The VIKOR model has been considered a viable tool for many decision-making applications in the past few years, given the advantages of considering the compromise between maximizing the utility of group and minimizing personal regrets. The q-rung interval-valued orthopair fuzzy set (q-RIVOFS) is a generalization of intuitionistic fuzzy set (IFS) and Pythagorean fuzzy set (PFS) and has emerged to solve more complex and uncertain decision making problems which IFS and PFS cannot handle. In this manuscript, the key innovation is to combine the traditional VIKOR model with q-RIVOFS to develop the q-rung interval-valued orthopair fuzzy VIKOR model. In the new developed model, to express more information, the attribute’s values in MAGDM problems are depicted by q-RIVOFNs. First of all, some basic theories and aggregation operators of q-RIVOFNs are simply introduced. Then we develop the origin VIKOR model to q-RIVOFS environment and briefly express the computing steps of this new established model. Thereafter, the effectiveness of the model is verified by an example of supplier selection of medical consumer products and through comparative analysis, the superiority of the new method is further illustrated. In view of the merits of the VIKOR model in considering the compromise between group utility maximization and individual regret minimization, in recent years, it has been recognized as a meaningful tool that can be applied to many decision areas. In previous literature, some traditional decision models have been applied to MADM problems, such as the ELECTRE model ,2,3,4, tBecause of the uncertainty and decision problem of decision support system (DSS), in the practical DSS problem, we often cannot give the accurate evaluation value of the alternative to choose the best one. To overcome this problem, in 1965, the fuzzy set theory defined by Zadeh , initialNevertheless, to describe more decision information, Yager  later deand Wang  put forwand Wang  defined and Wang  gave somand Wang  providedand Wang  came up and Wang  offered and Wang  gave theHowever, to date, it is clear that the VIKOR model with q-RIVOFNs information has not been studied. Therefore, it’s essential to take q-RIVOF-VIKOR model into consideration. The aim of our manuscript is to create an enlarged VIKOR model with the original VIKOR method and q-RIVOF information to settle MADM problems more effectively. Our manuscript is structured as: the definition, score function, accuracy function, operation rules, and some aggregation operators of q-RIVOFSs are briefly given in Based on the theorems of q-ROFSs and the interval values, the essential definition and theorems of q-RIVOFSs are retrospected in brief below.Definition 1  .Let:where the function defines the membership degree and the function defines the non-membership degree of the element to respectively, and, for every x ∈ X, it meets that  is the degree of indeterminacy membership. For convenience, we called  a q-RIVOFN.Definition 2 .Let be a q-RIVOFN, a score function S can be written as follows:Definition 3 .Let be a q-RIVOFN, an accuracy functioncan be written as follows:S and H, the order relation between two q-RIVOFNs will be obtained as below:According to Definition 4 .Let  and  be two q-RIVO FNs, assume that and  be the scores of  and and let and be the accuracy degrees of and respectively, then when when (1) if then (2) if Definition 5 .Let and be three q-RIVOFNs, and some basic rules about them are defined as follows:Definition 6 .Let be a list of q-RIVOFNs with weighting vector be thereby satisfying and then the q-RIVOFWA operator can be written as:Definition 7  .Let be a list of q-RIVOFNs with weighting vector be thereby satisfying and then the q-RIVOFWG operator can be written as:The VIKOR model, which firstly define by Opricovic and Tzeng , is a meStep 1. Establish the decision matrixes Step 2. Calculate PIS Step 3. According to the Formula (7) and the attribute weighting vector Step 4. Calculate the results of Step 5. Then according to Assume that there are Step 1. Give the q-RIVOFNs decision making matrixes Step 2. Calculate PIS For benefit attribute:For cost attribute:Step 3. On the basis of the Equations (17) and (18) and Step 4. Calculate the results of Step 5. According to 1 is the environmental improvement quality; ② G2 is the transportation convenience of suppliers; ③ G3 is the green image; ④ G4 is the environmental competencies. The five feasible medical consumption products suppliers The supplier terms of an enterprise is undoubtedly very important, and in the future will be an even more important influence on the quality of a vendor’s business, as it will affect the business of purchasing, production, inventory and sales, and so on. The relationship between suppliers and future enterprise is not a simple relationship between management and managed, suppliers will become a strategic partner companies, it is a win-win relationship. So supplier preliminary evaluation and selection is quite important. Medical supplies products have their own characteristics to distinguish it from other types of products, which can be distinguished from their production, transportation, marketing, and other aspects. It can be seen that supplier selection of medical consumption products is the classical MADM or MAGDM issue ,70,71,72Next, we make use of the VIKOR technique with q-RIVOFNs developed for medical consumption products supplier selection.Step 1. Give the q-RIVOFNs decision making matrixes Then according to q-RIVOFWA operator and q-RIVOFNs given in Step 2. Calculate PIS Step 3. According to the Formulas (19) and (20) and Then the results of Step 4. On the basis of Step 5. According to In this subsection, we shall compare our presented VIKOR model for q-RIVOFNs with other existing q-RIVOF decision making tools including q-RIVOFWA operator and q-RIVOFWG operator proposed by Wang, Gao, Wei and Wei  to explaAccording to the score function of q-RIVOFNs, the score results For q-RIVOFWA operator:For q-RIVOFWG operator:Then the ordering of alternatives by q-RIVOFWA and q-RIVOFWG operators is listed in Compare the results of the q-RIVOF-VIKOR model with q-RIVOFWA and q-ROIVFWG operators, the aggregation results are a little bit different in ranking of alternatives but the optimal scheme is the same. However, q-RIVOF-VIKOR model has the remarkable characteristics of considering the compromise between group utility maximization and individual regret minimization and can be more accuracy and valid in MAGDM problems.In this manuscript, the q-RIVOF-VIKOR model based on the traditional VIKOR model is presented. Firstly, we started with a review of the concept of q-RIVOFSs and introduced the score function, accuracy function, operation rules, and some aggregation operators of q-RIVOFNs. Furthermore, we combined the traditional VIKOR technique with q-RIVOFNs information, the q-RIVOF-VIKOR model is built and the calculational steps are detailedly given. The proposed model considers the compromise between group utility maximization and individual regret minimization, which is proved to be more accurate and effective. Finally, the new model is illustrated by taking the supplier selection of medical consumer products as an example, and the advantages of the new method are further illustrated by comparing q-RIVOF-VIKOR model with two q-RIVOFNs aggregation operators. In the future, the q-RIVOF-VIKOR model can be used in many other uncertain and fuzzy environments, such as risk analysis ,82,83,84


Scientific reports, 10.1038/s41598-020-65364-8 published online 26 May 2020.Correction to: The original version of this Article contained an error in the title of the paper, where the space between words “the” and “Amalda” was omitted. This has now been corrected in the PDF and HTML versions of the Article.


Leishmania infantum, little is known about how co-infections with or co-seropositivities for other pathogens can influence aggravation of this disease. Therefore, the objectives of this study were to evaluate the frequency of co-infections with or co-seropositivities for certain pathogens in dogs seropositive for L. infantum and their relationship with clinical signs, histological changes and L. infantum load. Sixty-six L. infantum-seropositive dogs were submitted to clinical examination, collection of blood and bone marrow, culling, and necropsy. Antibodies against Anaplasma spp., Borrelia burgdorferi sensu lato, Ehrlichia spp. and Toxoplasma gondii and Dirofilaria immitis antigens were investigated in serum. Samples from different tissues were submitted to histopathology and immunohistochemistry for the detection of Leishmania spp. and T. gondii. Quantitative real-time PCR was used to assess the L. infantum load in spleen samples. For detection of Coxiella burnetii, conventional PCR and nested PCR were performed using bone marrow samples. All 66 dogs tested positive for L. infantum by qPCR and/or culture. Fifty dogs (76%) were co-seropositive for at least one pathogen: T. gondii (59%), Ehrlichia spp., (41%), and Anaplasma spp. (18%). Clinical signs were observed in 15 (94%) dogs monoinfected with L. infantum and in 45 (90%) dogs co-seropositive for certain pathogens. The L. infantum load in spleen and skin did not differ significantly between monoinfected and co-seropositive dogs. The number of inflammatory cells was higher in the spleen, lung and mammary gland of co-seropositive dogs and in the mitral valve of monoinfected dogs. These results suggest that dogs infected with L. infantum and co-seropositive for certain pathogens are common in the region studied. However, co-seropositivities for certain pathogens did not aggravate clinical signs or L. infantum load, although they were associated with a more intense inflammatory reaction in some organs.In canine leishmaniosis caused by the protozoan  Leishmania (Leishmania) infantum is a zoonosis of important public health concern. From 1990 to 2019, there were 93,614 confirmed human cases of VL in Brazil, with an average of 3,120 new cases per year Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Under my review criteria, this is a well-written manuscript, which deserves to be considered for publication after the necessary explanations and adaptations as described below. My main concern is whether the number of dogs may be considered as numerically sufficient, together with the hypothesis that these animals might not be representative of dogs from other breeds.Line 27 – delete “visceral”, as canine leishmaniasis caused by L. infantum is a generalized disease – change accordingly throughout the manuscriptLine 32 – the word “euthanasia” presupposes that the dogs were sick – if this was not the case, use “culling” insteadLine 39 – “co-infected” may not be the right word for all the pathogens; for example, regarding T. gondii, seropositivity may not mean infection – change accordingly  for this and other agentsLine 54 – delete “biological” – including this word suggests that there are also mechanical vectors and this is not the caseLine 59 – whenever possible, display agents alphabetically – change accordingly throughout the manuscriptLine 60 – Isospora is now Cystoisospora; there is more than one species, so please write “Cystoisospora spp.”Line 64 – replace VL with leishmaniasisMaterials and methods – which criteria have determined a sample size of 66 dogs? Furthermore, this sample composition may not allow extrapolation of results to other breeds.Line 92 – not clear whether the owners provided signed consent for euthanasia  or to sample collection – please make clearerLine 112 – abbreviate as T. gondiiLine 174 – Leishmania spp.Line 181 – L. infantumLine 234 – Serological detection of antibodies to Anaplasma spp., B. burgdorferi and Ehrlichia spp., and of D. immitis antigensLine 239 – inform on the per cent sensitivity and specificity for each agentLine 242 – replace Diagnosis with DetectionLine 247 – which values for this IFAT?Line 254 – median was calculated, but minimum and maximum were just copied Line 290 – these dogs may not be infected but only seropositive; please, take this into account in the textLine 415 – rates are different from percentages; and this is the case of percentagesDiscussion – comparison with results from other studies could have included a statistical assessment.Reviewer #2: It is a very interesting research and very well presented. Please use the term leishmaniosis, instead of leishmaniasis .My main concern is that you have selected some common and important pathoges to test and if negative you present the dog as a single infection case. However, there are many other important pathogens to be present and contribute. For example sarcoptic mange or worms. I did not see other tests for them. I would suggest to revise the text and title using the phrase certain or common pathogens, which is closer to your work. Also please revise your conclusions accordingly.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  28 Jan 2021Dear Dr. Caldas Menezes,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.A well written manuscript, which deserves consideration. However, the authors should consider to not generalise their results. Also, seropositivity is not equivalent of infection.plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.Please submit your revised manuscript by 31.01.2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at Please include the following items when submitting your revised manuscript:• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocolsIf applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Angela Monica Ionica, Ph.D.Academic EditorPLOS ONEJournal Requirements:When submitting your revision, we need you to address these additional requirements.1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found athttps://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf andhttps://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdfRESPONSE TO REVIEWER #1Reviewers' comments:Reviewer's Responses to QuestionsComments to the AuthorReviewer #1: Under my review criteria, this is a well-written manuscript, which deserves to be considered for publication after the necessary explanations and adaptations as described below. My main concern is whether the number of dogs may be considered as numerically sufficient, together with the hypothesis that these animals might not be representative of dogs from other breeds.Line 27 – delete “visceral”, as canine leishmaniasis caused by L. infantum is a generalized disease – change accordingly throughout the manuscriptResponse:Line 27. The word “visceral” was deleted and the word “leishmaniasis” was replaced with “leishmaniosis”, as recommended by the reviewer#2. In addition, following the recommendations of both reviewers, the word “visceral” was deleted, the word “leishmaniasis” was replaced with “leishmaniosis” and the abbreviation “CVL” was replaced with “CanL” along the text, as indicated below:Line 101. “canine visceral leishmaniasis (CVL)” was replaced with “ canine leishmaniosis caused by L. infantum (CanL)”Lines 105, 292, 295, 446, 447, 453, 513, 530, 531 and 549. The abbreviation “CVL” was replaced with the abbreviation “CanL”.Lines 487 and 527. “VL” was replaced with “CanL”.Lines 27, 52, 64, 85 and 101. The word “leishmaniasis” was replaced with “leishmaniosis”.Line 472- “clinical VL caused by caused by L. infantum” was replaced by “CanL”Line 32 – the word “euthanasia” presupposes that the dogs were sick – if this was not the case, use “culling” insteadResponse: Lines 33, 92 and 93. The word “euthanasia” was replaced with “culling”.Lines 91, 117 and 274. The word “euthanized” was replaced with “culled”.Line 39 – “co-infected” may not be the right word for all the pathogens; for example, regarding T. gondii, seropositivity may not mean infection – change accordingly  for this and other agentsIn fact, we can’t prove the infection by the pathogens investigated in this study except for L. infantum. Therefore, the terms “co-infected”, “co-infection” and “co-infections” were replaced with “co-seropositive”, “co-seropositivity” and “co-seropositivities” along all the text, respectively, when refers to the results of this study or to the results of other authors when appropriate. We preferred these terms other than “seropositive”or “seropositivity” for the results of this manuscript, because all the dogs were seropositive for L. infantum. The term “monoinfected” was maintained, because we confirmed the infection with L. infantum by qPCR and/or culture in all the 66 dogs investigated in this study. These modifications were done in the following parts of the text:The title of the manuscript was altered to: “Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum”The short title “Co-infections in dogs naturally infected with L. infantum” was replaced with “Co-seropositivities in dogs naturally infected with L. infantum”The keyword “Co-infection” was replaced with “Co-seropositivity”AbstractLines 28, 29-30. The text “co-infection with other pathogens” was replaced with “co-infections with or co-seropositivities for”.Line 30. The text “infected with” was replaced with “seropositive for”.Line 39. The text “co-infected with” was replaced with “co-seropositive for”.Line 41. The text “co-infected dogs” was replaced with “dogs co-seropositive for certain pathogens”.Lines 42-43 and 44. The text “co-infected dogs” was replaced with “co-seropositive dogs”.Line 45. The text “co-infections with L. infantum and other pathogens” was replaced with “infected with L. infantum and co-seropositive for certain pathogens”.Line 46. The text “ these co-infections” was replaced with” “co-seropositivities for certain pathogens”.IntroductionLine 58. The text “Co-infections with” was replaced with “Co-infections with or co-seropositivities for”.Line 67. The text “co-infections with” was replaced with “co-infections with or co-seropositivities for”.Lines 68-70. The text “co-infected with other pathogens” was replaced with “co-infected with or co-seropositive for certain pathogens”.Lines 71-72. The text “of co-infections with pathogens” was replaced with” co-infections with or co-seropositivities for certain pathogens”.MethodsLine 264. The word “co-infected” was replaced with “co-seropositive”.ResultsLines 283-284. The text “The following pathogens causing co-infections and their frequencies were detected in the 66 dogs investigated:” was replaced with “The following frequencies of co-seropositivity for certain pathogens were observed in the 66 dogs investigated:”Lines 281, 286, 287, 294, 295, 297, Table 1, 309, 318, 321-326, 330-331, 336, 338, Table 2, 355, 361, 365-366, 370, Table 3, 372, 378, 385-386, Table 4, 392, 398, 401-402, 409, 411, 412, 414-415, 417, 421, 425 and 435. “The word “co-infected” was replaced with “co-seropositive”.Lines 321-326. The text “ for co-infection with” was replaced with “when co-seropositive for”.Line 367. The text “coinfecting pathogen” was replaced with “the co-seropositivity for certain pathogens”.Figures 1 and 3. The word “co-infected” was replaced with “co-seropositive” in the legend. DiscussionLines 442-444. The phrase “A high frequency of seropositivity for pathogens coinfecting dogs naturally infected with L. infantum was found, especially co-infection with T. gondii” was replaced with the “A high frequency of co-seropositivity for certain pathogens in L. infantum seropositive dogs with infection with this parasite confirmed by PCR and/or culture was found, especially co-seropositivity for T. gondii.Lines 442, 443, 462, 469 and 505. The word “co-infection” was replaced with “co-seropositivity”.Line 474 and 476. The text “co-infection with” was replaced with “co-infection with or co-seropositivity for”.Line 482. The text “infection of dogs with” was replaced with “seropositivity of dogs for”.Lines 483. The text “by serology” was deleted.Line 487 and 501. The texts “or co-seropositivities” or “or co-seropositivity” were added.Line 507. The text “this co-infection” was replaced with “this co-seropositivity or co-infection”.Lines 523-524. The text “frequency of co-infections among dogs with VL found in this and other studies .” was replaced with “frequency of co-seropositivities among dogs seropositive for and/or infected with L. infantum found in this study and in other studies .Lines 541, 548, 572, 577, 591 and 596. The word “co-infected” was replaced with “co-seropositive”.Lines 549-552. The text “Another hypothesis for the lack of aggravation of CVL due to co-infection with other pathogens in the present study was the use of serological techniques for the detection of most of the pathogens investigated. This methodology may have contributed to the classification of exposed animals, but without active infection, as co-infected when, in fact, serological tests may have detected memory antibodies.” was replaced with the text below:“Another hypothesis for the lack of aggravation of CanL due to co-seropositivity for certain pathogens in the present study is that the seropositive dogs may not be co-infected or infection with these pathogens is not active, but they were previously exposed to the pathogens and exhibited detectable memory antibodies.”Lines 566 to 568. The phrase “However, 12.5% of monoinfected dogs and 10% of co-infected dogs did not have a positive culture or IHC result, suggesting that infection with L. infantum was not active in these dogs” was deleted.Line 602. The text “why co-infections” was replaced with “why co-seropositivity for certain pathogens”.ConclusionsLines 605 and 607. The word “co-infections” was replaced with “co-seropositivities”.Other alterationsIn addition, due to the alterations in the terms and to make clearer that all the dogs investigated were infected with L. infantum and not only seropositive for this parasite, we reviewed the definition of the groups of dogs of our study, as follows:Co-seropositive: L. infantum seropositive dogs with confirmed infection with this parasite by qPCR and/or culture and antibodies against at least one pathogen other than L. infantum.Monoinfected: L. infantum seropositive dogs with confirmed infection with this parasite by qPCR and/or culture and without antibodies against certain pathogens other than L. infantum.These new definitions of the groups were described in lines 309-312, 355-358, 372-375 and 392-395.The term “monoinfected” was maintained, because we confirmed the infection with L. infantum by qPCR and/or culture in all the 66 dogs investigated in this study.The titles of Tables 1, 2, 4 and S1 were also modified as shown below:Tables 1, 2, 4 and S1. The text “Leishmania infantum-seropositive dogs” was replaced with “dogs infected with Leishmania infantum”.Table S1. The text “according to the co-infecting pathogen” was replaced with “according to the co-seropositivity for certain pathogens”.In addition, the following texts were included:AbstractLines 38 to 39. “All 66 dogs tested positive for L. infantum by qPCR and/or culture.”ResultsLines 279 and 280. “All 66 L. infantum-seropositive dogs investigated in this study had infection with L. infantum confirmed by qPCR and/or culture, as described below.” DiscussionLines 442-443“…co-seropositivity for certain pathogens in L. infantum-seropositive dogs with infection with this parasite confirmed by PCR and/or culture…”Line 54 – delete “biological” – including this word suggests that there are also mechanical vectors and this is not the caseResponse:Line 54. The word “biological” was deleted, as recommended.Line 59 – whenever possible, display agents alphabetically – change accordingly throughout the manuscriptResponse:We desplayed agents alphabetically whenever possible in the following parts of the text:• AbstractLines 33 to 34.• IntroductionLines 59 to 64.• Materials and methodsLines 115 to 116, 236 to 237 and 239-240.• ResultsLine 286.• DiscussionLines 491-492 and 559-560.• ConclusionsLine 605.Line 60 – Isospora is now Cystoisospora; there is more than one species, so please write “Cystoisospora spp.”Line 59. “ Isospora sp.” was replaced with ““Cystoisospora spp.”, as recommended. Line 64 – replace VL with leishmaniasisResponse:VL was replaced with “with leishmaniosis caused by L. infantum”. We replace “leishmaniasis” with “leishmaniosis” along the text, as recommended by the other reviewer (indicated above).Materials and methods – which criteria have determined a sample size of 66 dogs? The sample size included all dogs of the study population, i.e., all dogs that tested seropositive for L. infantum in the town of Barra Mansa and that were sent to INI-Fiocruz during the study period (n=66).In order to make clearer the criteria for sample size, the following text was included in the materials and methodsLines 94 to 96. “The sample size included all dogs of the study population, i.e., all dogs that tested seropositive for L. infantum in the town of Barra Mansa and that were sent to INI-Fiocruz during the study period.” Furthermore, this sample composition may not allow extrapolation of results to other breeds.Response:This is a limitation of the study. Considering the breed, the results of this study can only be extrapolated to other similar populations, composed mainly by mongrel dogs (85%). In addition, statistical analysis of the association of breed with the monoinfected and co-seropositive groups was not possible because of the small number of breed dogs (n=10).In order to make these limitations clearer in the text, the following texts were included:ResultsLines 290-291. “Statistical analysis of the association of breed with the monoinfected and co-seropositive groups was not possible because of the small number of breed dogs.”DiscussionLines 444-445. Considering the breed, the results of this study can only be extrapolated to other similar populations composed mainly of mongrel dogs (85%).Line 92 – not clear whether the owners provided signed consent for euthanasia  or to sample collection – please make clearerResponse:The owners provided signed consent for culling. We clarify this in the line 93.Line 112 – abbreviate as T. gondiiResponse:Line 116. Toxoplasma gondii was abbreviated as T. gondii.Line 174 – Leishmania spp.Response:Line 177. “Leishmania species” was replaced with “Leishmania spp.”Line 181 – L. infantumResponse:Line 184. “Leishmania infantum” was replaced with “L. infantum”.Line 234 – Serological detection of antibodies to Anaplasma spp., B. burgdorferi and Ehrlichia spp., and of D. immitis antigensResponse:Lines 236 to 237. The text “Detection of antibodies against Ehrlichia spp., Anaplasma spp. and B. burgdorferi and D. immitis antigens in serum” was replaced with: “Serological detection of antibodies to Anaplasma spp., B. burgdorferi and Ehrlichia spp., and of D. immitis antigens”Lines 238 to 240. The text “...to detect antibodies against E. canis/E. ewingii, A. platys/A. phagocytophilum and Borrelia burgdorferi and antigens of D. immitis…” was replaced with:“... for the serological detection of antibodies to A. platys/A. phagocytophilum, B. burgdorferi and E. canis/E. ewingii, and of D. immitis antigens...”Line 239 – inform on the per cent sensitivity and specificity for each agenteResponse:Lines 241-243. The following text was included, as recommended:“…has the following sensitivities and specificities, respectively: 90.3% and 94.3% for Anaplasma spp., 94.1% and 96.2% for B. burgdorferi, 99.0% and 99.3% for D. immitis, and 97.1% and 95.3% for Ehrlichia spp. Line 242 – replace Diagnosis with DetectionResponse:Line 245. “Diagnosis” was replaced with “Detection”.Line 247 – which values for this IFAT?Response:There is no information in the literature about the values of specificity and sensibility of IFAT for the detecion of anti-T. gondii antibodies in the serum of dogs . However, IFAT is considered to have good specificity and sensitivity and is recommended for the immunodiagnosis of T. gondii infection in dogs, where it can be used as the only test .References:Dubey JP, Murata FHA, Cerqueira-Cézar CK, Kwok OCH, Yang Y, SU C. Toxoplasma gondii infections in dogs: 2009-2020. Vet Parasitol. 2020; 287:109223.Leal PDS, Coelho CD. Toxoplasmosis in dogs: a brief review. Coccidia. 2014; 2: 2-39.Line 254 – median was calculated, but minimum and maximum were just copied Response:Lines 256 to 258. The text “For the description of continuous variables (number of inflammatory cells and parasite load), the median (50th percentile) and the minimum and maximum values were calculated.” was replaced with:”“For the description of continuous variables (number of inflammatory cells and parasite load), the median (50th percentile) was calculated and the minimum and maximum values are reported.”Line 290 – these dogs may not be infected but only seropositive; please, take this into account in the textResponse:The terms “co-infected”, “co-infection” and “coinfections” were replaced with “co-seropositive”, co-seropositivity” and “co-seropositivities”, respectively, along all the text, when refers to the results of this study or to the results of other authors, when appropriate. These alterations are explained in more details above.Line 415 – rates are different from percentages; and this is the case of percentagesResponse:Line 450. The word “rates” was replaced with “percentages”, as recommended.Discussion – comparison with results from other studies could have included a statistical assessment.Response:This study had a descriptive observational design. Therefore, we chose to compare the findings of our study with the others in the discussion, punctuating whenever possible, the differences in the characteristics of the compared population such as geographical location and study design. To compare specific measurements of our findings with those of other studies using statistical analysis, this study should be designed for systematic review and meta-analysis, with a review carried out with key terms in a systematic way. In addition, detailed results of specific measurements and variability described in the various studies will be necessary, which is outside the scope of this article.RESPONSE TO REVIEWER #2Reviewer #2: It is a very interesting research and very well presented. Please use the term leishmaniosis, instead of leishmaniasis .Response: Lines 27, 52, 64, 85, and 101. The term “leishmaniasis” was replaced with”leishmaniosis”.My main concern is that you have selected some common and important pathoges to test and if negative you present the dog as a single infection case. However, there are many other important pathogens to be present and contribute. For example sarcoptic mange or worms. I did not see other tests for them. I would suggest to revise the text and title using the phrase certain or common pathogens, which is closer to your work. Also please revise your conclusions accordingly.Response:The text was revised and the term “pathogens” was replaced with “certain pathogens”, when reffered to the pathogens investigated in this study.Therefore, the following modifications were done:The title of the manuscript was altered to: “Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum”The word “pathogens” was replaced with “certain pathogens” in the following parts of the text:Abstract. Lines 30, 41, 45 and 46.Introduction. Lines 69 and 72.Results. Lines 282, 284, 312, 358, 363, 367, 375 and 395.Discussion. Lines 442, 549 and 602.Legend of S1 Table. Line 816.AttachmentResponse to reviewers_PONE-D-20-37262.docxSubmitted filename: Click here for additional data file. 10 Feb 2021Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantumPONE-D-20-37262R1Dear Dr. Caldas Menezes,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Angela Monica Ionica, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #3: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #3: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #3: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #3: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #3: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: I am very happy to see that the authors have addressed all my comments and made a substantial effort to accommodate my suggestions.Reviewer #3: The manuscript PONE-D-20-37262R1 'Frequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Leishmania infantum' is a well-written and concise manuscript well worth publishing in PLOS ONE. The article presents complex data involving co-infections associated with CanL. The article is very clear and objective and the authors have adequately addressed all comments raised in the previous round of review. This Reviewer has very little else to comment.**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Reviewer #1: NoReviewer #3: No 22 Feb 2021PONE-D-20-37262R1 Leishmania infantumFrequency of co-seropositivities for certain pathogens and their relationship with clinical and histopathological changes and parasite load in dogs infected with Dear Dr. Menezes:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. 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Scientific Reports 10.1038/s41598-020-67594-2, published online 07 July 2020Correction to: The original version of this Article contained an error in the title of the paper, where “CRISPR” was incorrectly given as “CRISRP”. This has now been corrected in the PDF and HTML versions of the Article.


To recognize abnormal electroencephalogram (EEG) signals for epileptics, in this study, we proposed an online selective transfer TSK fuzzy classifier underlying joint distribution adaption and manifold regularization. Compared with most of the existing transfer classifiers, our classifier has its own characteristics: (1) the labeled EEG epochs from the source domain cannot accurately represent the primary EEG epochs in the target domain. Our classifier can make use of very few calibration data in the target domain to induce the target predictive function. (2) A joint distribution adaption is used to minimize the marginal distribution distance and the conditional distribution distance between the source domain and the target domain. (3) Clustering techniques are used to select source domains so that the computational complexity of our classifier is reduced. We construct six transfer scenarios based on the original EEG signals provided by the Bonn University to verify the performance of our classifier and introduce four baselines and a transfer support vector machine (SVM) for benchmarking studies. Experimental results indicate that our classifier wins the best performance and is not very sensitive to its parameters. The maturity of the brain–computer interface (BCI) technology has provided an important channel for the human to use artificial intelligence (AI) to explore the cognitive activities of the brain. For example, many AI methods have been proposed for an intelligent diagnosis of epilepsy instead of neurological physicians through electroencephalogram (EEG) signals  for epilepsy EEG signal recognition. First, it further explores the marginal probability distribution adaption between the source domain and the target domain from two aspects. One is that it additionally introduces conditional probability distribution adaption to further minimize the distribution difference. The second is that it preserves manifold consistency underlying the marginal probability distribution. Second, it can selectively leverage knowledge from multiple source domains.Data and Methods, we give the EEG data and our proposed method. In Results, we report the experimental results. Discussions about experimental results are presented in Discussions, and the whole conclusions are summarized in the last section.The following sections are organized as follows: in In this study, we download very commonly used epilepsy EEGThree feature extraction algorithms, that is, wavelet packet decomposition (WPD) Li, , short-tWavelet Packet DecompositionWavelet packet decomposition is used to extract time-frequency features from epilepsy EEG signals. More specifically, the epilepsy EEG signals are disassembled into six different frequency bands with the Daubechies 4 wavelet coefficients. Each band is considered as one feature. Short-Time Fourier TransformShort-time Fourier transform is used to extract frequency-domain features from epilepsy EEG signals. More specifically, the epilepsy EEG signals are disassembled into different local stationary signal segments, and then the Fourier transform is used to extract a group of spectra of the local segments, which are with evident time-varying characteristics at different times. Finally, six frequency bands are extracted from each group of spectra. Kernel Principal Component AnalysisKernel principal component analysis is used to extract time-domain features from epilepsy EEG signals. More specifically, the Gaussian function is chosen as the kernel to map the original features nonlinearly. Then six kinds of features are selected from the top six PC eigenvectors. We construct six online transfer scenarios from the EEG data after feature extraction . Each scM = 20 subject-specific objects from the target domain. The online calibration flowchart is shown in m0.In general, calibration in BCIs can be divided into two types, that is, offline calibration and online calibration . Lastly, we give the detailed algorithm steps of OS-JDA-MR-T-TSK-FC including how to select source domains.X, P(x)} in the transfer learning or domain adaption scenario consists of a d-dimensional feature space ∈ Rd and a marginal distribution P(x), and a task Γ = {Y, P(y|x)} in the similar scenario consists of a one-dimensional label space Y and a conditional distribution P(y|x), where y ∈ Y. Suppose that Ψs and Ψt are two domains derived from Ψ, they are deemed to be different when Xs ≠ Xt and/or Ps(x) ≠ Pt(x). Homoplastically, two tasks Γs and Γt derived from Γ are different when Ys ≠ Yt and/or Ps(y|x) ≠ Pt(y|x).A domain Ψ = {s having N-labeled EEG epochs t having M-labeled EEG subject-specific epochs s = Ψt, Ys = Yt, Ps(x) ≠ Pt(x), and Ps(y|x) ≠ Pt(y|x).Based on the above definitions, the target of OS-JDA-MR-T-TSK-FC is to train a predictive function on a source domain ΨOnline Transfer Learning FrameworkBecause the classic one-order TSK fuzzy classifier (1-TSK-FC)  for an unseen object x is formulated as the following form:k(x) is expressed asif parts, and then parts. Referring to the literature   in (2) as the following form:pg:pg to zero, that is, ∂Jorder−TSK−FS1−(pg)/∂pg = 0, we can compute pg analytically ast in the first term is the overall weights of the specific-subject objects. Generally, ωt should be larger than 1 so that more emphasis is given to objects in Ψs than Ψt. Therefore, we set ωt to ωt = max. λ1 and λ2 are regularization parameters. The first term contains two parts: the first is to measure the loss on Ψs, and the second is to measure the loss in Ψt. The second one is the joint distribution adaption term, and the third one is the manifold regularization term. Below, we will explain how to embody them formally.The . et al. . When cju et al.  and Zhanu et al. , we formObjective function of OS-JDA-MR-T-TSK-FCUnder the framework shown in (11), we specify each term to get the objective function of our online transfer TSK fuzzy classifier OS-JDA-MR-T-TSK-FC.s and Ψt; hence, the first term in (11) can be formulated asThe squared loss is taken as the loss function to measure the sum of squared training errors on both Ψs and Ψt are extracted in the same way, the joint distributions  between Ψs and Ψt are generally different. In order to meet practical requirements, we assume that Ps(x) ≠ Pt(x) and Ps(y|x) ≠ Pt(y|x). Therefore, a joint distribution adaptation should be designed to minimize the distribution similarity (distance) D between Ψs and Ψt.As all we know that even EEG epoch features in ΨD between Ψs and Ψt. As a result, D can be expressed ass,c belongs to Ψs and its objects are selected by {xi|xi ∈ Ψs ∧ yi = c}, and Ψt,c belongs to Ψt and its objects are selected by {xi|xi ∈ Ψt ∧ yi = c}, where c means the cth class in one domain. Also, for the source domain, Nc is used to denote the number of objects in the cth class, and for the specific-subject objects in the target domain, Mc is used to denote the number of objects in the cth class. Hence, D can be expressed asc is an MMD matrix defined as follows:D = D+D so that the joint distribution adaptation can be formulated asFirst, the projected MMD  and P(xj), then the corresponding Q(yi|ix) and Q(yj|xj) are considered as being similar. That is to say, for the objects in Ψs and the calibration objects in Ψt, if they are in a manifold, it is expected that their output  differences should be as small as possible. Therefore, the manifold regularization can be formulated as follows under geodesic smoothness,W = [wij]N+M)×(N+M)( is the graph affinity matrix in which each element is defined asν(xi) represents a set of v-nearest neighbors of object xi. L = [lij]N+M)×(N+M) by setting its derivative w.r.t pg to zero asBy substituting (14), (19), and (20) into our transfer learning framework shown in (12), we can obtain a transfer learning model, that is, OS-JDA-MR-T-TSK-FC asDifferent from most of the existing transfer models, OS-JDA-MR-T-TSK-FC can leverage knowledge from multiple source domains. However, as we know, too many source domains will improve computational complexity. Additionally, some source domains having significant differences with the target domain may bring some negative transfer knowledge. Therefore, according to Wu et al. , we adopvz,c to denote the mean vector of each class in the zth source domain, where z = 1,2,…, Z. Similarly, vt,c is used to denote the mean vector of each class in the target domain. The Euclidean distance between the zth source domain and the target domain can be computed asd, d, …, d} that contains Z domain distances. The distance set then is partitioned by k-means to k groups , and the source domains are selected from the cluster who has the smallest center.We use As a whole, the training of OS-JDA-MR-T-TSK-FC contains three parts: the first one is source domain selection, the second one is model training on a source domain combining with the target domain, and the last is classifier combination. Algorithm 1 shows the detailed training steps of OS-JDA-MR-T-TSK-FC.y from the space R to the space RC by that yij = 1 if y(xi) = j, and yij = 0 otherwise, where i = 1, 2, …, N + M, j = 1, 2, …, C, and C represents the number of classes. Thus, the label space becomes Rd+1 to Rd+1)×C. Its training set consists of the five source domains directly connected, and its testing set is the target domain. Therefore, BL1 is considered as a calibration-independent classifier, which does not use the subject-specific data in the target domain for training.Baseline 2 (BL2). It uses only subject-specific calibration EEG data in the target domain for training. Its testing set is the unlabeled data in the target domain. Therefore, BL2 is considered as a source domain-independent classifier, which does not consider the EEG data in the source domains at all.Baseline 3 (BL3). BL3 is trained on five training sets, receptively. Each set consisted of a source domain and the subject-specific data in the target domain. The five trained models are finally combined by a weight schema that is also used in Algorithm 1. Its testing set is the unlabeled data in the target domainTransfer support vector machine (TSVM) , λ1, and λ2 that should be fixed before a classification task. So, we should consider the robustness OS-JDA-MR-T-TSK-FC to them. The sensitivity analysis results are shown in From the objective function of OS-JDA-MR-T-TSK-FC, we see that there are three parameters, that is, ωWe observe from Moreover, the classification results in M, whereas the other four classifiers depend on M, and it is intuitive that they gradually perform better than BL1 with the increasing of M.BL1 does not use the specific-subject objects, so its accuracy is independent on M is set to 0. But BL1, BL3, TSVM, and OS-JDA-MR-T-TSK-FC can work because, except subject-specific objects, they also leverage training objects from the source domains. Compared with other algorithms, when M is too small, BL2 performs so badly because it cannot get enough training patterns from subject-specific objects.BL2 is only trained by the subject-specific objects. Therefore, BL2 becomes unusable when M is set to 0, TSVM always achieves the best performance. With the subject-specific objects gradually added into the training set, OS-JDA-MR-T-TSK-FC soon performs better than TSVM, which indicates that significant differences exist among the domains. Hence, a domain-dependent classifier, for example, TSVM is not very expected in our online transfer scenarios.When When one batch (four subject-specific objects are taken as a batch in our experiments) or at most two batches of subject-specific objects are added into the training set, the classification performance of OS-JDA-MR-T-TSK-FC becomes stable. That is to say, the number of subject-specific objects OS-JDA-MR-T-TSK-FC needs is very small. So, OS-JDA-MR-T-TSK-FC meets the practical requirements because subject-specific objects are very few in real-world applications.x1 is In addition to classification performance, interpretability is also a main characteristic of the proposed OS-JDA-MR-T-TSK-FC. From If the energy of an EEG signal band (band 1) is “high,” and the energy of an EEG signal band (band 2) is “a little low,” and the energy of an EEG signal band (band 3) is “low,” and the energy of an EEG signal band (band 4) is “low,” and the energy of an EEG signal band (band 5) is “low,” and the energy of an EEG signal band (band 6) is “low,”thenthe consequent of the first fuzzy rule can be expressed as:x1+0.4452x2−0.5342x3+0.2342x4−0.9734x5−0.3244x6.1 in the range of (Geng et al., 2 in the range of (Ghosh-Dastidar et al., From In this study, we propose a seizure classification model OS-JDA-MR-T-TSK-FC using an online selective transfer TSK fuzzy classifier with a joint distribution adaption and manifold regularization. We use epilepsy EEG signals provided by the University of Bonn as the original data and construct six transfer scenarios in three kinds of feature spaces to demonstrate the promising performance of OS-JDA-MR-T-TSK-FC. We also generate four baselines and introduce a transfer SVM model for fair comparison. The experimental results show that OS-JDA-MR-T-TSK-FC performs better than baselines and the introduced two transfer models. However, in this study, we only consider how to select the source domains. Recent studies show that dynamically selecting useful samples from the source domain can effectively induce the learning on the target domain. Therefore, in our future work, we will try to develop a mechanism, for example, classification error consensus to select most useful samples from the source domain.http://www.meb.unibonn.de/epileptologie/science/physik/eegdata.html.The original EEG data are available in YZ designed the whole algorithm and experiments. ZZ, HB, and WL contributed on code implementation, and LW gave some suggestions to the writing.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.


Correction to: Modern Pathology10.1038/s41379-019-0359-9This article was erroneously published with standard licence and copyright, but it is in fact published with open access, so has a CC BY licence and the copyright line is ‘The Author(s)’. This has been updated in the original article.


Nature Communications 10.1038/s41467-019-13286-z, published online 12 December 2019.Correction to: The original version of this Article contained an error in Fig. 4, in which samples marked with blue dots were labelled with the suffix ‘WD’ instead of ‘DD’. This has now been corrected in both the PDF and HTML versions of the Article.


The  Contingency tables and their analysis are of special importance for various diverse fields, like medical sciences, psychology, education, demography and social sciences. In these fields, categorical variables and their cross-classification play a predominant role, since characteristics of interest are often categorical, nominal or most frequently ordinal. For example, diagnostic ratings, strength of opinions or preferences, educational attainments and socioeconomic characteristics are expressed in ordinal scales. The origins of contingency table analysis (CTA) lie back in 1900 with the well-known contributions by Pearson and Yule, while, for the history of CTA before 1900, we refer to the interesting paper by Stigler [of Pardo . For logof Pardo .Here, we aim at highlighting a different structural role of els see ,5) while whileϕ-dThe organization of the paper is as follows. Preliminaries on log-linear models, divergence measures, association and correlation models for two-way tables are provided in X and Y, respectively, where Consider an X and Y /x (cf. ). Settingiven byIϕC=given byIϕC=For nd λ→−0,  convergeThe goodness of fit (GOF) of model  is usualsed (see ).In case of ordinal classification variables, the association models (AMs) impose a special structure on the underlying association and thus provide non-saturated models of dependence. AMs are based on scores Usually, the uniform (Linear-by-Linear (LL) AM. In case the vector Row effect (R) AM, while the Column effect (C) AM is defined analogously. Finally, when the row and the column scores are all unknown parameters to be estimated, (multiplicative Row-Column (RC) AM. Scores that are unknown need not necessarily to be ordered. Note that models LL, R and C are log-linear while the RC is not. The degrees of freedom (Uniform (U) AM.If ed, then  has justunknown,  is the Rtimated,  is the mM-th order is defined asM is the rank of In case the RC model is not of adequate fit, multiplicative row-column AMs of higher order can be considered. Such a model of ed model . The sumfined aslogmij=λ+imνjm in , Λ=λijXYman Recall that the e, model  is alterMM, while, for A popular, mainly descriptive method for exploring the pattern of association in contingency tables is correspondence analysis (CA). The detailed discussion of CA is beyond our scope. For this, we refer to the book of Greenacre . Correspstraints  subject  between , expressMpij=pi+p+Mpij=pi+p+M1pij=pi+p+ Goodman .AMs and correlation models were developed competitively and opposed to each other (cf. ), until Under the conditions of , namely aioannou  derived straints  with marthe formπij=πi+π+ied that  with marstraints∑iπi+αi=∑the formπij=πi+π+Model , with knegories,  becomes fined by  for M=1, models,  simplifiFor the power divergence (for ), model  becomesd Sarkar . Express, model  model (The RCrictions  with marM) model  is derivgiven byπij=πi+π+2, model  with μm=Models  and 21)21) can aFor )=xlogx,  becomes elled in  and fromRemark 1.Kateri and Papaioannou  studied In order to understand the role and nature of the scaled measures of local association , one canis case,  becomes ization,  for I=J=d Kateri . For theratio CI . Here, tRemark 2.The idea of viewing a model as a departure from a parsimonious reference model, with the property of being the closest to this reference model under certain conditions in terms of the KL divergence, can be adopted for other types of models as well, such as the quasi symmetry (QS) model and the logistic regression, lightening thus a different interpretational aspect of these models. Substitution of the KL divergence by the ϕ-divergence, leads further, in an analogous manner to AMs, to the derivation of generalized ϕ-divergence based classes of QS models , ordinalWe shall focus on the case of uniform local association and the corresponding fined by  or 22)  ϕ divergfined by . Model Umpare to  for the mphR-package. The standard U model, denoted in the sequel as UR for the example of R-function for constructing the design matrix The MLEs of the marginal probabilities are the corresponding marginal sample proportions ) models . It is s) models . In pracression2L(mv)=Cloression2L(mv)=ClopressionL(mv)=CloUnder Ursion of , is giver is the sample correlation between the row and column scores (compare to (Another option ismeasures . For thetimators  and 29)(29)θ¯(ϕ)stimator  takes thmpare to ).Under the Udel (see  and Sectdel (see ).Remark 3.For model UTomizawa  proposed Salicrú  extendedTomizawa  as well Remark 4.For square contingency tables with commensurable classification variables, analogous to the measure of departure from Ua et al.  introduca et al.  a measura et al.  extendeda et al. ,24 is alWe revisit a classical data set of Grizzle , providedel see ,19). Our. Our0 model (see  and prov [0 (see ). The maThe stimates  for TablWe observe that all considered U not see . The maxObserve in  that theWe revealed the role of Crucial quantities are the  Agresti .It is important to realize that, due to the scale difference, The conditional test of independence  can be btel (see ). It con also of , is inteR-package mph.Throughout this paper, we assumed a multinomial sampling scheme. For the models considered here, the other two classical sampling schemes for contingency tables  are inferentially equivalent. Furthermore, for ease of presentation, we restricted here to two-way tables. The proposed models extend straightforwardly to multi-way tables. For two- or higher-dimensional tables, the subset of models that are linear in their parameters  models  and can


Saccharomyces cerevisiae plays an important role in nutrient storage. Arginine, in particular, accumulates in the vacuole of nitrogen-replete cells and is mobilized to the cytosol under nitrogen starvation. The arginine import and export systems involved remain poorly characterized, however. Furthermore, how their activity is coordinated by nitrogen remains unknown. Here we characterize Vsb1 as a novel vacuolar membrane protein of the APC (amino acid-polyamine-organocation) transporter superfamily which, in nitrogen-replete cells, is essential to active uptake and storage of arginine into the vacuole. A shift to nitrogen starvation causes apparent inhibition of Vsb1-dependent activity and mobilization of stored vacuolar arginine to the cytosol. We further show that this arginine export involves Ypq2, a vacuolar protein homologous to the human lysosomal cationic amino acid exporter PQLC2 and whose activity is detected only in nitrogen-starved cells. Our study unravels the main arginine import and export systems of the yeast vacuole and suggests that they are inversely regulated by nitrogen.The vacuole of the yeast  Saccharomyces cerevisiae is an important storage compartment for diverse nutrients, including the cationic amino acid arginine, which accumulates at high concentrations in this organelle in nitrogen-replete cells. When these cells are transferred to a nitrogen-free medium, vacuolar arginine is mobilized to the cytosol, where it is used as an alternative nitrogen source to sustain growth. Although this phenomenon has been observed since the 1980s, the identity of the vacuolar transporters involved in the accumulation and the mobilization of arginine is not well established, and whether these processes are regulated according to nutritional cues remains unknown. In this study, we exploited in vitro and in vivo uptake assays in vacuoles to identify and characterize Vsb1 and Ypq2 as vacuolar membrane proteins mediating import and export of arginine, respectively. We further provide evidence that Vsb1 and Ypq2 are inversely regulated according to the nitrogen status of the cell. Our study sheds new light on the poorly studied topic of the diversity and metabolic control of vacuolar transporters. It also raises novel questions about the molecular mechanisms underlying their coordinated regulation and, by extension, the regulation of lysosomal transporters in human cells.The lysosome-like vacuole of the yeast  Saccharomyces cerevisiae is the counterpart of the lysosome and has proved to be a valuable model for studying this organelle Please let us know if you have any questions while making these revisions.Yours sincerely,Widmar TannerGuest EditorPLOS GeneticsGregory P. CopenhaverEditor-in-ChiefPLOS GeneticsConcerning the aspects reviewer 2 raised as major concerns, it would suffice if the authors state more clearly that they present no direct evidence for the genes under discussion representing transport proteins.Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1: This is a description of a well carried out study of how vacuoles help cells store and mobilize organic nitrogen. Two important gene-function relationships are identified, and kinetics and/or other important characteristics of their encoded transporters are described. Furthermore, the authors show the nitrogen status to have strong regulatory effects on the two transporters in a way that makes biological sense, although the mechanisms are yet obscure.The manuscript is also well-written, creating a flow where generally the results from one experiment suggest questions that are addressed in the next one. This makes it easy and pleasant to read.I have one general comment. The exchange function of Ypq2 is pointed out in a slightly wrong way. At places, the reader will think that Ypq2 has two states, one in which it is a uniporter and another where it exchanges molecules; that is in my view unlikely and would be a complicated assumption. All observations agree with the highly efficient exchange reaction being a side reaction that will always take place when there is Arg on both sides. It requires no energy, and there is no reason to think that it has any important biological consequence. Such exchange is also named counterflow and is a priori expected to be very commonly occurring. Let us assume that the function important for fitness in nature is uniport (export in the case of Ypq2) function and not exchange. All you need to explain the occurrence of a much higher rate of exchange than rate of net transport is a kinetic bottleneck in the conformational shift of the transporter without ligand between the inward-open conformation and the outward-open conformation. Such bottlenecks should a priori be expected to commonly arise in evolution on a seemingly random basis in various steps of transport cycles including the conformational shift mentioned, and indeed counterflow is often found in transporters when it is looked for, also when it has no apparent biological significance. Not only uniporters exhibit counterflow; good old LacY also has it  Lessons from lactose permease. Annu Rev Biophys Biomol Struct 35:67–91). I encourage you to state this or parts of it, perhaps in the Discussion. I have below indicated a few places in the manuscript where changes should be made with this view; there may be others. Nevertheless, I fully agree that the exchange function is an important part of the story that contributes to the flow guiding the reader.I have many minor comments:P1 Italicize “VSB1”.Line 4 Consider changing “, and” into a semicolon.15 Insert a comma after “nutrients” and another one after “arginine”, in both cases to indicate a non-defining relation.76 Change “VTC” into “vacuolar transporter chaperone (VTC)”.77 Change “mM)” into “mM phosphate groups)”.103 Consider changing “seemed” into “appeared”.125 I suggest changing “are” into “appear”, since the experiments described immediately above concern a single amino acid only.136-140 First, you do not at this point of the story need to indicate one interpretation as being simpler than others. Second, if you wish to list possible explanations here, you need to mention a third, and in my mind even simpler, possible explanation, namely the one you reach later in the manuscript: [14C]Arg uptake can take place through Ypq2 by exchange with intravacuolar amino acid(s).149 Change “w-t” into “w-t (23344c)” here and perhaps other places.171 Change “Unsurprisingly, cold” into “As expected, non-radioactive” in order to make text understandable to readers unfamiliar to the convention “hot”-“cold”. Furthermore, change “neutral amino acids such as” into “the neutral amino acids”. I agree that it is likely that other neutral amino acids behave the same, but you should not conclude it unless of course you tried them, in which case you should describe it.180 and 205 Insert “primarily” or “mainly” after “is”.191 Change “previously described,” into “described for intact vacuoles,”.199 Change “not.” into “not, although ATP stimulated the uptake.”.204 Insert “into vacuole-derived vesicles” after “uptake”.210-212 This is an overstatement. Instead, it would be correct to say “Appreciable Ypq2 activity does require, however, the presence of at least one of its substrates inside the vacuole, since little 14C-Arg was taken up by vacuolar vesicles devoid of Arg or His .”, of course with “14” in superscript.213-215 This is also an overstatement; it gives a wrong impression of the content of the three references given. None of the two first references mention a switch. In each case, a mitochondrial carrier is described which has a high kcat for exchange and a much lower yet significant kcat for uniporter activity. It is expected, and not a consequence of a shift of mechanism, that the relative significance of the latter becomes larger when the concentration ratio across the membrane increases. The third reference, on GLUT1, does describe a switch, but also in this case is the statement misleading. The effect of lowering the external glucose concentration has a large delay and occurs indirectly via the ATP/AMP energy system, a fact that in my view makes this reference less relevant; there is nothing that points to a similar mechanism for Ypq2. Most unprepared readers who do not read the three cited papers will erroneously think that the papers show cases where a high concentration ratio of a ligand (transport substrate) directly causes a change of transporter mechanism. This is not so; in references 30 and 31 one just sees a consequence of different experiments emphasizing different capabilities of the protein in question. The wording must be corrected, see my general comment and e.g. my comment to line 977.223-225 Modify wording. I do not see that the hypothetical pH profile itself argues for the model, but it is indeed important to point out as an explanation for the effect of ATP, based on evolutionary considerations. I agree that, otherwise, readers might at this point think that the effect of ATP suggests that the pmf can drive transport through Ypq2, which I also agree appears unlikely.261 Insert “than” after “more”.297 I encourage you to reserve the name with SGD.457 Consider changing “our observations show” into “we find”; the first could to some readers mean all observations described in the manuscript.461 Consider changing “is” into “shows up as”.533 Make “4” subscript.595 “adopt” is misleading, see my general comment, and reconsider reference 32, see my comment to lines 213-215.600 Also here, please rephrase slightly and avoid “switch”.611 Insert “the” before “protein”.657-659 The nomenclature for PS buffer must be clarified. Strictly interpreted, the layer e.g. immediately over the lysate should contain 15+8 percent Ficoll, but I think it only contains 8.665 Change “O” to lower case, italic, since it means ortho; otherwise some readers will think it refers to binding to an oxygen atom. Another common name that you can choose is 1,10-phenanthroline. You can also add a vendor or producer and a catalog number.669 and 996 Insert comma before “and”, since a new independent sentence starts.691 Consider changing “Corrected for” into “normalized to”.704-705 Specify the scintillation liquid. Counting efficiency depends on the identity of the scintillation liquid, the sample solvent, and the mixing ratio.736 Change “m” into “nm”.804 Delete “ (80-)”.819 Change “enzymology” into “Enzymology”, and consider changing “177-96 p” into “pp 177-96”. Check full reference list for similar cases.821 Change “Vacuolar” into “vacuolar” and “Cellular” into “cellular”. Check full reference list for similar cases.831 Make the “+” superscript. Check full reference list for other cases of possible subscripts and superscripts.955 Change “Arg uptake into” into “Arg uptake through Ypq2 into isolated”.955-957 Specify in this experiment whether ATP was present or absent.956-957 and 959 Change “ (see text for the strains used)” into “. Values for the ypq1-2-3Δ strain were subtracted from those for the ypq1-3Δ strain”, of course with the genotypes in italics.958 Insert “of Ypq2” after “selectivity”.963 Change “Vacuoles were” into “Vesicles were”.965, 1049, 1052 and 1069 and Table 1 header: Make “14” superscript.967, 971, 1007, 1010 and 1046 Change “ctrl” into “Ctrl”.967, 971, 1008 and 1011 Consider changing “cold” into “non-radioactive”.977 Change “Ypq2 switches into an exchange mode of transport” into “exchange will dominate, and net transport will slowly cease”.1025 Change “w-t and vsb1Δ” into “w-t (SL073) and vsb1Δ (SL074)”.1032 Change “ctrl” into “control (Ctrl)” or “untreated (Ctrl)”.1085 Insert space between number and unit. Check full manuscript for similar cases.Figures: In micrograph panels, either insert an indicated 10-micrometer bar (one per figure is sufficient) or indicate the length of sides of the panels.Fig. 1A: Explain in the legend the scissors depicted in right panel.Fig. 4A: Please delete this. The text is easy to understand by itself and can even be improved, and the two drawings make more confusion than explanation. There are at least two problems with them. First, the exchange reaction has no consequence on any concentrations; it is the uniporter function for both directions that assures that an equilibrium is approached. Second, the membrane potential and the negative charges of the polyphosphates make it unlikely that a cation like Arg will have the same concentration on both sides at equilibrium.Fig. 4C: The ordinate axis of the right panel needs some explanation for non-experts on survival curves to understand. Move, e.g., the explanation on s from Materials and methods to the figure legend.Fig.5A: Most genotype designations here end correctly with a delta, and that was intended also under the rightmost open bar. However, on my screen the character is erroneously a framed cross.Fig. 5D: This is the first time the authors present CMAC. A brief explanation, at least of the purpose and principle, is needed in the legend.Reviewer #2: The manuscript from Cools et al. focuses on the principal proteins that control arginine transport from and to the vacuoles in yeast, and provide evidence that this transport is regulated by nutrient requirements of the cell. The authors identify a novel vacuolar transmembrane protein, Vsb1, and provide compelling evidence that Vsb1 is as a major contributor to drive arginine transport into the vacuole. The authors also address the role of another transporter, Ypq2, a previously described exchanger of arginine  at the vacuole. The authors propose a model where Vsb1 imports arginine to the vacuole under nutrient rich conditions, and Ypq2 mediates the export to the cytosol during nitrogen starvation.The authors present an overall convincing and elaborate study. They show by an elegant assay the contribution of each transporter to arginine uptake into the vacuole in vivo. Their method is based on the selective permeabilization of the plasma membrane followed by the osmotic rupture of the vacuole, thus allowing the recovery of the cytoplasmic and vacuolar pools of arginine.Although the authors propose that Vsb1 is the main transporter of arginine import into the vacuole, they were not able to demonstrate Vsb1-dependent transport in vitro or its transport activity per se. Similarly, they propose that Ypq2 acts as an exporter  during starvation conditions, yet they only show impaired uptake if the protein is deleted. Direct transport activity of these proteins by reconstitution was not shown, though also admitted by the authors. It is important to note that measurements in the Wiemken and collaborators study, as also in this work includes the whole set of transporters at the vacuole, so it is difficult to assign the observed exchange rates to only one transporter My major concerns are listed below:1. The authors claim that Ypq2 is a transporter (Lines 177-179); however, neither they nor others have shown direct activity of this protein, they probe activity in system (intact vacuoles) containing all or most of vacuolar protein. It would be important to stress this in their manuscript.2. Along the same line, the authors show that Ypq2-dependent uptake is dependent on the intra-vacuolar concentration of arginine  and propose that transport is bidirectional, and only unidirectional during starvation, although this was not assessed. Interestingly, they are able to detect activity even in the control condition that, although it is low, it is still stimulated by ATP. Can the authors rule out that the proposed exchange reaction is carried out by more than one transporter and that Ypq2 is rather the importer?3. The authors propose that Ypq2 would mobilize Arg from the vacuole to the cytosol during nitrogen starvation . They convincingly show that Ypq2 is required for intracellular consumption of arginine during starvation; however, they do not show direct Ypq2-dependet export from the vacuole. To address this a bit more directly, the authors could measure the efflux of radioactive arginine that was previously incorporated into vacuoles before to demonstrate Ypq2-dependent export of this amino acid. Additionally, the export could be assessed under conditions of nutrient rich and starvation conditions.4. The assumption that Ypq proteins are arginine transporters seem to be supported by the fact they homologues to the PLQ2 mammalian transporter. For PLQ2 it has been shown that mutation in P55 abolished arginine uptake. To demonstrate homologous activity, the authors could mutate the equivalent position in Ypq2 to test if the mutant is able or not to rescue the uptake phenotype they observe.5. The authors claim that Vsb1 is a transporter, but do not show transporter activity. Although I do not expect them to demonstrate activity in vitro, I find it important that they remain cautious with their interpretation as also in their statements along the manuscript, as they refer to this protein as an essential transporter.6. Figure 7: The analysis using bafilomicin A was performed in wt cells, so it is difficult to claim that the block in V-ATPase function specifically affects the activity of Vsb1. Please rephrase.7. Along their entire manuscript the authors mix up a few things in my view. They observe under different conditions (N-depleted/repleted) different requirements of their transporters, and do so by comparing deletion mutants with wild-type. They then conclude that each of the transporters is regulated (in abstract and throughout the manuscript). However, they have no evidence at all that this is the case. Results from Figure 8 do not strictly show that these proteins are inactive in each condition, they are just not required. I agree that nitrogen regulation may be likely, but they do not show regulation of either transporter, and they should thus rephrase these passages accordingly.8. I found the entire discussion too long and too excessive. The repeat many aspects of their results section. I recommend to condense it strongly (by ½) to make to more accessible.Minor issues:1. Line 124: “…the intact yeast vacuoles isolated via the above methods…”. The authors refer to the “above methods” as if they would explain them, but they do not describe the isolation method itself, so it would be suitable to say “our method” or “the used method”.2 .Paragraph between lines 112-115 can be interpreted as they could not establish the pH measurement when incubated with the inhibitor. “ the pH measured in isolated vacuoles became more acidic in the presence of ATP , this acidification was partially lost upon addition of the K+/H+ ionophore nigericin, and it could not be established if the vacuoles were pre-incubated with the V-ATPase inhibitor bafilomycin A ”3. Figure 7C: It would be helpful for readers if the authors name of the protein fused to GFP (Can1) also in the figure.4. Many experiments have been repeated only twice. Particularly, Figures 3B and 6D show the same control condition but the effect of ATP is only evidenced in Fig 3B. 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Critical Care [We have read with attention the commentary from von Düring et al. questioning our research letter on safe doubling of ventilator capacity recently published in cal Care . Our pubIn fact, we argue for the idea of ventilating two patients with a single ventilator, not against. This is a serious misunderstanding of our work proposing a safe way to ventilate two patients at once, so as von Düring et al. note, a second life can be saved.in-series (one-after-the-other) breathing circuit (Fig. We proposed a safe, innovative method based on a simple uit Fig. . It direWhile we agree with von Düring et al. when they say “The objective of ventilator sharing is to save a second life by buying time to find a second ventilator” , but we safe last resort proposal for last resorts, rather than a desperate measure.So yes, we believe our in-series setup is a