Objective: to describe clinical and laboratory characteristics; to assess predictors for death in HIV-infected patients with Kaposi sarcoma (KS).We performed a retrospective study of HIV-infected patients diagnosed with KS in one infectious diseases hospital in Romania, between January 2008-November 2013. KS diagnosis was established on physical examination, skin biopsy, and for visceral involvement upper gastrointestinal endoscopy, bronchoscopy and computed tomography. KS was staged according to the AIDS Clinical Trials Group (ACTG) [We identified 27 HIV-infected patients with KS. The median age was 42 years (IQR 34-52) and 18 (67%) were male. The median CD4 count at HIV diagnosis was 195 cells/cmm (IQR 55-313), while at KS diagnosis the median CD4 count was 101 cells/cmm (IQR 41-270). Eighteen (67%) patients had a CD4 count <200 cells/cmm. The median HIV viral load at the time of KS diagnosis was 120,000 copies/mL . HIV infection was diagnosed before KS in 19 patients (70%), with a median time between HIV and KS diagnosis of 7 months (IQR 0-58). The most frequent KS localization was the lower limb in 16 (59%) patients and 7 (26%) patients had disseminated KS. Oral, gastrointestinal and pulmonary involvements were seen in 10 (37%), 4 (15%) and 3 (11%) patients respectively. Concomitant opportunistic infections were diagnosed in 20 (74%), while other malignancies in 3 (12%) patients. According to the ACTG classification 16 (59%) patients had poor risk KS. Fifteen (56%), 6 (22%), 1 (4%) and 5 (18%) were in stage 1, 2, 3 and 4, respectively according to the Mitsuyasu classification. Six (22%) patients received specific KS treatment: three local radiotherapy and three systemic therapy . The overall mortality was 41% with a median duration between KS diagnosis and death of 6 months (IQR 2-15). Gastrointestinal involvement (p=0.019), poor-risk KS in ACTG classification (p<0.001) and stage IV Mitsuyasu (p=0.006) were associated with death in univariate analysis.The mortality rate in this study was high, due to poor immunological status, extended KS and high incidence of opportunistic infections, but also due to the lack of specific systemic treatment.


EHP in August 2014. Historically, the Editor-in-Chief of EHP had two major functions: providing scientific and strategic vision, and managing day-to-day office operations. This dual role derived from EHP’s unique position as a government publication staffed by federal personnel, in which the head of the journal was also the head of an administrative branch. But the dual role is outdated and has not allowed the journal to grow in a way that best serves the field of environmental health sciences.As many of you may recall, Hugh A. Tilson retired as Editor-in-Chief of EHP has developed a new position to manage many of the operational duties once handled by the Editor-in-Chief. We are pleased to announce that Shaun Halloran has been hired as EHP ’s inaugural Operations Manager. Mr. Halloran was formerly Manager of Journals Production and Senior Manager of Production for the American Society of Civil Engineers, where he was responsible for more than 35 journals. As Operations Manager, Mr. Halloran serves as deputy to the Editor-in-Chief, overseeing manuscript production to ensure efficient and timely publication of the journal. EHP staff are excited about working with Shaun and bringing his experience to bear on journal operations.Managing operations to ensure manuscripts are processed and published in a timely fashion is a full-time job all by itself. Therefore, EHP is seeking a new Editor-in-Chief. The job responsibilities for this position have been updated: the Editor-in-Chief will still function as the chief executive for the journal, but he or she will focus primarily on bringing a strategic vision to and managing all scientific aspects of the journal to ensure that EHP continues to publish articles of the highest quality.Now that the Operations Manager position has been filled, The new Editor-in-Chief will need to be an internationally recognized leader in the environmental health sciences community and should have substantial experience in reviewing manuscripts and serving on editorial boards. The position will be posted on the USAJOBS website 30 March–17 April 2015 and will be filled within the next few months.EHP will help reduce our current backlog of manuscripts. We have made other recent changes that will help even more. Specifically, we have revised our process for final review of manuscripts, which will decrease the time from provisional acceptance to publication. Additionally, we have obtained a limited accessibility accommodation that will facilitate meeting our requirement, under Section 508 of the Rehabilitation Act, to ensure that all our electronic content is accessible to people with disabilities. As a federally funded publication, EHP is fully committed to 508 accessibility, yet producing an accessible document from an author’s manuscript can be surprisingly difficult and time-consuming, depending on the complexity of the paper. The result has been a significant strain on EHP ’s resources. With the new accommodation, EHP staff have developed an efficient strategy for providing accessible documents to readers who need them. We expect this to expedite publication.We are confident that filling these positions at We understand the frustration some authors have experienced with the delay in publication of their papers. We believe the steps we have taken will help alleviate this frustration. By hiring an Operations Manager, we expect to improve the efficiency of our production processes. We expect additional improvement with our new streamlined policies for final review of papers and meeting readers’ accessibility needs.EHP want to thank everyone, especially our authors and Associate Editors, for their ongoing support for the journal. As we continue making strides in addressing problematic issues, we look forward to your continued support.The editors and staff at


In renal transplantation, treatment options for antibody-mediated rejection are limited. Here, we report a case of severe AMR treated with eculizumab. A 50-year-old woman known for end stage kidney disease secondary to IgA nephropathy received a kidney transplant from a 50-year-old deceased donor. At 5 months after transplantation, she presented with acute graft dysfunction and biopsy showed a severe antibody-mediated rejection associated with thrombotic microangiopathy. Despite an aggressive conventional immunosuppressive regimen, signs of rejection persisted and the patient was treated with 3 doses of eculizumab. Following the therapy, markers of TMA improved and graft function stabilized. However, ongoing signs of rejection remained in the repeated biopsy. In kidney transplantation, eculizumab is an expensive treatment and its role in the treatment of antibody-mediated rejection remains to be determined. The management of antibody-mediated rejection (AMR) represents a challenge in kidney transplantation, as the treatment options are limited and sometimes ineffective . Here, wA 50-year-old Asian woman known for end stage kidney disease secondary to IgA nephropathy received a kidney transplant from a 50-year-old deceased donor. The pretransplant crossmatch (CDC) was negative despite a PRA value of 25% and a complete HLA mismatch (in B and DR). The cold ischemia time was 7 hours and the patient was induced with basiliximab and methylprednisolone. Subsequent maintenance immunosuppression consisted of tacrolimus, mycophenolate, and prednisone.μmol/L (1.48 mg/dL)).After transplant, the recipient had delayed graft function requiring three dialysis sessions. Ultimately, the clinical course was favorable and she was discharged with a good renal function  was not a recognized entity in 2013; she was only treated with intravenous pulses of methylprednisolone and the creatinine stabilized at 160 μmol/L. A few weeks later, an analysis revealed the presence of a donor specific antibody (DSA) (DQA0302) with MFI of 1800. This DSA was present before the transplant (MFI 1600) but we were not aware of its presence at the time of transplant. The DSA titers decreased mildly after initial treatment (MFI 900).One week after discharge, the patient was readmitted for graft dysfunction with a rise in creatinine to 194 μmol/L) but, this time, there were signs of thrombotic microangiopathy (TMA): thrombocytopenia, decreased haptoglobin, increased LDH, decreased fibrinogen, and hemolytic anemia. A second biopsy was performed and it showed an acute active type 2 AMR with lesions of TMA, peritubular capillaritis, and glomerulitis (negative Cd4) associated with a grade IA cellular rejection , signs of AMR (TMA) still persisted in a repeat biopsy (3rd biopsy) and DSA remained elevated (MFI 1300). We decided to treat the recipient with 3 doses of eculizumab   . Followi 900 mg) . Howeverμmol/L) and haptoglobin gradually decreased, but platelets were normal and there was only mild anemia. All infectious complications resolved.The last graft biopsy (8 months after transplant) showed ongoing AMR and progression of chronic lesions  . HoweverIn AMR, the complement system is activated and constitutes one of the main mechanisms responsible for endothelial damage. Eculizumab is a monoclonal antibody that specifically binds to factor C5 and inhibits the terminal pathway of the complement cascade, preventing endothelial injury . ClinicaIn our case, the diagnosis of AMR was not done initially despite the presence of glomerulitis and peritubular capillaritis because C4d-negative AMR was not a recognized entity before 2014. Therefore, the patient was not aggressively treated after the first biopsy. Once the diagnosis of AMR was made, she had CMV infection that limited the immunosuppressive treatment. However, she probably could have benefited from a more aggressive and longer conventional treatment. By the time eculizumab was introduced, it was probably too late as AMR had become extremely severe. Furthermore, because eculizumab is an expensive medication and not officially approved for AMR treatment, the patient only received 3 doses. Despite improved TMA blood parameters and transient stabilization of renal function following the use of eculizumab, signs of severe AMR persisted in the biopsies. The overall treatment failed to decrease the titer of DSA and because of the heavy immunosuppressive burden she developed several potentially fatal infections.Stegall et al. used eculizumab for the prevention of AMR in a group of 26 recipients with very high immunological risk . These rInterestingly, at 1 year after transplant, the incidence of transplant glomerulopathy was also lower in the eculizumab group (7% versus 28%). Recently, Orandi et al. published a study using eculizumab alone or combined with splenectomy as rescue therapy for AMR. Of the 14 patients, 5 recipients treated with this combination had no graft loss. However, inquisitively, 80% of patients treated with eculizumab without splenectomy experienced graft loss [In the literature, there are more than a dozen case reports on the use of eculizumab for treatment of AMR. In the majority of these cases, eculizumab is described as being effective in reversing the rejection and improving graft function –11. HoweIn kidney transplantation, eculizumab is an expensive treatment and its role in the treatment of antibody-mediated rejection remains to be determined.


In recent years, there is an increasing demand for sharing and integration of medical data in biomedical research. In order to improve a health care system, it is required to support the integration of data by facilitating semantic interoperability systems and practices. Semantic interoperability is difficult to achieve in these systems as the conceptual models underlying datasets are not fully exploited. In this paper, we propose a semantic framework, called Medical Knowledge Discovery and Data Mining (MedKDD), that aims to build a topic hierarchy and serve the semantic interoperability between different ontologies. For the purpose, we fully focus on the discovery of semantic patterns about the association of relations in the heterogeneous information network representing different types of objects and relationships in multiple biological ontologies and the creation of a topic hierarchy through the analysis of the discovered patterns. These patterns are used to cluster heterogeneous information networks into a set of smaller topic graphs in a hierarchical manner and then to conduct cross domain knowledge discovery from the multiple biological ontologies. Thus, patterns made a greater contribution in the knowledge discovery across multiple ontologies. We have demonstrated the cross domain knowledge discovery in the MedKDD framework using a case study with 9 primary biological ontologies from Bio2RDF and compared it with the cross domain query processing approach, namely SLAP. We have confirmed the effectiveness of the MedKDD framework in knowledge discovery from multiple medical ontologies. KEGG, DrugBank, MGI, HGNC and several of NCBI databases into ontologies using Semantic Web technologies. Bio2RDF contains over 2.5 million triples and 0.19 million outlinks and 0.19 million inlinks  is defined a share pattern of any two predicates Pi and Pj ii) [CA] for a Connectivity pattern of any two predicates.The direct association describes the direct relationship between Definition 9: Share Association Given predicates Pi and Pj in a directed RDF schema graph G. Let C(Pi) and C(Pj) denote the entities (subjects or objects) that are directly connected to Pi and Pj regardless of the direction. l is the reachability test for the given predicates Pi, Pj. SA indicates the probability-based association matrix for a share pattern between Pi and Pj.Definition 10: Connectivity Association For a Connectivity pattern of any two predicates Pi and Pj, CA defines the probability-based association for a Connectivity pattern between Pi and Pj based on the Share Pattern. For the given Share Associations SA and SA and the distance between the predicates l, the connectivity association can be computed as follows:The definition is influenced by the chain matrix multiplication problem (a kind of dynamic programming) of determining the optimal sequence for performing a series of operations. After we get the similarity score for all pairs of predicates, we use the formula in Eqs  and 4)  4) to geDefinition 11: Predicate Association Matrix Given the total number of predicates n and the probability-based association score for cross domain share patterns SA and Cross Domain Connectivity Patterns CA between predicates Pi and Pj, PA indicates an association matrix for all pairs of predicates Pi and PjThere are various different approaches in clustering heterogeneous information networks. In , we desiIn this paper, we designed a new algorithm, called the Predicate-based Hierarchical Agglomerative Clustering (PHAL), for topic discovery from the heterogeneous information network of the multiple domains. PHAL is a hierarchical bottom-up clustering algorithm by applying Hierarchical Agglomerative clustering (HAC)  to the hPhase 1: Hierarchical Agglomerative Clustering This phase focuses on clustering predicates from the heterogeneous information network of the given datasets using Hierarchical Agglomerative Clustering [topics  in a hierarchical structure similar to the topics shown in ustering . This alPhase 2: Construction of Topics Starting at Level Mid Given the tree from Phase 1, we first compute the mid-level of the tree . The topics at the mid-level Mid are assigned to FinalTopicSet. If there is no topic at the level Mid, then go upward until find any topic groups on the subsequent level of the Mid  in the hierarchy. Among 43 topics shown in Mid.InitialMap. Starting from the level Mid—1, we start traversing the tree upward to construct topic groups with each topic at the the subsequent level of the Mid level  and assign it to FinalTopicSet. Repeat this step at Mid—2 until reaching the tree root. In addition, we have made a special topic group  that is a collection of the singleton topics whose size is 1. Topics 12-43 in This phase illustrates the constructing process of topics for the remaining topics, which do not belong to the topic groups Phase 3: Hierarchical Topic Refinement There are some cases such that relevant concepts are disconnected. This is due to the hard partition in which a predicate was not allowed to join more than one topic. To handle the issue, a refinement process is conducted to construct a more complete topic model with the respective predicates and their neighborhood. More precisely, for any two pairs of predicates, if they form a Connectivity pattern and then we include their intermediate predicates to the topic and update those topics in FinalTopicSet. From this refinement process, a predicate may join more than one topic group that results into fuzzy clustering.Algorithm 1 Hierarchical Heterogeneous ClusteringInput: X = {x1, …xn}Output: Topic Set T = {t1, …, tk}Phase 1: Hierarchical agglomerative clustering/* L = 0Define level X as a topic, save them in InitialMap with level L = 0 Consider each element in L, X〉 to InitialMap Put pair 〈Whiletruedo ifThe active set InitialMap only has one itemthen     breakelse  L from InitialMap   Extract all topics at current level p and q ∈ X with the best distance computed using formula    Choose pair M = M ∪ {p, q} //Save all pairs to set M   L = L + 1 //Update the level   foreach pair of elements p and q in set Mdo   p and q into a new topic u    Merge u to set X     Add L, X〉     Update InitialMap with 〈end   end  end Phase 2: Construction of Topics Starting at Mid Level/* L determined from Phase 1Get the tree height Mid = Roundup (L/2) Compute the middle level of the tree  //Construct the topics while traversing the tree upward until it reaches the tree rootwhile Mid > 0 do ifThere is at least one topic at level Mid of InitialMapthen  T = {T1, …, Ti} at level Mid by checking 〈Mid, T〉 from InitialMap   Extract topics Z containing all the remaining topics   Define set    //Initializing the topic indexT| + 2 // Excluding T1 and the initial topics T = {T1, …, Ti}   index = |T   FinalTopicSet = FinalTopicSet + foreach topiczi in Zdo   ifzi.size = 1 then    zi to the special topic Topic1     Add Topic1      Update FinalTopicSet with the special topic else    zi to Topicindex     Add Topicindex      Update FinalTopicSet with       index++end    end      breakelse  Mid = Mid-1   end  endreturn FinalTopicSetAlgorithm 2 Hierarchical Topic RefinementInput: FinalTopicSet = {t1, …, tk}Output: FinalTopicSet =Phase 3: Hierarchical Topic Refinement/* foreach topic t in FinalTopicSetdofori and pj in topic tany two predicates pdo ifpiandpji, pj) = 2are connected through a Connectivity pattern & d = 3are connected through a Connectivity pattern & d. The share patterns in the lower level are part of the Connectivity patterns in the higher level. From these results, we confirm that the CDNP patterns play a significant role in integrating data and finding cross domain topics from heterogeneous information networks.Predicate and Concept Ranking: The predicates (the primary component in MedKDD) and their associated concepts are ranked based on their in-degree and out-degree. From this analysis, we found out the roles of ontologies for cross domain collaboration in heterogeneous information networks. Among 374 concepts, top concepts such as clinv:Resource, kv:Resource, dv:Resource, phv:Resource are shown in dv:source and dv:calculated.properties are from three ontologies such as DrugBank, ClinicalTrials, and PharmGKB. These predicates and concepts are mainly from the primary ontologies including ClinicalTrials, KEGG, DrugBank, and PharmGKB.Cross Domain Predicate and Concept Ranking: The contents of cross domains were ranked based on the in-degree/out-degree of cross domain concepts and predicates. We observed the cross domain rankings with predicates and concepts were different from the non-cross domain rankings. However, the ontologies playing important roles are similar. PharmGKB , KEGG, and DrugBank . kv:pathway, clinv:arm.group and dv:x.kegg are top three cross domain predicates of KEGG, ClinicalTrials, and DrugBank, respectively.Topic Ranking with Cross Domain Features: These patterns are ranked according to primary features such as cross domain predicates, predicate popularity (in-degree/out-degree of the predicates), and domain verity (the number of domains in which the patterns are captured). Topic Ranking with Cross Domain Neighborhood Patterns: Topics are ranked based on the CDNP patterns. Provider, Consumer, Reacher, CD and NCD patterns) is very similar to the ranking computed by the predicate popularity, cross domain predicate, and variety shown in For the given nine ontologies in OWL shown in The comparative analysis will provide valuable insight into the effectiveness of the Cross Domain Neighborhood Patterns (CDNP) and the CDNP-based topic discovery model. The evaluation of the proposed model has been conducted using practical examples of the cross domain predicate patterns and topic discovery. We show the patterns are useful in knowledge discovery from multiple ontologies through evaluation and validation of the proposed model compared to other approaches in knowledge discovery from diverse domains.N is the number of nodes (concepts and predicates) and E is the number of edges (links between nodes). The association score were computed by the Predicate Association formula The case studies involve the comparative analysis with the HPKM and PHAL algorithms and experiments with the both algorithms to confirm the effectiveness of the proposed method. For the given nine ontologies shown in Resource appears 92 times and pubmed_vocabulary:Resource appears in all 9 domains. This indicates that concepts like Resource are mainly used for a high level mapping between different domains. Thus, these concepts are too abstract to be of practical use of such data. For the data integration, data normalization was performed to map 30 Semanticscience Integrated Ontology (SIO) concepts to domain concepts. In addition, about 45% (149 of 330 predicates) are named with a prefix x. This indicates that the predicates are also too abstract to provide meaningful relationships between concepts. After clustering, the size of predicates became doubled and the concepts quintupled. All the predicates except sider_vocabulary:reported.frequency are fully contributed to the integration of cross domains and discovery of relevant patterns. Through the normalization and clustering, relevant concepts and predicates were integrated and clustered according to their contexts.As seen in OMIM:Uniprot}, where the symbol → represents a path from one concept to another within a single domain and the symbol ⇒ represents a path from one concept to anther across domain. Similarly, SLAP also has the ability to retrieve the information on the association between drugs and targets. However, SLAP does not support the information retrieval for any other association besides the drug and target association.We have conducted a comparative analysis with the Semantic Link Association Prediction (SLAP)  that wasDrugBank . In order to demonstrate the knowledge discovery process with multiple datasets, the top five drug instances such as NADH, Beta-D-Glucose, Flavin adenine dinucleotide, Pyridoxal Phosphate, and Citric Acid shown in m targets and then m targets relate to n genes, where First, we have conducted several queries that are designed to retrieve the association among the key concepts in dv:Drug(SIO_010038), dv:Target(SIO_010423) and dv:Gene(SIO_001121) as shown in dv:Drug (SIO_010038) → dv:target → dv:Target(SIO_010423) → dv:x-genecards → dv:Gene(SIO_001121) across these two topics. bind, hasGo, hasSubstructure, hasPathway, hasTissue, and PPI. Thus, some of information could not be retrieved from the query processing. However, the GraphKDD framework does not put any restriction on this query processing so that it has a capability to find any associations for a given query on drug, target, and gene.In MedKDD, among 43 topics discovered from the Bio2RDF ontologies, there is a path between Topic 16 and Topic 27 through the common concepts such as NADH, L-Glutamic Acid, Pyridoxal Phosphate, Ethanol, and Zonisamide) were also selected according to the number of the instances associated with target, gene and OMIM resource. A cross domain query was designed with the following paths such as i) {dv:Drug (SIO_010038) → dv:target → dv:Target(SIO_010423) → dv:x-genecards → dv:Gene(SIO_001121)}; ii){DrugBank:〈dv:Drug (SIO_010038) → dv:target → dv:Target(SIO_010423) → dv:x-uniprot〉 ⇒ omimv:Uniprot}; iii) {omimv:Resource → omimv:x-uniprot → omimv:Uniprot}. OMIM resources for the top five drug instances. OMIM resources. Similar to the first case, for the query across two domains DrugBank and OMIM, MedKDD retrieved all the relevant information while SLAP could not retrieve any information except partial information about drug and gene association.Second, for cross domain knowledge discovery, top five drug instances  or across domains (DrugBank and OMIM). The SLAP prediction of drug and gene interactions was strictly limited to the association between chemical compounds and targets. This result implicates the effectiveness of the MedKDD framework in discovering knowledge even across topics or across domains compared to the cross domain query processing approach, namely SLAP.Based on top five CDNP patterns , we analyzed the collaboration across domains as shown in Case 1:ProviderPatterns in Domain Collaboration Five domains  are involved in the collaboration of the Provider pattern. In this collaboration, we found that DrugBank and KEGG are a Provider, CTD is a Balancer, and PharmGKB is a Consumer as well as a Bridger. ClinicalTrials is its Consumer. Provider pattern. provider pattern graph of Topic 25.Provider pattern in Topic 25. This pattern describes the collaboration of two predicates, namely phv:x-hgnc and kv:x-hgnc to integrate information from three domains. Specifically, PharmGKBResource links to KEGGGene  through HGNCGene symbol. Provider pattern of Topic 25.Case 2: Domain Collaboration withConsumerPatterns Five domains, namely KEGG, OMIM, DrugBank, CTD, and PharmGKB, are involved in this case. We found that CTD is a Consumer of KEGG, OMIM and PharmGKB. DrugBank are a Balancer with KEGG. Consumer pattern, CTD. Consumer pattern graph of Topic 15.Consumer pattern in Topic 15. This Consumer pattern shows the collaboration between predicates mgv:x-ensembl-protein and kv:x-uniprot as a Consumer of the PharmGKB concept , SIO_001077:Gene. The collaboration is established across three domains such as KEGG, MGI and PharmGKB. In this pattern, due to the collaboration of these two Consumer predicates, the Uniprot concept Resource is linked to the Ensemble concept Resource through PharmGKB concept Gene . Consumer pattern in Topic 15.Case 3: Domain Collaboration withReacherPatterns Only two predicates from two domains, namely PharmGKB and ClinicalTrials, are involved in the Reacher pattern. From this pattern analysis, we found that PharmGKB plays a Provider and ClinicalTrials a Consumer from this collaboration. Reacher pattern between PharmGKB and ClinicalTrials. Reacher pattern graph of Topic 22.Reacher patterns in Topic 22. This Reacher pattern was formed with the predicates kv:pathway and dv:x-kegg across four domains . Through the collaboration of these two predicates in this pattern, the PharmGKB concept Drug  is linked to the KEGG concept Resource and the KEGG concept Resource is linked to the CTD concept Pathway . Reacher pattern of Topic 22.Case 4: Domain Collaboration withDirectional ConnectorPatterns From the pattern analysis with top 40 predicates, all nine domains have the Directional Connector (DC) patterns. DC patterns with 54 links among these domains. We have found that CliniclalTrials, DrugBank and SIDER play the role of Provider and CTD, HGNC, KEGG, MGI, OMIM, PharmGKBConsumer. Furthermore, KEGG, PharmGKB, SIDER, HGNC play the role of Bridger. The connection among the domains were established through the Bridger pattern. DC pattern graph of Topic 16.DC pattern of Topic 16, three predicates such as dv:x-hgnc, hv:x-omim and omimv:x-mgi were used to connect concepts across five domains . In this pattern, the KEGG concept Enzyme  links to the HGNC concept Resource. The HGNC concept Resource links to the OMIM concept Resource, and the OMIM concept Resource links to the MGI concept Resource. We found all the paths within the bounded context  determined by the DC patterns. One of them is the path 〈SIO_010343:Enzyme → dv:x-hgnc → hv:Resource → hv:x-omim → omimv:Resource → omimv:x-mgi → mgv:Resource〉. DC pattern of Topic 16.Case 5: Domain Collaboration withNon-Directional ConnectorPatterns In the Non-Directional Connector (NDC) pattern discovery, all the 9 domains are involved. NDC patterns. These 9 ontologies are connected with 72 links, which means all of them are fully connected. Interestingly, all of them have the same number of in-degree and out-degree, so that they are well balanced. Thus, no Bridge pattern is required in this collaboration. NDC pattern graph of Topic 23.NDC pattern in Topic 23. This NDC pattern is composed with four predicates such as mgv:x-refseq-transcript, ctdv:pathway and ctdv:disease that are used to connect nine different domains . Specifically, in this pattern, those three predicates are used to connect six concepts such as KEGG Gene , Refseq resource, KEGG Resource, CTD Chemical, KEGG Pathway  and CTD Chemical-disease-association. NDC pattern in Topic 23.Case 6: Domain Collaboration with Topics The 43 topics discovered from 9 domains are shown in DrugBank, MGI, SIDER, respectively. Second, the number of topics that were discovered from KEGG, DrugBank, ClinicalTrials is 35, 29, 28, respectively. They are three highest topic numbers among 9 domains. On the other hand, CTD and HGNC contain less than 10 topics, which are two smallest ones. In terms of the size of these domains, ClinicalTrials, DrugBank and KEGG are the top three biggest ones while HGNC and CTD are the bottom two smallest ones. Thus, we found that the size of domains  is strongly related to the number of topics in these domains.There are many efforts that have been made for semantic annotation of heterogeneous data and perform knowledge discovery on biomedical data –37. MostWe now discuss existing work on knowledge discovery. For relation extraction, ontologies are helpful for extraction of relations in the form of thesaurus, dictionary, or general corpus , for extA variety of research have been conducted in systematical and computational knowledge discovery with cross domain datasets. HeteSim is a general framework that was designed for relationship discovery and linking detection from heterogeneous networks , 55. TheHowever, our approach is different in that, first of all, we focus on a more general approach for graph structural pattern analysis and topic discovery from heterogeneous information networks. In addition, we have combined an unsupervised learning algorithm with a pattern discovery technique to provide a more systematic way of knowledge discovery from multiple domains.Our approach for finding roles in ontology collaboration is related to existing work in ontology matching, alignment, classification and mapping . OntologPattern based knowledge analysis has been conducted in many aspects of biomedical research. van Leeuwen  proposedOur work is motivated by previous work that emphasised the importance of ontological relations. Tartir et al.  pointed In our study, we hypothesize that an association measurement based on predicate neighborhood patterns would be more effective in finding relevant information than a concept-based measurement. Our approach defined a new model of predicate-based patterns and neighboring closeness for an automatic knowledge discovery. In this paper, we fully focus on the discovery of cross domain patterns from the heterogeneous information network representing different types of objects and links in multiple biological ontologies. The MedKDD framework was designed to effectively discover topics from multiple ontologies by partition them into smaller topic graphs and constructing a topic hierarchy. The topic hierarchy was constructed based on the analysis of the discovered patterns and participating graphs into smaller sub-graphs. To our knowledge, there is no existing work that aim to discover cross domain topics based on predicate-oriented neighborhoods patterns discovered from multiple ontologies and use the discovered topics for knowledge discovery across domains.In this paper, we presented the MedKDD framework for knowledge discovery and semantic interoperability through the discovery of the Cross Domain Neighborhood Patterns (CDNP) from the heterogeneous information network of the multiple medical ontologies. In MedKDD, we developed the bottom-up hierarchical clustering (HPAL) algorithm and discovered cross domain topics from the given multiple ontologies. We demonstrated that cross domain cohesive topics can be dynamically discovered from heterogeneous information networks of multiple ontologies and used for cross domain knowledge discovery. The MedKDD framework was evaluated using a case study with nine ontologies of Bio2RDF and compared with the cross domain query processing approach, namely SLAP. Overall, the experimental results confirm that the MedKDD framework is effective in the cross domain knowledge discovery from heterogeneous information networks of multiple ontologies.Future work will include the development of Apache Spark framework that is an extension of Hadoop for parallel and distributed knowledge discovery processing from heterogeneous information network . For the


Ojeok-san (OJS) is a traditional herbal formula consisting of 17 herbal medicines that has been used to treat various disorders. In this study, quantitative analytical methods were developed using high-performance liquid chromatography equipped with a photodiode array detector to determine 19 marker compounds in OJS preparations, which was then combined with chemometric analysis. The method developed was validated in terms of its precision and accuracy. The intra- and interday precision of the marker compounds were <3.0% of the relative standard deviation (RSD) and the recovery of the marker compounds was 92.74%–104.16% with RSD values <3.0%. The results of our quantitative analysis show that the quantities of the 19 marker compounds varied between a laboratory water extract and commercial OJS granules. The chemometric analysis used, principal component analysis (PCA) and hierarchical clustering analysis (HCA), also showed that the OJS water extract produced using a laboratory method clearly differed from the commercial OJS granules; therefore, an equalized production process is required for quality control of OJS preparations. Our results suggest that the HPLC analytical methods developed are suitable for the quantification and quality assessment of OJS preparations when combined with chemometric analysis involving PCA and HCA. Zingiberis rhizoma recens, and Allii fistulosi bulbus. Traditionally, OJS has been used to treat disorders such as fever, anhidrosis, headache, whole body pain, contracture of the nape and neck, vomiting, abdominal and heart pain, and menstrual irregularities [Ojeok-san (OJS) is a traditional herbal formula used in Korean medicine that consists of 17 compositional herbal medicines: Atractylodis rhizoma, Ephedrae herba, Citri Unshius pericarpium, Magnoliae cortex, Platycodonis radix, Aurantii Fructus Immaturus, Angelicae gigantis radix, Zingiberis rhizoma, Paeoniae radix, Poria sclerotium, Angelicae dahuricae radix, Cnidii rhizoma, Pinelliae tuber, Cinnamomi cortex, Glycyrrhizae radix et rhizoma,Recent studies have reported on the therapeutic effects of OJS against lumbago and inferior limb pain , primaryCluster analysis is a data analysis method used to assign similar objects belonging to the same group and is used in a variety of practical applications like bioinformatics, using chemometric analyses, such as principal component analysis (PCA) and hierarchical clustering analysis (HCA) , 9.PCA is an unsupervised pattern recognition technique and is a useful tool for visualizing similarities or differences in multivariate data . PCA canRecently, herbal formulas have been manufactured by pharmaceutical companies in diverse dosage forms, such as powder, granules, and tablets, as these are more convenient and easier to take than traditional decoction forms. However, the compositional herbal ratio or the origin of herbal components of a herbal formula may differ between different companies, and so the formula produced by each company may contain a variety of chemical constituents –19. SuchTherefore, in this study, we developed analytical methods for the quantification of 19 marker compounds in a laboratory-produced water extract and in commercial granules of OJS using HPLC–PDA. In addition, chemometric analysis data were combined with the quantitative results and employed to assess the quality of OJS preparations via the Pearson correlation coefficient and PCA and HCA data.1), chlorogenic acid (3), ferulic acid (6), benzoic acid (8), neohesperidin (12), and cinnamic acid (15) used were obtained from Sigma-Aldrich . The protocatechuic acid (2) and nodakenin (9) used were purchased from ChromaDex  and NPC BioTech , respectively. The albiflorin (4), paeoniflorin (5), liquiritin (7), naringin (11), cinnamaldehyde (17), and glycyrrhizin (19) used were purchased from Wako Pure Chemical Industries . The hesperidin (10), ononin (13), oxypeucedanin hydrate (14), byakangelicin (16), and benzoylpaeoniflorin (18) used were purchased from Chengdu Biopurify Phytochemicals .The HPLC-grade acetonitrile and water used were purchased from JT Baker Inc.  and the guaranteed reagent grade acetic acid used was obtained from Junsei . The gallic acid  using an electric extractor . The extracted decoction was filtered through a standard sieve  and freeze-dried to make OJS water extract powder denoted as “OJS01.”Dried herbal drugs consisting of OJS were mixed and extracted using a 10-fold volume of distilled water (w/v) at 100°C for 2 h under pressure  before being injected into the HPLC system.Powdered OJS01 (200 mg) and commercial OJS granules  were dissolved in 10 mL of distilled water and the solutions were filtered through a 0.2 3), and a photodiode array detector (SPD-M20A). Separation was conducted on a Gemini C18 column . The column temperature was set at 40°C. The mobile phase consisted of water containing 0.1% formic acid (A) and acetonitrile (B). The composition of the mobile phase was 6%–20% (B) for 0–20 min, 20%–25% (B) for 25–30 min, 25%–40% (B) for 30–40 min, 40%–46% (B) for 40–50 min, and 46%–87% (B) for 50–55 min, held for 5 min and then reequilibrated to 6% (B) until the end of the analysis. The flow rate was 1.0 mL/min and the injection volume was 10 μL. The detection wavelengths of all standards and samples were in the UV at 230, 250, 260, 270, 275, 280, 290, 310, 325, and 335 nm.The HPLC system used was a Shimadzu LC-20A  chromatograph equipped with a solvent delivery unit (LC-20AT), an autosampler (SIL-20AC), a column oven (CTO-20A), a degasser , which was calculated using the equation RSD = (standard deviation/mean) × 100. The precision was measured three times in a single day (intraday precision) and over three consecutive days (interday precision).The accuracy of the method used was evaluated through the recovery test. Both low and high concentrations of the marker compounds were added to the samples. The recovery was calculated as follows: recovery (%) = /spiked concentration) × 100.The relationship between OJS samples was evaluated using the Pearson coefficient of the amounts of the marker compounds. To cluster the OJS sample, PCA and HCA were performed based on the rows (OJS samples) and columns (the amounts of the 19 marker compounds). The evaluation of the Pearson coefficient and the clustering analysis (PCA and HCA) were carried out using the open-source software package R (v. 3.0.2).18 column was employed for the simultaneous determination of the 19 marker compounds in the OJS water extract, as it has been the most frequently used technique in the chemical analysis of herbal medicines [The mobile phase modifier, gradient ratio, and UV detection wavelength were considered as the main factors for optimizing the conditions for the HPLC analysis of the OJS water extract. A Cedicines , 21. TwoVarious ratios of the components of the mobile phase (A : B) were tested using gradient elution, and the optimal separation was observed at the following gradient conditions: 6%–20% (B) for 0–20 min, 20%–25% (B) for 25–30 min, 25%–40% (B) for 30–40 min, 40%–46% (B) for 40–50 min, and 46%–87% (B) for 50–55 min, held for 5 min and then reequilibrated to 6% (B).18 column for quantitative analysis , which ranged in value from 0.9993 to 1.0000 for each compound. The LOD and LOQ values were 0.004–0.090 μg/mL and 0.012–0.272 μg/mL, respectively  and commercial OJS granules (OJS02–OJS10). There was wide variation observed in the contents of the marker compounds in the 10 OJS samples. While OJS01 contained the 19 marker compounds, the commercial OJS granules showed lack of one or more of the following compounds: protocatechuic acid, chlorogenic acid, ferulic acid, nodakenin, hesperidin, neohesperidin, and cinnamaldehyde.Moreover, variation in the content of these compounds was apparent between the OJS samples: 2.8–16.6-fold for gallic acid, 1.1–3.5-fold for protocatechuic acid, 1.4–37.0-fold for chlorogenic acid, 2.0–64.0-fold for albiflorin, 2.1–8.7-fold for paeoniflorin, 1.1-fold for ferulic acid, 5.9–57.1-fold for liquiritin, 1.1–7.8-fold for benzoic acid, 5.0–224.5-fold for nodakenin, 5.5–624.9-fold for hesperidin, 1.6–8.5-fold for naringin, 2.4–73.0-fold for neohesperidin, 3.5–13.8-fold for ononin, 3.8–71.8-fold for oxypeucedanin hydrate, 3.7–21.3-fold for cinnamic acid, 5.0–124.0-fold for byakangelicin, 1.5-fold for cinnamaldehyde, 3.8–112.6-fold for benzoylpaeoniflorin, and 3.1–8.6-fold for glycyrrhizin .This result implies that the water extract and commercial granules of OJS were not chemically equivalent because of the variation in the content of the marker compounds.r2), which is a measurement of the distance between two samples and shows the degree of their relationship: a stronger correlation is observed when r2 is closer to a value of 1 [r2 for OJS01 was the lowest, followed by OJS04, while the values of the other OJS samples were in the range 0.5 < r2 < 0.8  produced in the laboratory showed little correlation with the commercially manufactured OJS granules from a chemical perspective. This result demonstrates that the low correlation between the OJS samples, particularly the laboratory-produced water extract and the commercial granules, can presumably be ascribed to the different ratios of the compositional herbal medicines, herbal resources, or extraction procedures of the OJS preparations between different pharmaceutical companies.Therefore, verification of the herbal resources, using an identical combination ratio, or using a valid extraction process, is required to produce chemically equalized OJS preparations that can guarantee an equivalent therapeutic efficacy.18 column was precise, accurate, and reliable and was successfully applied to the simultaneous determination and quantification of 19 marker compounds for the quality assessment of OJS samples. The content of the marker compounds varied between the OJS samples. Moreover, a laboratory-produced OJS water extract was not closely related to the commercial OJS granules, which also showed a wide distribution in the results of chemometric analyses, such as the Pearson correlation coefficient, PCA, and HCA. Our results suggest that HPLC–PDA combined with chemometric analysis can be a useful strategy for the quality evaluation of OJS samples from different origins. It is necessary to produce chemically equalized OJS preparations for better quality samples.The analytical method developed using an HPLC-PDA with a reversed-phase C


Early and unambiguous detection of bovine tuberculosis (bTB), a significant disease of cattle worldwide, is necessary to control the spread of infection to other animals and humans. Current testing strategies are laborious, time consuming and heavily reliant on host responses that do not distinguish bTB from other mycobacteria. We report the presence of a pathogen signature, liparabinomannan (LAM), as a potential biomarker for bTB infection.Fifty-five animals  from a well characterized bovine serum repository were screened for the presence of LAM using a commercially available ELISA. Analysis showed that LAM had a sensitivity of 100% and a specificity of 91.7% for bTB detection .LAM detection easily separated bTB infected animals from bTB exposed and negative controls. We propose that pathogen related markers, such as LAM, should be included with current testing strategies as a battery diagnostic for bTB.The online version of this article (doi:10.1186/1756-0500-7-559) contains supplementary material, which is available to authorized users. M. bovis functional genome usage during infection identified the release of mycobacterial specific proteins, peptides, DNA and lipids that are shed or secreted into host fluids[M. bovis specific peptides  identified in bTB infected sera as biomarkers for detection of subclinical infection[M. bovis peptides were capable of detecting bTB infected animals and Pks5 had little to no cross-reactivity to other mycobacterial species[M. bovis diagnostics is likely to follow a battery testing model, which includes multiple pathogen signatures, such as peptides and lipids. Therefore, we hypothesized that the major mycobacterial cell wall glycolipid, mannosylated lipoarabinomannan (ManLAM), also circulates in the serum and may be utilized as a biomarker for bTB infection. We validated the presence of ManLAM in randomly selected samples from a well characterized cattle serum repository including confirmed cases of M. bovis (n = 10), bTB exposed  and negative controls (n = 33). Based on sensitivity and specificity analyses, we propose that ManLAM may aid current diagnostic tools for bTB infection.Successful control of bovine tuberculosis (bTB) is achieved through the interplay of best management practices and diagnostics. Early dt fluids, 8. We aM. bovis .Field samples from bovine tuberculosis infected and exposed cases were generously provided by the National Veterinary Services Laboratory  serum repositories. All field samples were collected from a single herd in California. Bovine tuberculosis disease status for each animal was validated using a combination of bacterial culture, antemortem caudal fold tests (CFT) and lesion histology at necropsy. Johne’s disease history for this herd was indeterminate. Bovine tuberculosis exposed cases were defined as animals that had contact with bovine tuberculosis infected animals but remained bovine tuberculosis negative . Negative controls were collected from a bovine tuberculosis free dairy herd in Minnesota. Negative controls tested negative for M. tuberculosis strain H37Rv purified mannosylated lipoarabionmannan (ManLAM)  or M. smegmatis purified non-mannose-capped lipoarabinomannan (AraLAM)   were diluted 1:5 in PBS and analyzed for the presence of LAM using the human LAM ELISA kit  per manufacturer’s instructions. A LAM standard curve was included using kit controls and two-fold dilution series of ManLAM spiked sera. The standard curve was plotted using GraphPad Prism software . The optical density was read at 450 nm with a wavelength correction at 570 nm. All samples were read in three wells. LAM ELISA was repeated twice.Randomly selected field sera samples (bTB positive n = 10 and bTB exposed n = 12), negative controls (n = 33), and negative controls spiked with either Optical densities for each animal were averaged across the replicates. Receiver operating characteristic (ROC) curves were compared for 1) positive versus within-herd negative exposed (exposed) and 2) positive versus negative controls using the area under the ROC curves (AUC). Optimal cutoff values were determined by maximizing specificity and sensitivity by plotting the true negative rate against the true positive rate. ROC curves and specificity-sensitivity plots were created in SPSS® .M. bovis infection, mycobacterial specific proteins and lipids, including those from the bacterial cell wall, are shed into host fluids. Several studies have shown the presence of lipoarabinomannan, a major cell wall glycolipid found on pathogenic mycobacteria, in sera and urine[M. bovis infected animals. Fifty-five animals  were screened for the presence of LAM using a commercially available ELISA. A LAM concentration curve was created and cross-reactivity to another glycolipid, non-capped lipoarabinomannan (AraLAM), found in environmental mycobacteria was assessed  were scses n = 1) were scM. smegmatis cultures and detected no significant changes to OD  and bovine sera spiked with LAM (0–300 ng/mL) showed a positive signal. Bovine sera spiked with non-capped lipoarabinomannan  did not react. All samples were conducted in triplicate. (PDF 59 KB)Additional file 1:


Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causal agent of all forms of Kaposi’s sarcoma (KS), including AIDS-KS, endemic KS, classic KS and iatrogenic KS. Based on Open reading frame (ORF) K1 sequence analysis, KSHV has been classified into seven major molecular subtypes . The distribution of KSHV strains varies according to geography and ethnicity. Xinjiang is a unique region where the seroprevalence of KSHV is significantly higher than other parts of China. The genotyping of KSHV strains in this region has not been thoroughly studied. The present study aimed to evaluate the frequency of KSHV genotypes isolated from KS tissues in Classical KS and AIDS KS patients from Xinjiang, China. ORF-K1 of KSHV from tissue samples of 28 KS patients was amplified and sequenced. Two subtypes of KSHV were identified according to K1 genotyping. Twenty-three of them belonged to subtype A, while five of them were subtype C. More genotype A than genotype C strains were found in both Classical KS and AIDS KS. No significant difference was found in the prevalence of different genotype between Classical KS and AIDS KS. Kaposi’s sarcoma (KS) is a tumor, originally described by Moritz Kaposi in 1872 . Classicet al. [Kaposi’s sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), is a gamma-2-herpesvirus or rhadinovirus. KSHV was first identified in a biopsy tissue from a patient with AIDS-related Kaposi’s sarcoma (AIDS-KS) by representational difference analysis in 1994 by Chang et al. .As a large double-stranded DNA virus, KSHV has approximately 90 identified open reading frames, of which over 60 show homology with other rhadinoviruses and 15, designated K1–K15, were unique to KSHV when its genome was first sequenced . ORF-K1,Xinjiang Uygur Autonomous region is the largest province in northwestern China, and located on the ancient Silk Road as an important staging post over a thousand years ago. Xinjiang borders on Russia, Kazakhstan, Kyrgyzstan, Tajikistan, Pakistan, Mongolia, India and Afghanistan. Ethnic groups in Xinjiang are diverse and distinct, the main ethnic groups are the Uygur (45.7%) and the Han (39.7%), other ethnic minorities include Kazakh, Mongolians, Hui, Kirgiz, Manchu, and Xibo. Classic KS are rarely seen in the Han Chinese, but are seen more frequently in the Uygur ethnic group, more than one patient has been diagnosed histopathologically as having KS every year at the Affiliated Tumor Hospital of Xinjiang Medical University in Urumqi, the capital city of Xinjiang Uygur Autonomous Region. We have previously shown that the overall seroprevalence of KSHV was 19.2% in the general population in Xinjiang, which was substantially higher than the 9.5% seroprevalence of KSHV in the control subjects from the general population in Han Chinese in Hubei Province . Our datet al. (2001) had reported that KSHV strains from seven patients with classical KS in Xinjiang were classified as subtype C [n = 18), and several by subtype A (n = 4) [Dilnur ubtype C . Zhang ret al. 01 had reTwenty-eight KS classical tissue examples were collected from Xinjiang Uygur Autonomous Region, China. Seven of them were peripheral blood mononuclear cells examples and the rest were formalin-fixed paraffin-embedded tissues (FFPET). All the specimens were collected between 1997 and 2009. Eighteen of them were from Uygur patients, eight of them were from Kazakh Patients, one from a Hui patient and one from a Han patient. Permission to conduct the study and informed consent was obtained in accordance with a protocol approved by the Ethics Committee of Wuhan Institute of Virology, Chinese Academy of Sciences.DNA from peripheral blood mononuclear cells of KS patients was extracted with a Blood & Cell Culture DNA Mini Kit according to manufacturer’s instructions .DNA from FFPET was extracted as the following steps. Tissues were incubated at 55 °C with 1 mL xylene for 10 min, and then centrifuged at 8000 rpm for 5 min, supernatant was collected. The supernatant was further centrifuged at 8000 rpm for 5 min to totally remove the paraffin. The remaining dimethylbenzene was removed by using anhydrous ethanol. Then the supernatant was added with 250 μL cell lysis buffer  and kept at 55 °C over night. Equal volumes of phenol, chloroform, and isoamyl alcohol (25:24:1) were added to the supernatant. The supernatant was clarified by centrifugation at 8000 rpm for 2 min at 4 °C. Then 2.5 times the volume of pre-cold anhydrous alcohol was added to precipitate DNA. 30 min later, the mixture was centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was discarded. After slightly drying, 20 μL TE (pH8.0) was added to dissolve DNA, and kept in −20 °C.Fragments (363 bp) of the VR1 region of KSHV ORF-K1 were amplified by nested-PCR. Primers were designed as described . BrieflyThe purified K1 gene fragments were cloned into pGEM-T vector for constructing recombinant plasmid pGEM-T-K1 and transformed into DH5α competent cells. Then, the recombinant plasmid was extracted from DH5α cells using TIANprep Mini Plasmid Kit  and sent for sequencing by Shanghai Majorbio Biotech Co., Ltd. Twenty-eight of the unique sequences (GenBank Accession numbers: KM582680–KM582707) and 19 other KSHV strains obtained from GenBank were used to construct the phylogenetic tree and were analyzed with a DNAStar package and aligned with Clustal W in Bioedit (version 7.0.0). Phylogenetic trees were constructed by neighbor-joining analysis by Phylip (version 3.68) and MEGA (version 4.0.2), respectively. The statistical reliability of the NJ tree was evaluated using 1000 bootstrap samples. TreeView (version 1.6.6) was used to see the trees constructed by Phylip. The sequences of 19 other KSHV strains consisted of 1 strain of subtype A1 XJ-27 (FJ853386); 1 strain of subtype A2: Ema-7 (AF130305); 1 strain of subtype A3: IT-268 (GU097421), 1 strain of subtype A4: BCBL-B (AF133039),US114 (GU097431) ; 1 strain of subtype A5: KE229 (GU097433); 3 strains of subtype B: UG-65 (FJ884618), UG-85 (FJ884620), MP10 (AF387367); 2 strains of subtype C2: XJ-6 (FJ853368), XJ30 (FJ853388); 1 strain of subtype C3: XJ-20 (FJ853379), AF17170531; 2 strains of subtype D: TKS10 (AF133043), ZKS3 (AF133044), 2 strains of subtype E: Sio1 (AY329025), Tupi1 (AF220292); and 2 strains of subtype F: AF178810, FJ884616.All the information of cases was analyzed by descriptive statistics. MS Excel was used to calculate the mean, standard deviation, coefficient of variation, and median of data. Fisher's exact test was used to test the associations of sex ratio and KSHV genotype between two groups and the associations of KS subtypes and KSHV subtypes. One-way analysis of variance (ANOVA) was used for statistical evaluation of differences between the two groups.p < 0.001; ANOVA). , while female patients were 4/28 (14.29%). Classic KS cases were twenty-three, while AIDS-associated KS were five. Classic KS has a male/female ratio of 6.67 (20/3), and AIDS-associated KS has Male/Female Ratio of 4.0 (4/1). Compared to AIDS KS, the male/female ratio in Classic KS is higher but without significant difference by Fisher’s exact test. The mean ages for Classic KS and AIDS-KS were 54.39 and 23.80 years old, respectively. AIDS associated KS patients were younger than Classic KS ( ANOVA). .Total DNA were extracted from FFPET and plasmid samples. 363 bp of ORF-K1 VR1 region of KSHV were amplified by nested PCR from each sample. PCR products were detected by 1.5% agarose gel electrophoresis see . DNA extThe 363 bp K1 sequences of the 28 KSHV isolates from Xinjiang Uygur Autonomous Region, China were aligned with 19 previously reported sequences in the database of NCBI using Bioedit (version 7.0.0). Phylogenetic trees were constructed by neighbor-joining analysis by MEGA (version 4.0.2) and Phylip (version 3.68), respectively. From the MEGA phylogenetic trees , the XinTraditionally, herpesviruses are considered to be co-evolved with their hosts throughout vertebrate evolution. KSHV is an ancient human virus and spread together with its migrating human hosts . Many stIn China, KS cases are rare, only reported in Xinjiang Region  and TaiwCo-migration of viruses with the human population is common. KSHV subtypes A and C are predominant in the Mediterranean, Middle Eastern and Asian regions ,22, whicClassic KS was only found in Xinjiang Minority, but not often seen in Han population. More epidemiologic study should be done to investigate the risk factors, including environmental and human genetic factors.


It is increasingly recognized that molecular chaperones play a key role in modulating the formation of amyloid fibrils, a process associated with a wide range of human disorders. Understanding the detailed mechanisms by which they perform this function, however, has been challenging because of the great complexity of the protein aggregation process itself. In this work, we build on a previous kinetic approach and develop a model that considers pairwise interactions between molecular chaperones and different protein species to identify the protein components targeted by the chaperones and the corresponding microscopic reaction steps that are inhibited. We show that these interactions conserve the topology of the unperturbed reaction network but modify the connectivity weights between the different microscopic steps. Moreover, by analysing several protein-molecular chaperone systems, we reveal the striking diversity in the microscopic mechanisms by which molecular chaperones act to suppress amyloid formation. Molecular chaperones are recognized to interfere with protein aggregation, yet the underlying mechanisms are largely unknown. Here, the authors develop a kinetic model that reveals the variety of distinct microscopic mechanisms through which molecular chaperones act to suppress amyloid formation. The great variety of cellular functions on which life depends reflects a multiplicity of carefully regulated interactions between macromolecules, notably involving proteins and their substrates and cofactors. Understanding the mechanisms underlying such interactions is a fundamental objective of molecular biology, not least because it opens up therapeutic opportunities of correcting dysfunctional cellular behaviours linked to disease. For example, in the context of the malignant growth of tumours, enzymes involved in cell signalling, particularly kinases, are widely investigated as targets for intervention and for the design of drug discovery programmes. Another intriguing example of dysfunctional cellular behaviour involves the pathogenic aggregation of normally soluble peptides and proteins into aberrant insoluble species known as amyloid fibrils234in vitro and in vivo studies in the last decade have demonstrated that molecular chaperones play important roles in the specific suppression of amyloid formation and of the toxicity that is associated with this process891011121314Molecular chaperones are vital components of the protein quality control system, which assist in the synthesis, folding, trafficking and degradation of proteins67The challenge arises at a fundamental level from the large variety of potential interactions between a given molecular chaperone and the different protein species that can be present during the aggregation process . Indeed,1617192021222324An established route to interrogate conventional chemical systems to define reaction mechanisms consists of performing kinetic experiments and comparing the results to rate laws derived from canonical molecular mechanisms22192027In this work, we demonstrate a strategy based on chemical kinetics that is designed to go beyond the initial documentation of the inhibitory effects of particular species and their magnitudes. In this approach, the mechanism of action of a given molecular chaperone is defined in precise terms including: (1) identifying the species with which it interacts, (2) the specific microscopic processes that are perturbed, and (3) the extent to which the rate constants of the distinct steps are affected. The core of the strategy consists in building on a previous kinetic approach to develop a new model that describes pairwise interactions between molecular chaperones and the different protein species. We show that these interactions conserve the topology of the unperturbed aggregation network and modify the connectivity weights between the different microscopic reaction steps . We applkn, k+, k− and k2, respectively2Supplementary Equations 1–3)2129M) of the type2129The formation of protein aggregates, such as amyloid fibrils, is the consequence of a series of microscopic events, including primary nucleation, fibril elongation, fibril fragmentation and secondary nucleation processes, which can be associated with reaction rate constants C±, D and κ are functions of a limited number of combinations of the microscopic rate constants: knk+, kn/k− and k2k+ We illustrate the potential of this analysis by considering examples of the inhibition of specific molecular chaperones on the aggregation kinetics of two different amyloidogenic systems . The firk+), keeping constant both the fragmentation (k−) and the nucleation rate constant (kn). This result indicates clearly that in this case the molecular chaperone inhibits specifically the elongation process.The second example  involvesβ42 ) is:Molecular chaperones can sequester misfolded monomers or oligomers in solution, or bind to fibril surfaces or fibril ends, as illustrated in Ci the concentration of the free binding sites of the molecular chaperones, and mbound is the concentration of monomers bound to the molecular chaperones.where Binding to soluble monomers reduces their free concentration, thereby decreasing the rates of all nucleation steps and the elongation processes. In analogy with the situation in the absence of inhibitors2129Keq and the equilibrium concentration of free chaperone  are similar to those measured independently by surface plasmon resonancekon=5 × 103 M−1 s−1 and koff =2 × 10−4 s−1), and only an additional minor contribution coming from the binding to fibril ends was required to describe the kinetic profiles at different chaperone concentrations. The binding constant estimated by model simulations, Keq=2 × 108 M−1, corresponds to a binding free energy of ΔG0=−47 kJ mol−1, similar but slightly larger than the free energy evaluated in the previous system for binding to fibril ends.We next focus on the molecular chaperone proSP-C Brichos discussed above that was shown in the semi-empirical approach to inhibit Areaction  . The kinetic analysis shows that these two molecular chaperones affect different microscopic processes involved with the aggregation of the Aβ42 peptide. In particular, this analysis, together with experimental evidence of the binding of the molecular chaperones to the aggregatesThe examples described above represent cases where a molecular chaperone modulates mainly a single microscopic event. In other more complex situations, a molecular chaperone can influence several microscopic processes simultaneously. Two examples of such a situation are reported in Another remarkable implication of the kinetic analysis is concerned with the topology of the reaction network in the presence of biological perturbations. The reaction network can be defined as the series of microscopic events which contribute to the overall aggregation mechanism, and the topology of the network describes the way through which these different microscopic processes are connected . The pre3132In addition, we note that the potential alternative reaction mechanisms with respect to nucleation-polymerization models could be captured by the kinetic approach based on population balance equations discussed here, provided that sufficient experimental information is available to enable fitting of the data to a given model.In situations where the association and dissociation rates of binding of the molecular chaperone are much faster than the microscopic aggregation reactions of the peptide or protein, the system can be considered to be at equilibrium, and we can derive here simple expressions for the effective microscopic reaction rates as a function of molecular chaperone concentration by analogy with the closed expressions obtained in the context of the inhibition of enzymatic catalysis in the presence of ligandsFor example, in the case of binding to fibril ends, the effective elongation rates can be readily derived as :k+ is the elongation rate constant in the absence of inhibitor, Keq the equilibrium binding constant and Ci the free chaperone concentration, which can be approximated as the total concentration of molecular chaperone in the case of a large excess of the latter relative to the number of fibril ends. For the system involving the prion Ure2p and the molecular chaperone Ssa1 (inset of Keq required to describe the apparent elongation rate constants with Keq) and of the free energy of binding (ΔG0).where inset of , we compin vitro and in vivo studies indicates that, in addition to their role in assisting protein folding678910111213In vitro studies suggest that these effects could be the consequence of several possible interactions not only with the monomeric species but also with the aggregated forms of the misfolded proteins1617Increasing evidence from β42 aggregation by DNAJB6, large effects are evident even at highly low sub-stoichiometric ratios of DNAJB6 to Aβ42, because the molecular chaperone interacts with small oligomeric species which are intermediates to the fibril formation. This behaviour could reflect the ability that molecular chaperones have developed to recognize specific hydrophobic patches of particular misfolded species to allow high efficiency at low concentrations.By applying this analysis to a set of systems involving different amyloidogenic proteins and molecular chaperones, we have provided a detailed mechanistic description of a variety of the modes of action through which molecular chaperones can suppress amyloid fibril formation. The analysis has shown that these naturally occurring inhibitors of protein aggregation are able to exert a protective function through a variety of diverse microscopic mechanisms, as summarized in The application of the approach described in this paper to other systems will increasingly provide more insights into the modes of action of molecular chaperones in suppressing the formation of amyloid forms of proteins and its consequences for the biological systems. These primary results, however, already indicate that molecular chaperones have evolved to prevent the formation of amyloid fibrils by developing the capacity to interact with different misfolded protein species and hence to be able to perturb all the microscopic steps of the aggregation process. The identification of the microscopic mechanisms of the action of molecular chaperones against amyloid formation is not only relevant in the context of understanding how living systems are able to suppress under normal circumstances the effects of protein misfolding and aggregation, but also in the context of the rational design of potential therapeutic strategies targeted against clinical disorders resulting from protein aggregation373940134445β peptide, Aβ(M1-42), UniProtKB ID P05067 and aa 672–713, with an N-terminal methionine corresponding to residue 671, (MDAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) was recombinantly expressed in E. coli BL21 DE3 star PLysS and purified essentially as described previouslyαB-crystallin (UniProtKB ID P02511) was recombinantly expressed and purified as described previously15Human Aβ42 monomer. ThT fluorescence was recorded under quiescent conditions at 37 °C, using a Fluostar Omega or Optima plate reader  with a 440-nm excitation filter and a 480-nm emission filterβ42 and the DNAJB6 molecular chaperone, and for Aβ42 and the proSP-C Brichos are taken from refs Aggregation kinetics were monitored using a ThT fluorescence assay based on the enhanced quantum yield for ThT fluorescence as the dye binds to amyloid fibrils. All experiments were performed in an assay buffer  with 10 μM ThT in microplate wells . Samples were prepared on ice in low binding tubes using freshly prepared An different chaperone concentrations Ci were simulated individually according to Msim(ti) and Mexp(ti) are the simulated and the experimental total fibril mass fraction at time ti, respectively. A similar procedure was followed to estimate the association and dissociation rate constants according to n different concentrations of chaperone were globally fitted by defining a least-squared error function: Msim,j(ti) and Mexp,j(ti) are, respectively, the simulated and the experimental total fibril mass fraction at time ti at a given concentration j of molecular chaperone.For a given chaperone-protein system, the aggregation profiles in the absence and presence of different molecular chaperone concentrations were simulated according to the procedure described in the How to cite this article: Arosio, P. et al. Kinetic analysis reveals the diversity of microscopic mechanisms through which molecular chaperones suppress amyloid formation. Nat. Commun. 7:10948 doi: 10.1038/ncomms10948 (2016).Supplementary Figures 1-3, Supplementary Methods and Supplementary References.


However, how these composite nanofibers promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unknown. Here, gelatin/β-TCP composite nanofibers were fabricated by incorporating 20 wt% β-TCP nanoparticles into electrospun gelatin nanofibers. Electron microscopy showed that the composite β-TCP nanofibers had a nonwoven structure with a porous network and a rough surface. Spectral analyses confirmed the presence and chemical stability of the β-TCP and gelatin components. Compared with pure gelatin nanofibers, gelatin/β-TCP composite nanofibers caused increased cell attachment, proliferation, alkaline phosphatase activity, and osteogenic gene expression in rat BMSCs. Interestingly, the expression level of the calcium-sensing receptor (CaSR) was significantly higher on the composite nanofibrous scaffolds than on pure gelatin. For rat calvarial critical sized defects, more extensive osteogenesis and neovascularization occurred in the composite scaffolds group compared with the gelatin group. Thus, gelatin/β-TCP composite scaffolds promote osteogenic differentiation of BMSCs in vitro and bone regeneration in vivo by activating Ca2+-sensing receptor signaling.Calcium phosphate- (CaP-) based composite scaffolds have been used extensively for the bone regeneration in bone tissue engineering. Previously, we developed a biomimetic composite nanofibrous membrane of gelatin/ Calcium phosphate (CaP) ceramic materials have been used traditionally in research into bone regeneration and clinical repair of bone defects because of their favorable biocompatibility and osteoconductivity –3. Howev in situ is of crucial importance for optimizing the design of scaffold materials for bone regeneration applications. Extracellular calcium ions are important to enhance the proliferation and phenotype expression of osteoblast cells [In recent years, electrospun CaP/polymer nanofibrous composites have been recognized as beneficial for the attachment, proliferation, and osteogenic differentiation of osteoblasts –16, as wst cells , 21. Prest cells  or humanst cells  is conceβ-TCP composite nanofibers with different contents of β-TCP nanoparticles using the electrospinning technique. The results demonstrated that attachment, spreading, proliferation, and differentiation of human osteosarcoma MG-63 cells increased with increasing content of β-TCP nanoparticles and continuous release of Ca2+ into the medium [β-TCP led to significant bone formation compared with that of the pure electrospun gelatin scaffolds [Previously, we successfully prepared gelatin/e medium . In addicaffolds . Howeverβ-TCP composite nanofibers on the osteogenic differentiation of rat BMSCs and examine the underlying mechanism in vitro and in vivo. Initially, we assessed the cell attachment, proliferation, and spreading and alkaline phosphatase (ALP) activity of rat BMSCs on gelatin/β-TCP compared with pure gelatin nanofibers. We then detected mRNA levels of osteogenic specific genes and calcium-sensing receptor (CaSR) as a calcium-signaling molecule. Subsequently, we investigated the efficacy of gelatin/β-TCP to induce new bone regeneration and related CaSR expression by surgically creating a critical-sized calvarial defects model in rats.The objective of the present work was to analyze the effect of electrospun gelatin/β-TCP solution is shown in β-TCP nanoparticles  was dispersed in deionized water containing 2% w/v sodium citrate. Then 20% (w/v) of gelatin  was added into the β-TCP suspension solution. The contents of β-TCP were set as 20 wt% of the gelatin. Electrospinning was then performed using the following variables: applied voltage 20 kV, solution feeding rate 0.3 mL/h, collecting distance 12 cm, and ambient conditions of 40°C. To prepare scaffolds for cell culture, the electrospun nanofibrous membranes were chemically cross-linked according to our previous research [The detailed procedure for the electrospinning of gelatin/β-TCP nanoparticles in the gelatin nanofiber matrix was investigated by transmission electron microscopy (TEM) using a Hitachi H-800 machine. The crystal and chemical structures of the composite nanofibers were examined by X-ray diffraction  and Fourier transform infrared spectroscopy  spectrometry, respectively.The surface morphology and internal structure of the composite nanofibers were observed using a scanning electron microscope . The distribution of 4 cells/well) were seeded onto experimental scaffolds in 12-well plates and incubated at 37°C in a humidified atmosphere with 5% CO2. After 1 day of culture, the samples were fixed in 2.5% glutaraldehyde and serially dehydrated with an increasing ethanol gradient, air-dried in a hood, and sputtered with gold before observation under SEM . Cytoskeletal organization was observed under a confocal laser scanning microscope . Nuclei were stained with 4′,6-diamidino-2′-phenylindole  and actin filaments were stained with rhodamine phalloidin  after culturing for 24 h. The cell spreading areas were measured using Image J software  employing a random sampling method. Cell proliferation was assayed using a CCK-8 kit  at 1 day, 3 days, and 7 days of culture, with the absorbance being read at a wavelength of 450 nm, using an enzyme linked immunosorbent assay reader .Rat BMSCs (5 × 10n = 6) were continually cultured in wells supplemented with osteogenic medium containing 50 mg/mL ascorbic acid-2-phosphate, 100 nM dexamethasone, and 10 mM β-glycerolphosphate. At 4, 7, and 14 days, the ALP activity of the adherent cells was assessed using an Alkaline Phosphatase Assay Kit , according to the manufacturer's instructions. The absorbance was measured at a wavelength of 405 nm, and values of ALP activity were read off a standard curve based on standard samples provided in the kit.Rat BMSCs/scaffolds (RUNX-2), collagen type I (COL1A1), bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and calcium-sensing receptor (CaSR), are listed in After osteogenic induction culturing for 7, 14, and 21 days, total RNA was extracted from each sample using the TRIZOL reagent , following the manufacturer's instructions. The RNA was then reverse transcribed to generate cDNA using the Reverse Transcription System . Real-time RT-PCR was performed using the SYBR Green Detection System with an ABI PRISM 7500 Real-Time PCR System . All reactions were carried out in triplicate. The primer sequences of the osteogenic genes, including runt-related transcription factor 2  and the dorsal cranium was exposed. Two critical-sized full thickness bone defects (5 mm diameter) were prepared in each rat at the center of each parietal bone, using a saline-cooled trephine drill . Each deAt 4 and 12 weeks after implantation, calvaria samples were harvested intact and fixed in 4% paraformaldehyde for 24 h at 4°C. The specimens were examined using micro-CT scanning, as previously described . Files wµm thickness. Hematoxylin and eosin (H&E) staining and Masson's trichrome staining were performed separately on tissue sections, according to the manufacturer's protocols, and images were captured under a light microscope .Tissue processing and sectioning were carried out as previously described . BrieflyImmunohistochemistry for OCN and CaSR was performed as previously described , 28. Bri t-test for independent samples. Differences between groups of *P < 0.05 were considered statistically significant and **P < 0.01 was considered highly significant.All quantitative data were expressed as means ± standard deviation (SD). Statistical analyses were performed using the SPSS 19.0 software . Statistical differences were determined using Student'sβ-TCP composite nanofibers. All the electrospun nanofibers showed a nonwoven structure, with an interconnected porous network. Pure gelatin nanofibers were continuous, smooth, and homogeneous  and β-TCP (PO43−: 950–1100 and 550–620 cm−1) were detected clearly. In our previous study, electrospun gelatin/β-TCP composite nanofibers with 20 wt% β-TCP possessed remarkable effects in terms of the bioactivity of osteoblasts-like MG-63 cells in vitro [ in vivo [β-TCP composite nanofibers with 20 wt% β-TCP were employed to further investigate that how the process of osteogenic differentiation of BMSCs and bone defects repair in situ was promoted by these composite nanofibers.in vitro  and guid in vivo . Therefoβ-TCP nanofibrous scaffolds for 24 h. Generally, cells attached onto the scaffolds displayed a flat and well-spread morphology, and the actin filaments were organized in well-defined stress fibers throughout the cells. Interestingly, rBMSCs seeded on composite nanofibrous exhibited more apparent cellular processes  (PLA)/20% TCP accelerates osteogenic differentiation of human adipose-derived stem cell compared with neat electrospun PLA scaffolds [To evaluate the effect of nanofibers on the early osteogenic differentiation ability of rBMSCs, ALP activities were quantified at days 4, 7, and 14 after cell seeding. As shown in us study  and otheus study , 33. Thecaffolds .2+ release behavior from composite nanofibers with different β-TCP contents in cell culture medium was estimated by refreshing the medium every 2 days. The results showed that the concentration of Ca2+ increased with the content of β-TCP loading and the highest Ca2+ concentration was reached in 20 wt% β-TCP loading [2+ released from these composite nanofibers. The expression levels of osteogenic genes of rBMSCs on nanofibrous scaffolds were evaluated in the osteogenic induction culture, as shown in  RUNX-2, COL1A1, BMP-2, and OCN on gelatin/β-TCP composite scaffolds were higher than those on pure gelatin nanofibers. This promotion effect could be ascribed to the Ca2+ released from composite nanofibers.In our previous study, the Ca loading . In this2+ and osteogenic differentiation of BMSCs, we examined the expression of CaSR in rBMSCs. Interestingly, the expression of CaSR in the composite materials group was significantly higher than that of pure gelatin group. Agarose gel electrophoresis of the PCR products showed a similar trend in the quantitative data. This result implied that CaSR may contribute to osteogenic differentiation of BMSCs mediated by composite scaffolds containing β-TCP. CaSR is reported to act as a sensor, thus transducing the Ca2+ signaling to intracellular gene expression to regulate cell function [ in vitro and in vivo [2+ medium [2+. In our work, the activation of CaSR in promoting osteogenic differentiation of rBMSCs may be a comprehensive effect regulated by both the structural property of the nanofibers and the sustained Ca2+ release.To examine the relation between released Cafunction , and has in vivo , 35–37. + medium . This maβ-TCP nanofibrous scaffolds and confirm the activation of CaSR signaling in vivo, the calvarial defect in rat was chosen as the experimental animal model because it is a common model and has been adopted widely by many researchers [β-TCP composite scaffolds and used the right defect implanted with pure gelatin scaffolds as a control. β-TCP composite scaffolds group was significantly elevated compared with the pure gelatin group (P < 0.05) at 4 and 12 weeks (Figures β-TCP group compared with that in pure gelatin group but not significantly.To investigate the guided bone regeneration ability of electrospun gelatin/earchers , 39, 40. Figures . The bonβ-TCP composite scaffolds expressed a higher level of OCN than the pure gelatin groups at 4 and 12 weeks after implantation (β-TCP composite nanofibers have a positive effect in guiding bone regeneration. The present results were consistent with another research [β-TCP sponges did not significantly improve bone formation compared with pure gelatin [The newly formed tissues within the calvarium defect were further analyzed by histological staining, as shown in antation . Thus, tantation . Collectresearch  and our research . However gelatin . This diβ-TCP composite nanofibrous scaffolds on CaSR expression, we examined the expression of CaSR in bone regeneration region after 12 weeks of implantation. As shown in β-TCP group (β-TCP composite scaffolds promote bone regeneration in situ by activating Ca2+-sensing receptor signaling.To assess the effect of implantation with gelatin/CP group  comparedCP group  and the CP group . These rβ-TCP composite scaffolds, which have compositional and structural features close to natural bone ECM, supported rBMSCs adhesion, spreading, and proliferation and ALP activity. Furthermore, gelatin/β-TCP composite scaffolds induced osteogenic differentiation of BMSC in vitro by activating Ca2+-sensing receptor signaling. Finally, the gelatin/β-TCP composite exhibited more extensive osteogenesis and higher CaSR expression in vivo compared with pure gelatin nanofibers. This study highlighted the great potential of the gelatin/β-TCP composite nanofibers in the practical application in orthopedics and dentistry, such as guided bone regeneration membranes in periodontal pockets.In the present study, the nanofibrous gelatin/


OxyVita Hb is a new generation hemoglobin based oxygen carrier (HBOC) produced through modification of a zero-linked polymerization mechanism using activators which incorporate cross-linked bovine tetramer hemoglobin into “super-polymeric” macromolecules (Average molecular weight = 17 MDa) for the purpose of oxygen delivery when whole blood or packed red cells are not available. This molecular design approach was generated in order to address several fundamental biochemical and physiological weaknesses of previous generations of HBOCs. Observation during pre-clinical and clinical studies provided evidence that these early generation acellular HBOCs were directly associated with loss of retention within the circulatory system, extravasation across endothelial tissue membranes due to their small molecular size leading to arterial and venous vasoconstriction with coupled increases in mean arterial pressure (MAP). The inherent increase in molecular size and structural stability of the OxyVita Hb is a direct response to addressing these serious weaknesses that have occurred during the evolution of HBOC development within the past two decades. The nature of the zero-linked synthetic route eliminates any chemical linkers remaining in the product, eliminating side reaction concerns, such as reversibility and decomposition due to weak chemical bonds, dependency on temperature and pressure, and residual toxicity Mosin vivo, the quest for the a safe and efficacious acellular HBOC for clinical applications has provided opportunities to pursue a variety of pathways in the molecular design to address the human physiological conditions under which they must function  is used to activate the side chain carboxylate groups on the hemoglobin surface, resulting in a highly reactive and short-lived ide bond . Some ino groups . One advAn extensive description of this zero-linked polymerization process as applied to the production of the original “Zero-Linked Bovine hemoglobin” (ZLBvHb) as originally described and produced by Professor Enrico Bucci and co-workers can be found in reference . EarlierAfter the initial development of this zero-linked polymeric hemoglobin and with the acquisition of the license to manufacture this HBOC, OXYVITA, Inc., modified some of the preparation procedures to produce a more homogeneous molecular weight polymer with an average M.wt. of 17 MDa. Using anion-exchange (DEAE) and size exclusion chromatography (Fractogel 20–40) purification and isolation of discrete molecular weight fractions were achieved for this zero-linked polymerization process. Recent pre-clinical studies ,23 have 2.2.The powder form of OxyVita Hb is produced from the lyophilization of the liquid form of the protein. Recent acquisition of a new Virtus, Inc. lyophilization instrument allows for the production of this product under carefully controlled processing. The new instrument has the capabilities of monitoring (CFR/211 compliant software) the essential steps associated within the freeze-drying process as well as automatically sealing of the product, thus reducing the chances of any endotoxin introduction during the course of the operation. In creating the powder form of OxyVita Hb, consideration has been given to the number of different formulations that include the need for proper buffering, essential electrolytes and osmolarity of the final product for infusion applications. Currently work is proceeding to improve re-constitution time (solubility) for rapid application within a variety of clinical situations.3.3.1.The differentiating structural and chemical properties of OxyVita Hb are due fundamentally to the unique chemical and physical methods incorporated into its production as described in the methods section. OxyVita Hb is synthesized through a polymerization reaction of purified (ββ)cross-linked tetrameric bovine hemoglobin using controlled activators which are removed after the initial phase of synthesis. This activation process is primarily associated with the carboxylic surface residues of the tetramers which allow for the formation of the “zero-linked” molecular species. Selected modulation of these reactions allows for specific lysine residue involvement due to the differential pKs exhibited by these residues at pH 6.7. This approach allows for the elimination of any chemical linkers remaining within the product, eliminating possible side chain reaction concerns, such as reversibility and decomposition due to weak chemical bonds, dependency on temperature and pressure, and residual toxicity.This production method utilized by OXYVITA, Inc. allows for enhanced manufacturing and quality control resulting in sustained identical batch to batch preparations with a mean molecular weight of 17 MDa as determined by dynamic light scattering. No more than 5% methemoglobin is present in each preparation. Given the unique chemistry associated with the use of reaction activators, variation of concentration of components involved, time and temperature of polymerization, this distinct polymerization process allows for the production of a range of molecular weight molecules that may find different clinical applications in the future. In addition, this polymerization process allows for the use of any mammalian blood (tetrameric hemoglobin) as the starting raw material for a zero-linked polymeric hemoglobin.3.2.These pseudo-peptide bonds initiated by the activiators between the tetrameric hemoglobin molecules within OxyVita Hb play a crucial role in the overall conformational stability of these large polymeric molecules. Structural integrity and biological functionality depend upon the integrity of both intramolecular and intermolecular bonds within the tetrameric units and between the multiple tetrameric units  that go to form this “super-polymeric” HBOC. Given the inherent secondary and tertiary structure of bovine hemoglobin  and the fact that each tetramer is cross-linked between the β-82 lysine residues, this formation of a large number of amide bonds between adjacent tetramers, clearly suggests that this large polymeric hemoglobin will possess increased conformational stability and be very resistant to molecular unfolding. This resistance to conformational unfolding ought to provide increased protection to the heme-iron moieties responsible for the transport and reversible binding of molecular oxygen within the circulatory system and oxygen delivery within the cellular/tissue environment.Studies on the structural integrity of this “super-polymeric” HBOC were carried out by isothermal unfolding studies at 37 °C using urea as the perturbant of the secondary and tertiary structure of these large molecules . Figure +2) is essential for the reversible binding/release of molecular oxygen in vivo. As evident in +2 → heme-Fe+3) are reflected by a blue shift in wavelength maximum within the Soret region. In contrast to LtHb and ArHb, wherein the Soret wavelength maximum blue shifts are 413 nm → 400 nm and 412 nm → 398 nm, respectively, OxyVita Hb undergoes little change in the Soret maximum, 410 nm → 408 nm over the entire range of increasing urea concentrations. This significant blue shift, indicative of methemoglobin formation (heme-Fe+3), is associated with an increase in the extent of heme exposure during unfolding within these natural acellular hemoglobins [An additional advantage of using the Soret spectral region for evaluating conformational changes in hemoglobin is that it also allows for analysis of the redox state of the hemoglobin associated with the unfolding process. Maintenance of the heme-iron complex in the reduced state  ∼1. Less than 5% methemoglobin is present in the final product.Further studies ,25 usingThe uniqueness of the chemical methods of synthesis of OxyVita Hb and its resultant molecular size have had profound impacts on some of the most important issues in the field of HBOC development. For years the question of loss of retention within the circulatory system associated with leakage of earlier HBOCs was associated with stress on the glomerulus and kidneys. More recently the nature of acellular HBOC extravasation and its impact on vasoconstriction and observed increase in mean arterial blood pressure (MAP) have been linked to complex interactions with nitric oxide (NO). During the past several years an understanding of these critical interactions has led to a better appreciation of the role of molecular size and its impact on the binding of NO and the concomitant physiological changes associated with any NO binding. The fact that increased molecular size can reduce the extent of acellular extravasation due to the molecules' inability to cross-over arterial or venous membranes which exhibit various pore sizes and bind with NO can lead to a reduction in vasoconstriction and help maintain MAP. In the case of OxyVita Hb the radius of this molecule allows for greater retention time in the circulatory system with a half-life of 8–12 times longer that smaller radius HBOCs. To date, all animal studies carried out with OxyVita Hb have resulted in no observable vascular extravasation as determined by its absence in the renal hilar lymph after exchange transfusion experiments . Additio4.This review has addressed the reasons as to why OxyVita Hb, a new therapeutic oxygen carrier and HBOC, is fundamentally different from previous generations of HBOCs. As our understanding of the relationship between the structure and functional behavior of the earlier HBOCs evolved, it soon became apparent that many of the structural characteristics incorporated into the modified HBOCs still resulted in problems of extravasation and vasoconstriction, oxidative degradation, and safety concerns when these HBOCs were used in many pre-clinical and clinical testing studies. OxyVita Hb was designed to address these serious concerns and the fundamental aspects of OxyVita Hb's development and success to date within the earlier pre-clinical studies are based upon its unique chemical technology and the recognition that molecular size, hydrodynamic properties, negative surface charge  and disc