, since in both models bursts were found to encode almost no information about the instantaneous amplitude Min of the minimum negative stimulus inside the same window. Finally, the positive charge entering both models was only weakly discriminated by bursts of different spike count.Among all the tested stimulus features, the stimulus' phase at burst onset was the one with highest mutual information . Samengo and Montemurro  observedIT and ISag. The ISag current slowly rectifies prolonged (lasting >400 ms) hyperpolarizing membrane voltage deflections and has little effect on the timescales of burst stimulus selectivity. The IT current has a strong influence on burst stimulus preference. The timescale of this current is controlled by a voltage-dependent variable τh which has an average value of 42 ± 18 ms. For the IFB model, the timescale of the IT current is given as τ+h = 100 ms. The timescales of bursting currents therefore closely match the timescales of amplitude discrimination in the IFB and MC models.The values of the stimulus amplitude, slope, and phase used to calculate the mutual informations reported in Figures n-ETAs in Figure n-ETAs that show the greatest n-dependent shape. Similarly, the area of poor discrimination is due to a n-independent depolarizing burst trigger, which can be seen in Figure n-dependent areas of n-ETAs . It is useful to compare these features with s Figure . In boths Figure . Howevern and different instantaneous stimulus features was measured. The features were chosen due to their simplicity and their biological relevance, and it is important to notice that they are not independent from one another. For instance, the amplitude at a given time depends on previous values of amplitude and slope, while the phase at a given time depends on the amplitude at previous times. Thus, the distributions in Figure In Figure n of the bursts they trigger. This problem is solved by multi-discriminate analysis (MDA)  when distinguished by the number of spikes k time points is represented by a single point in a k-dimensional space. Stimuli evoking bursts of n spikes should share some common structure and cluster together in this space. The MDA method finds linear axes in k-dimension space that best separate different n-triggering stimulus clusters. The obtained axes are ranked, with the first axis V1 pointing along the direction of maximum discrimination, followed by V2 along the second optimal direction, and so on  so only the stimulus directions where different V1 and V2) estimated for MC model bursts. Figures n-spike bursts.Figures n = 1 differs from the first eigenvector of all other bursts  far outperforms the discrimination of instantaneous features. We also verified that these results are qualitatively unchanged when using input signals with half or double the correlation time used in Figure For the MC model, the first MDA vector Figure  includess Figure . In thisn Figure . Here, dThe second MDA vector of the MC model Figure  shows seV1 and second V2 MDA vectors , and those of bursts containing multiple spikes (n>1). Once again, the resulting information (0.43 bits/event) markedly outperforms the discrimination of instantaneous stimulus parameters. The second MDA vector V2 (B3) shows non-zero structure spanning from −250 to 30 ms after onset. This interval coincides with the second ETC eigenvector where n-dependent structure is apparent .The increased information values obtained from MDA implies that Tstart and Tend. For each window, we project the stimulus on the obtained optimal axis, and calculate the mutual information, as shown in Figure Tstart < Tend. Figure Tend < 0. Windows in region (b) are entirely located after burst onset, since even Tstart > 0. Windows in region (c) are astride burst onset, since Tstart < 0 and Tend > 0.The directions obtained with MDA, and consequently, the information values derived from projecting the stimulus on the first MDA vector, depend on the location of the time window used to represent the stimuli. In order to determine the optimal window, we now repeat the analysis, systematically sweeping the location of the first and last times defining the window, n-spike triggering stimuli onto the first V1 discriminant vector obtained from MDA. Information maps for the V2 vector are given as supplemental information. The data processing inequality , the window shrinks and the information necessarily falls down to zero. The same happens when Tstart is increased and approaches Tend. The optimal window is the one encompassing all informative events, and no uninformative events. For the MC model, the optimal windows are around Tstart ≈ −70 ms, and Tend ≈ 20 ms. For the IFB model, the corresponding values are −200 and 30 ms. In both models, n-burst events are more sensitive to the stimulus preceding burst onset than those coming after.Information maps for the MC and IFB models are plotted in panels B and C, respectively. The information is calculated after projecting all In the supplementary information we analyze the information maps for the tonic firing MC-T (A) and IFB-T (B) models. Removing the burst currents diminished the number of generated bursts, but did not eliminate them completely: Tonic firing may occasionally still produce packets of high-frequency discharges. The amount of information encoded in the remaining tonic bursts, however, dropped drastically. Interestingly, tonic bursts lost the ability to discriminate pre-onset stimulus features, but retained the post-onset features. Hence, the information encoded by time windows located in different regions of the maps of Figure n-spike burst code. We report that these bursts reliably modify their internal spike count (n) in response to different stimulus deflections that span up to 300 ms prior to burst onset. Moreover, we show that this code can induce stimulus-modulated correlations that are similar to the ones found in in-vivo thalamic neuron responses , which existed only when bursts were present in the response. Work by Montemurro et al.  and a more complex multi-conductance (MC) model with stochastic stimuli. The two models produced responses containing significant amounts of information through stimulus-modulated spike correlations . Therefore, assuming bursts encode instantaneous stimulus properties may underestimate their capacity to encode stimuli. In support of this idea, we found that n-spike bursts discriminated instantaneous stimulus features (particularly amplitude and phase) over a whole range of times. This suggests that burst size is tuned by multiple stimulus factors that cannot be quantified by a single 1-dimension feature. To analyse stimulus discrimination without this limitation, we used MDA to reduce the stimulus dimensionality into linear axes (discriminant vectors) along which the stimulus distributions evoking different n-spike bursts were most separable , UK. Grant code: BB/D526561/1. We also thank the Agencia Nacional de Promoción Científica y Tecnológica and the Ministerio de Ciencia y Tecnología of Argentina.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Objective: Medically unexplained symptoms are physical symptoms, which cannot be explained by organic causes. This study aimed to investigate mental health in patients with medically unexplained physical symptoms. Method: One hundred outpatients who were admitted to the Electro Diagnosis Clinic of Imam Khomeini hospital, Ahvaz/Iran, participated in this study. Data were collected using physical examination, paraclinical examinations, and SCL-90-R, and analyzed through multivariate analysis of variance  (MANOVA), Chi-square test and Fisher’s exact test. Results: The findings revealed significant differences between clients with medically explained and unexplained symptoms in obsessive compulsive and somatization (p<0.05). Differences in depression, anxiety, phobia, psychosis, aggression and paranoia were not significant (p>0.05).Conclusion: The present study suggested an association between some psychological problems and somatic symptoms. Therefore, screening for psychological impairments can improve clinical outcomes. Many patients have a range of persistent bodily complaints that have no identified pathology and cannot be corresponded to any organic disease . These symptoms, which are called medically unexplained symptoms (MUS), are common in all ages and all field of medicine around the world . PrevaleTreatment of patients with MUS can be frustrating . BecauseThere is evidence to suggest that psychological trauma relate to vulnerability to more medical symptoms, and psychological factors play an important role in development of MUS . Between 26 to 58% of patients with MUS have been reported to have co morbid anxiety or/and depression , 11, 12 Although several studies support the link between psychological problems and MUS, there are some lines of evidence that challenge this association; for example, Morriss et al. (1999) did not find an association between depression and the reporting of pain, psychophysiological syndromes and medically unexplained symptoms in patients with chronic fatigue syndrome . JacksonBecause of these controversial findings, and since the association between medically unexplained symptoms and mental health has received little attention in Iran, the present study was aimed to compare the mental health of patients with medically unexplained physical symptoms and those with a medical diagnosis in Ahvaz, Southwest Iran.This study was conducted at the Electro Diagnosis Clinic of Imam Khomeini Hospital affiliated to Jundishapur University of medical sciences in Ahvaz, Iran. The sample of the study consisted of 100 patients (18 years and older) who referred to the Electro Diagnosis Clinic by endocrinologists, orthopedists, neurologists or neurosurgeons. The clinical appraisals by these specialists showed that the patients’ physical complaints had no medical explanations. Therefore, they referred to the center for paraclinical appraisals.Paraclinical appraisals including imaging (CT and MRI), the specific neurologic examination and EMG and NVC were done. Paraclinical assessments were prescribed by a specialist and no additional cost was imposed to the patients.After describing the procedures and purposes of the study, informed consent was obtained from the participants. To obtain demographic data about ethnicity, gender, age, level of education and marital status, each participant was asked to complete the Persian version of Symptom Distress Check List (SCL-90-R) as well as a questionnaire. Exclusion criteria included the presence of intense pain, mental retardation (IQ < 70), and dissatisfaction with the cooperation.The study was approved by the Ethics Committee of the University (No: Eth-105). Data were collected over a period of 12 months from 2014 to 2015.According to the clinical and paraclinical appraisals and specialist diagnosis, patients were divided into two groups: Group I: Patients with medically unexplained or poorly explained symptoms Group II: Patients with medically explained symptomsInstruments:-      Physical Examinations-      Paraclinical Tests including Imaging (CT and MRI), EMG, NCV-     Symptom Distress Check List (SCL-90-R): SCL-90-R is a self-report scale which has ten subscales including obsessive-compulsive (OC), somatization (SOM), phobic anxiety (PHOB), interpersonal sensitivity (INT), depression (DEP), anxiety (ANX), anger-hostility (HOS), psychotism (PYS), paranoid thought (PAR) and extra scale (ES). The scale has a general symptom score. Higher scores represent more problems .Statistical analysisWe used Kolmogorov–Smirnov test to determine if the data had normal distribution. Data were normally distributed into two groups for total score of SCL-90 and its subscales . Data were analyzed using multivariate analysis of variance (MANOVA), Chi-square test and Fisher’s exact test. The probability level of 0.05 was accepted as statistically significant. Statistical analyses were carried out using SPSS version 16. Among the participants, 19 (19%) had medically unexplained symptoms and 81(81%) had symptoms with identified pathology. The mean age of the participants was 37.42 years (±14.03) for the patients with MUS and 44.76 years (± 12.45) for patients with clinical diagnosis. There was a significant difference between the two groups in age .Frequencies of the participants’ demographic features are listed in The mental health of the patients with MUS and patients with medical diagnosis was compared using multivariate analysis of variance (MANOVA). There were significant differences between the two groups in obsessive-compulsive  and somatization .Differences in aggression, paranoia, depression, anxiety and phobia were not significant at p<0.05. This study was conducted to investigate mental health in patients with medically unexplained physical symptoms and was the first to report mental health in patients with painful somatic symptoms in different parts of body including the head and neck, chest, back, abdomen, hip, lower limb and upper limbs in the Iranian population. Pervious researches , 20 in IThe results revealed significant differences in obsessive-compulsive and somatization between the two groups of patients with and without medically unexplained physical symptoms. Differences in depression, anxiety, phobia, psychosis, aggression and paranoia were not significant. However, Zarghami et al. 2006) found impairment in all dimensions of mental health measured by SCL-90-R in patients with unexplained LBP than in those with well-defined LBP . In addi06 found In contrast to our results, many studies showed depression and anxiety in chronic pain patients with MUS  -28. NeveWe found no significant difference between the two groups in gender, marital status, ethnicity and level of education. Maiden et al. (2002) also reported no association between unexplained symptoms and educational level .But Verhaak (2006) found that patients with MUS are more often female and less educated than the average patients or patients with identified diagnosis . ZarghamPrevalence of MUS has been shown to have a wide range of 1.6–70%, 2.4–87%, and 4.6–18%, in the younger, middle (50–65 years) and older (≥65 years) age groups, respectively . In the This gap might be somewhat due to the features of the participants, methodological differences in the assessment of psychological factors, lack of agreement in defining MUS, or cultural differences in somatic presentations of psychological problem. In this study, we used paraclinical examinations, while a few previous studies had used paraclinical assessments. The association between psychological factors and medically unexplained manifestations in the current study may improve the screening of mental impairments in routine health examinations.Psychological interventions could be included as an effective part of mental health improving interventions considered for patients with MUS. Therefore, a collaborative working relationship between physicians, psychologists and psychiatrists should be established to manage health care users with medically unexplained symptoms.In conclusion, our results revealed that some psychological problems might relate to vulnerability to somatic symptoms. Therefore, screening for psychological impairments can improve clinical outcomes. Several limitations should, however, be taken into account in interpreting our findings. It is possible that somatic manifestation of psychological problems is a cultural phenomenon . Therefo Lack of psychiatric interview was another limitation of this study, so it is recommended for future studies. Finally, the present study included the relatively small sample size, so conducting further studies with a larger sample size is highly recommended.In spite of these limitations, our findings had some important implications for physicians in management of their patients. They should be aware of the relationship between mental health and somatic symptoms.


There was a linear decrease in dressing percentage (DP) with source × level interaction (p ≤ 0.001), while there was a linear increase in breast yield and thigh yield with increasing dNa supplementation (p ≤ 0.001). Chicks fed 0.35% NaHCO3 and 0.44% dNa Na2SO4 supplemental salts had lower abdominal fat (p ≤ 0.04). Chicks that received increasing levels of dNa (from 0.17 to 0.44%) showed increasing gizzard weight (p ≤ 0.002) and decreasing spleen weight (p ≤ 0.02). When both salts were supplemented at 0.26% dNa, the chicks showed their lowest bursa weight (p ≤ 0.001). Consequently, chicks at higher dNa showed an increase in breast and thigh meat yield, and increasing capacity of their digestive organ. The higher levels of dNa should be tested with other cations and anions to fully understand acid base homoeostasis.The effect of sodium and sodium salts on carcass and body organ characteristics of broilers under a four phase feeding program were investigated. A basal diet (0.08% dNa with NaCl) was formulated and one of two sources of dNa (NaHCO Rondon et al.[.[+, K+, or Cl−. Recently, Mushtaq et al.[3) as the best supplemental salt for Na+ and HCO3−[2SO4) induced severe blood acidosis when it was compared with other sulphate sources in growth trials; hence it was suggested that the acidic properties of sulphates are directly linked to supplemental salts [Mongin  describeon et al. and Borg et al.[. reportedaq et al., 7 testeaq et al., 8–10. Mand HCO3−–16. Sodial salts .3 in water) under normal but not under cyclic environmental conditions [+ and K+ in the breast and thigh muscles of broilers from 4 to 8 wks old under high temperature (32°C). The author, moreover, observed that breast muscles had significantly lower Na+ and higher K+ concentrations than thigh muscles. Pourreza and Edriss [A significant effect of temperature on abdominal fat was found for treatments  were given one of eight dietary treatments replicated four times; under this system each replicate contained 40 birds (without considering the sex). Each replicate pen was equipped with a separate overhead, transparent, and volume-graduated 20 L water bottle linked to a nipple drinker line. Water bottles were cleaned and filled with fresh water on a daily basis. One flat bottom round feeder was provided for each experimental pen. Birds were housed in environment control systems where variation in temperature and relative humidity were recorded and maintained according to the production manual . ContinuBirds were vaccinated against Newcastle Disease (ND) plus Infectious Bronchitis viruses at 4 d, Infectious Bursal Disease virus at 8 d and again at 14 d; Hydropericardium Syndrome virus at 18 d, and ND-Lasota strain at d 22.3) or sodium sulphate (Na2SO4). The levels of dNa corresponded to DEB values of 200, 240, 280, and 320 mEq/kg, respectively. The experimental period was divided into four phases: pre-starter (1 to 10 d), starter (11 to 20 d), grower (21 to 33 d), and finisher (34 to 42 d) which met or exceeded the nutrient specifications recommended by the Hubbard management guide , BW gain  and feed-to-gain ratio  were recorded at the end of the experiment. The feed was withheld for 6 h before weighing the birds to ensure the emptying of the digestive tract of the bird. Mortality was recorded on a daily basis and dead bird was weighed prior to removal to correct FG.At the end of 42 d, two birds were randomly selected from each replicate and subjected to carcass and body organ evaluation. The feed was withheld for 6 h before slaughter to ensure emptying of the digestive tract. Carcass and body organ responses were evaluated in terms of dressing percentage, breast, thigh, abdominal fat, gizzard, proventriculus, heart, liver, kidney, spleen, pancreas, bursa, gallbladder, intestine, and lung weights, and for the shank and intestine lengths ; therefore, supplied water was analyzed for these electrolytes. Water characteristics were also recorded twice (morning and noon) daily to check pH by pH meter (LT-Lutron pH-207 Taiwan) and dissolved oxygen by DO meter . Moreover, temperature, electrical conductivity (EC), total dissolved solids (TDS), and salinity were recorded by the Combo meter . A statistical significance of 0.05 was used unless stated otherwise.Water was evaluated on a daily basis for quality parameters including temperature, pH, EC, TDS, DO, and salinity Table . At the 2SO4 at 0.26% showed higher BWG at day 42. However this change in BWG was not sufficient to positively influence feed:gain (FG).All growth responses were unaffected by dietary treatments, except for sodium level and salt interaction on BW gain  and thigh (p ≤ 0.001) meat yield increased with increasing supplementation of dNa  was observed by Mushtaq et al. [The abdominal fat pad was affected with source × level interaction Table . The abdq et al. . In the 3) and 0.35% dNa (Na2SO4). Intestinal weight reflects the gut’s capacity to absorb nutrients, which reflects better health; therefore, higher levels of dNa showed a lower carcass yield.The interaction (level × source) effect was found to change intestinal weight (p ≤ 0.001); the lowest weight (51.6 vs. 51.1 gm) was recorded at 0.44% . This suggests the bicarbonate buffer system mainly determines blood acid–base balance for optimal production performance and functions under regulatory control of the kidneys [Organ weights of the proventriculus, heart, liver, pancreas, gall bladder, lungs, and intestinal and shank lengths were measured and found to be non-significant (data not shown). In contrast, weights (% of dressed weight) of gizzard, kidney, spleen and bursa significantly changed Table . Gizzard kidneys . Kidney  kidneys  or the p kidneys , 36. In + and Cl−), keeping a constant DEB level, and changing salt sources is suggested.Birds showed increased breast and thigh meat yield, and increasing capacity of the gizzard at higher levels of dietary sodium. In contrast, a reduced dressing percentage with increasing supplementation of dietary sodium was unclear. Lower levels of dietary sodium were sufficient for supporting immune organs (bursa and spleen). Therefore a verification of requirements by changing other electrolytes (K