Stage 1 is labeled Mild KS, withdisease limited to skin, flat oral mucosa lesions, and/or flesh coloredsubcutaneous nodules, with fewer than 10 lesions total. Stage 2 is labeledLymphadenopathic KS, including patients with lymph node involvement, nodularoral lesions, facial edema, conjunctival lesions, exophytic mass, or 10 to 19hyperpigmented skin/oral lesions . Stage 3 is labeled Woody Edema KS, defined as woody edemawith or without any of the characteristics of stages 1 or 2; this stage issubdivided into two parts: 3A, edema that involves less than 10% of estimatedbody surface area (BSA), and 3B, edema that involves more than 10% of estimatedBSA . Stage 4 is labeled Visceral and/or Disseminated Skin/Oral KS, definedas clinical pulmonary or abdominal visceral involvement and/or ≥ 20hyperpigmented skin/oral lesions in a widespread distribution (excludingflesh-colored subcutaneous nodules), with or without any of the criteria of theother stages.12 (3)Although the majority of patients with stage 2 disease had lymphadenopathy, thisstage does not require lymph node involvement. (4) Because presentation withmoderate-severe cytopenias was not associated with inferior survival, cytopeniaswere not incorporated into the classification.12The following are important footnotes to the proposed staging classification: (1)Coalescing or confluent hyperpigmented skin lesions localized to an anatomicregion count as one lesion per cluster. (2) Detailed working definitions forvisceral KS in the absence of bronchoscopy and endoscopy have been describedpreviously.16Induction failure in our cohort was defined as the inability to achieve morethan 90% reduction in size (subjectively determined) of all lesions afterinduction-phase treatment with BV (four cycles). Treatment regimens, supportivecare, and statistical analyses have been described previously.12 Ethics committee approvalswere obtained in Malawi and by the collaborating institution in the UnitedStates.ACTG TIS staging criteria definitions were evaluated for comparison on the basisof published guidelines.Sixty-eight patients had their diseases fully staged by using these criteria. Nopatients had stage 1 disease; 37 (54%) had stage 2; 14 (21%) had stage 3; and 17(25%) had stage 4 disease. Clinical characteristics and treatment responsediffered distinctly by stage .StatistP < .001). The two who survivedwith stage 4 disease were treated with intensified chemotherapy regimens thatincluded doxorubicin plus BV (ABV), and they remained alive on lifelong cART at17 and 27 months, respectively, from their dates of KS diagnoses.When applied to this cohort, the pediatric-specific staging classificationdetermined significantly different outcomes for both EFS and OS . With a median follow-up time of 28 months , patients who had stage 2 KS demonstrated a probability of 2-year EFSand OS of 73% and 75%, respectively, compared with patients who had stage 3 KS or stage 4 KS . Among the 15 deaths inpatients with stage 4 disease, all were attributed to refractory/progressive KS.Three patients received ABV (after disease failed to respond to BV) and survived4, 9, and 12 months, respectively. The remaining 12 patients received treatmentbefore the availability of doxorubicin; the median time to death in this groupwas 3 months . All four deaths in patients withstage 3 disease were a result of non-KS causes.A comparison of the Lilongwe pediatric KS staging classification with the ACTG Tstaging classification is depicted in Appendix Figs A1A and A1B. Althoughanalysis of survival to compare T1 versus T0 stages demonstrated a statisticallysignificant difference in EFS, the difference in OS was not significant.12 In this retrospective cohort, the proposed stagingclassification differentiated clinical patterns of disease associated withcontrasting survival outcomes. Thus, the classification presents a potentialplatform for the development of risk-stratified treatment protocols insub-Saharan Africa.Because of the clinical heterogeneity that is inherent to all childhoodmalignancies, risk stratification is an essential element that guides theformation of treatment paradigms in pediatric oncology worldwide. Distinctheterogeneity exists in pediatric KS as well, which provides the impetus for theformation of pediatric-specific systems.The relevance of this risk-stratification system stems from prognostication ofwhich patients can achieve favorable survival with the minimallymyelosuppressive BV chemotherapy regimen versus which patients will require amore intensive chemotherapeutic strategy. Given the limited capacity to deliveroptimal supportive care for patients with cancer throughout Africa, it iscritical to identify patients— especially patients with underlying immunesuppression from HIV—whose disease can be treated successfully with lessmyelosuppressive regimens.12 The clinical presentation of patients classified withstage 2 disease is characterized by a high rate of KS lymphadenopathy, aclinical presentation that can be difficult to identify in the absence ofprototypical skin, oral, or edematous KS lesions. Heightened awareness oflymphadenopathic KS is important, because these children often present ascritically ill with severe cytopenias. Given that the reconstitution of cellularimmunity may require several months with cART, we feel that the timelyinitiation of BV chemotherapy in addition to cART is critical in this group andhas a high potential to achieve a durable CR.The pediatric-specific staging classification highlights the most common clinicalfeature of childhood KS in central and eastern Africa: lymph nodeinvolvement.17Compared with the ACTG staging system used worldwide for HIV-related KS inadults, the proposed pediatric-specific system divides the T1 population intothose with woody edema  and those withvisceral disease . In addition, children withdisseminated skin disease, whose disease would be considered T0 in the ACTGsystem because of the absence of visceral involvement, had disease defined asstage 4 in the proposed pediatric classification because of the ≥ 20hyperpigmented skin/oral lesions. These patients experienced extremely highmortality rates, contrary to descriptions of adult KS in whom the T0presentation of more than 50 skin lesions is not uncommon and is not necessarilyassociated with poor survival.17 Stage 3disease is characterized by an indolent course, so the combination ofchemotherapy and cART more often achieved stable disease control, rather thanCR, and improved quality of life. Although a minority (n = 4) of patients withstage 3 disease achieved CR with BV , it is notable that all fourpatients presented with less than 10% of their BSA affected by edema, which mostoften was localized to the anterior aspect of the inner thigh. Thus, studiesthat involve larger cohorts may consider subdivision of stage 3 into A and Bsubstages on the basis of the extent of estimated BSA involved. In ourexperience with extensive woody edema, BV seemed to provide similar partialremission/stable disease status compared with more intensive regimens (ABV orpaclitaxel), although small patient numbers preclude definitive analysis.Ultimately, the most reasonable goal may be to optimize quality of life with theleast intensive chemotherapy possible.Woody edema is the defining characteristic of patients classified with stage 3disease; it tended to occur in older children and adolescents , which resembled a common clinical presentation of adults with KS inMalawi.Stage 4 disease was typified by an 88% mortality rate; all deaths were attributedto KS. Severe CD4 suppression was common in this group, but it was not adefining characteristic. In our experience, patients rarely survived more than 6months unless the chemotherapy regimen was intensified to include doxorubicin.With time, as the clinical pattern of stage 4 disease became apparent to ourteam, earlier intensification to ABV was instituted and benefited two patients,who remain alive and in CR. However, it is difficult to predict whether theintensification of chemotherapy to ABV or paclitaxel will consistently achievelong-term disease control. A limitation lies in the challenge of prospectivediagnosis of visceral disease in the absence of endoscopy and bronchoscopy inlow-income settings.Mild KS with fewer than 10 skin/oral/subcutaneous lesions was designated stage 1in our classification. No patient in the cohort fit this category, and theexplanation for the rarity of this phenomenon is uncertain. There exists bias ina study performed at a tertiary care facility, where access to care is limitedto referral patterns and outreach networks. Potentially, HIV-infected childrenwith mild KS do not get referred or identified if cART alone  induces remission.Alternatively, we hypothesize that mild KS is uncommon in children in regionswhere HHV-8 is endemic. Nonetheless, we feel that it is important to includethis group as a possible clinical scenario, because these patients theoreticallycould be spared chemotherapy and treated with cART alone.4 We alsorecognize that, because KS can involve virtually any organ, the proposedclinical staging paradigm is not exhaustive and may evolve.The biggest limitation to this proposed system lies in the use of the sameretrospective cohort to derive and evaluate the staging classification. Aprospective, multicenter, and preferably international study is required tovalidate the proposed staging classification. However, because of the void ofestablished treatment paradigms and the urgent clinical need in the face ofcontinued high numbers of pediatric patients with KS, we feel that it isimportant to preliminarily establish working staging and risk-stratificationsystems.The proposed Lilongwe pediatric KS staging classification differentiates clinicalpatterns of disease that may provide a platform for risk stratification and mayguide therapeutic strategies. It identifies groups of patients  who can achieve favorable survival with the minimallymyelosuppressive BV chemotherapeutic regimen as well as those  who require alternative (and likely intensified) regimens topotentially improve survival outcomes. In addition, the classificationestablishes a potential, albeit uncommon, subgroup of patients who theoreticallycould be treated with cART alone (without chemotherapy). Prospective validationof this staging classification is the next step to define optimal treatmentparadigms and improve OS outcomes for children and adolescents with KS.


Medulloblastoma is the commonest malignant neoplasm of the central nervous system (CNS) in children  which arMagnetic resonance imaging (MRI) is the preferred first-line imaging modality for patients with suspected brain tumors including medulloblastoma. Traditionally, pre-operative MRI has been used to arrive at a presumptive diagnosis based on lesion characteristics and extent of tumor spread. However, it is now being increasingly appreciated that imaging identifies and reflects underlying disease biology. Radio-genomics represents an exciting avenue of research that aims to correlate imaging-phenotypes with molecular/genetic markers . In its Several features from pre-operative MRI have been used to find associations with specific molecular subgroups. In a large single-institution cohort of 111 patients  with knoTissue-based information remains the gold-standard for histo-morphology and molecular subgrouping in medulloblastoma that cannot be replaced by any imaging-based classification. However, this tissue-based information is not available pre-operatively to the operating neuro-surgeon, but only after surgical resection of the tumor. The prognostic benefit of increased extent of resection becomes highly attenuated after taking molecular subgroup affiliation into account. In a retrospective multi-institutional cohort  involvin


Metalloporphyrins which form the core of many bioenzymes and natural light harvesting or electron transport systems, exhibit a variety of selective functional properties depending on the state and surroundings with which they exist in biological systems. The specificity and ease with which they function in each of their bio-functions appear to be largely governed by the nature and disposition of the protein globule around the porphyrin reaction center. Synthetic porphyrin frameworks confined within or around a pre-organized molecular entity like the protein network in natural systems have attracted considerable attraction, especially in the field of biomimetic reactions. At the same time a large number of macrocyclic oligomers such as calixarenes, resorcinarenes, spherands, cyclodextrins and crown ethers have been investigated in detail as efficient molecular receptors. These molecular receptors are synthetic host molecules with enclosed interiors, which are designed three dimensionally to ensure strong and precise molecular encapsulation/recognition. Due to their complex structures, enclosed guest molecules reside in an environment isolated from the outside and as a consequence, physical properties and chemical reactions specific to that environment in these guest species can be identified. The facile incorporation of such molecular receptors into the highly photoactive and catalytically efficient porphyrin framework allows for convenient design of useful molecular systems with unique structural and functional properties. Such systems have provided over the years attractive model systems for the study of various biological and chemical processes, and the design of new materials and molecular devices. This review focuses on the recent developments in the synthesis of porphyrin assemblies associated with cyclodextrins, calixarenes and resorcinarenes and their potential applications in the fields of molecular encapsulation/recognition, and chemical catalysis. Nature shows a magnificent example of porphyrin usefulness, whereby this basic macrocyclic framework nature is able to perform the most essential life processes- activate and/or transport molecular oxygen in animals and convert sunlight in plant photosynthetic systems. The selectivity and specificity exhibited by pophyrins in each of their biological forms confirms it to be greatly influenced by the nature of the protein network around the porphyrin center ,2,3. NowIn this context, the new millennium has witnessed tremendous development in the generation of some novel porphyrin systems by incorporating into them some suitable foreign host material, called molecular receptors. Molecular receptors are macrocyclic, three-dimensional host molecules with guest inclusion capabilities to ensure strong and precise molecular recognition ,27,28,29Macrocyclic oligomers such as calixarenes, resorcinarenes, spherands, cyclodextrins and crown ethers are found to be attractive molecular host systems possessing excellent recognition ability. Construction of novel ensembles comprising porphyrins and such macrocyclic cavitants is one of the efficient strategies for enzyme-like molecular recognition. The combination of the unique catalytic and photosensitizing efficiency of metalloporphyrins with the receptor characteristics of these cavitants for selected species is basic foundation for such molecular recognition. Last two decades have witnessed highly active research work on this concept and achieved much progress in the synthesis and applications of porphyrin-cavitant systems.In this review we attempt to cover the developments in the synthesis and application of porphyrin-attached molecular receptors which have appeared in the last 5 years. We are concentrated only on three major 3-dimensional molecular host materials—cyclodextrins, calixarenes and resorcinarenes. Different modes of incorporation of porphyrins into these cavitants are discussed, which include porphyrins covalently bonded to the cavitants, electrostatic self-assembly of charged porphyrins with cavitants and porphyrins encapsulated within the macrocyclic cavities.1A), seven (1B) and eight (1C) glucose subunits, respectively (The cyclodextrins (CDs) are a family of cyclic oligosaccharides linked by α-1,4-glucopyranose units. The family is made up of three major and well-known CDs: α-, β-, and γ-CD, containing six -phenyl],10,15,20-tris(4-hydroxyphenyl)-porphyrin (CD-THPP) porphyrin (p-THPP) as a nucleophile zinc(II)) film on an indium-tin oxide (ITO) electrode was prepared in which the self-aggregation of the porphyrin film was suppressed by encapsulation of the porphyrin unit in the cavity of cyclodextrin 2O]2C6H3}9 porphyrinato]Fe(III), TPPSFeCl, TPPS4FeCl, and 2-hydroxypropyl-β-cyclodextrin, HP-β-CD is reported by Khavasi et al. -10,15,20-triphenylporphine (TDPyP) with supramolecular aggregates of cationic amphiphilic β-cyclodextrin (SC6NH2) can act as singlet oxygen generation system having very high quantum yield (ΦΔ = 0.90). Although the yield of 1O2 generation was comparable to that obtained after TDPyP incorporation into cationic unilamellar liposomes of N-[1-propyl]-N,N,N-trimethylammonium chloride (DOTAP) , SC6NH2-bound TDPyP was more active than DOTAP-bound TDPyP in photosensitizing the inactivation of the Gram-positive methicillin-resistant bacterium Staphylococcus aureus (MRSA) triazolyl}malonic ester-bridged bis(permethyl-β-cyclodextrin) having an attached fullerene. Efficient photoinduced electron-transfer between porphyrin and C60 moieties are observed in these new fullerene bridged bis(permethyl-β-cyclodextrin)-porphyrin Co(II) (CoTPP) and a protein in the presence of a cyclodextrin derivative, heptakis-4-pyridyl]porphyrin (MnTPyP-M-Ad) and cyclodextrin-based telluronic acid (2-CD-TeO3H) through host-guest interaction in aqueous solution. The self-assembly of the adamantyl moieties of Mn(III) porphyrin and the β-CD cavities was also demonstrated along with this work. In this enzyme model, the Mn(III) porphyrin center acted as an efficient active site of SOD and tellurol moiety endowed GPx activity. The SOD-like activity of the new catalyst was found to be exhibit 2.56% of the activity of the native SOD. Besides this, enzyme model also showed a high GPx activity, and a remarkable rate enhancement of 27-fold compared to the well-known GPx mimic ebselen was observed. More importantly, this supramolecular artificial enzyme showed good thermal stability too [A new type of artificial smart bifunctional enzyme with both superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities was successfully constructed by the self-assembly of [tetra(1-(1-adamantylmethylketone)-4-pyridyl)porphyrinato]Mn(III), (MnTPyP-M-Ad), with four suspensory adamantyl moieties and β-cyclodextrin-terminated temperature-sensitive copolymer through host-guest interaction in aqueous solution . The Mn(lity too .O-β-cyclodextrin)-phenyl],10,15,20-tri(4-hydroxyphenyl)-porphyrin is reported by Oliveri et al. Fe(II) (Fe[II]TPPS) and a per-O-methylated β-cyclodextrin dimer having a pyridine linker (Py3CD). This hemo-CD binds O2 reversibly in aqueous solution. Encapsulation of the iron center of Fe(II)TPPS by two cyclodextrin truncated cones is supposed to be the determining factor for binding of O2 to the ferrous center of the porphyrin Fe(III) and a per-orphyrin . An anioobalamin .15A,B). They exist in a cup-like shape with a defined upper and lower rim and a centre annulus. Calixarenes have come a long way in offering tailor made candidates for diverse applications in the field of host-guest chemistry [The calixarenes are a class of cyclooligomers formed via a phenol-aldehyde condensation -substituted calixarene-bis-porphyrin was also reported which has been found to form a stable 1:1 complex with dimethyl maleate in toluene [3]2+ enantiomers in a porphyrin-(bis)calixarene assemblies is demonstrated by D’Urso et al. [Synthesis of a Pacman heterobis(porphyrin) containing an [octaethylporphyrinato]zinc (ZnOEP) energy donor and a (triarylporphyrinato)zinc (ZntPP) energy acceptor around a crown[6]-dipropoxy-calix[4]arene spacer is described . Remarkaswitches . A Zinc  toluene . Inductio et al. .It has been observed that several calixarene-porphyrin-tweezer systems are capable of size-selective complexation with fullerenes. Designing effective host molecules for such fullerene selectivity is highly important to reduce costs due to laborious fullerenes purification and its isolation procedures and thus to facilitate their application in chemistry and material science. Also porphyrin-fullerene architectures can lead to molecular assemblies ideally suited for devising integrated, multicomponent model systems to transmit and process solar energy ,99. The et al. reported an oxidative charge transfer  for the C60 inclusion complexes, while a reductive charge transfer  is operative in the endohedral metallofullerene host-guest complexes,  20B  tweezer 20A  was demom et al. . This twole) 20B  conjugat21 P(Zn) is then able to accommodate 1 equivalent of primary amine inside the calixarene cavity and is the thermodynamically favored one too. The use of a diamine with a C-12 aliphatic chain led to the formation of a heterodipotic complex highlights the possibility of generating cooperative interactions between the three components; calixarene-guest-porphyrin similar to interactions governing enzymatic catalysis. Synthesis and conformational analysis of some porphyrin derivatives substituted with calyx[4]arene subunits were also carried out recently by Holler et al. .23 , were synthesized via a high-yielding “3+1” condensation between meso-triphenylporphyrin and readily available new fluorodinitrobenzene-containing trimers were synthesized and photophysical properties were discussed by Jiao et al. [Wu and coworkers synthesized a new fluorogenic oxacalix[6]arene system 23  bearing 23  porphyrin 25  which prConstruction of multilayers comprising calixarene and porphyrin units is another important research area ,113,114 H,23H-porphine (26A) and p-tert-butylcalix[8]arene (26B) have been studied by Roales et al. arene matrix mentioned above -21porphine , 26A and[8]arene , 26B havs et al. . Althouged above . The disZn and Mn derivatives of 5,10,15,20-tetrakisporphyrins (EHO) have been mixed with calix[8]arene to enhance to the quality of the porphyrin films and their response to amine gases and also to increase the film lifetime . ZnEHO set al. employed a water-soluble calixarene -25,26,27-tripropoxy-28-(oxy)calix[4]arene]) as a templating agent for the assembly of a tetraanionic porphyrin ([tetrakis(4-sulfonatephenyl)porphyrinato]Cu(II)) Cu(II)) . The selet al. arene (C4TsTc) were used as key components for building up discrete supramolecular entities starting from the formation of the template species MTMPyP:C4TsTc . The stepwise addition of further amount of porphyrin allows the facile noncovalent synthesis of discrete supramolecular systems (2:4 and 3:4) which can be built up just by programming the right stoichiometric addition of the proper porphyrin. The authors reported that the electrochemistry of each of these supramolecular complexes is different from that of the parent components. This anomalous behavior can be explained only considering each of these supramolecular complexes as a unique entity, in which internal electronic communication might occur. A highly nanoporous material has been obtained by self-assembly of the polyanionic calixarene 5,11,17,23-tetrasulfonato-25,26,27,28-tetrakis(hydroxycarbonylmethoxy)calix[4]arene (C4TsTcn-) in the cone conformation and the tetracationic meso-tetrakis(4-N-methylpyridyl)porphyrin (H2TMPyP) building blocks [et al. [p-sulfonatocalix[5]arenes) and cationic porphyrins, respectively, are self-assembled to form two well-defined nano-architectures with 2-D netlike and 1-D linear topological structures, in which an unambiguous PET process is observed in nanoscale region arenes. The synthesis of the unlocked compound 28B , 1,4-naphthoquinone (NQ), phenanthrenequinone (PAQ) and dichlorodicyanobenzoquinone (DDQ)—to these porphyrin-resorcin[4]areneconjugates.29B  irradiation (in conjunction with Adler conditions) to make this important capsule and observed significant improvement in product yield relative to the traditional refluxing protocol.et al. [Self-assembled (1:1) supramolecular resorcinarene-Zn porphyrin capsules containing coordinated bifunctional ligands such as 4,4'-bipyridine were synthesized by Stefanelli et al. . The forCyclodextrin/calixarene/resorcinarene macrocyles can be used as preorganized three dimensional molecular receptors and functional materials by combination with porphyrins or metalloporphyrins. Owing to the cooperative contribution of the structural units, such molecular assemblies find unique application in catalysis, sensor devices, light harvesting or model structures for biomimetic research. The forthcoming research on these porphyrin incorporated molecular receptors is expected show an exponential growth because we are at a stage when researchers around the World are actively involved in biomimetic studies and nanosynthesis.


Kidney stone (KS) disease has high, increasing prevalence in the United States and poses a massive economic burden. Diagnostics algorithms of KS only use a few variables with a limited sensitivity and specificity. In this study, we tested a big data approach to infer and validate a ‘multi-domain’ personalized diagnostic acute care algorithm for KS (DACA-KS), merging demographic, vital signs, clinical, and laboratory information.We utilized a large, single-center database of patients admitted to acute care units in a large tertiary care hospital. Patients diagnosed with KS were compared to groups of patients with acute abdominal/flank/groin pain, genitourinary diseases, and other conditions. We analyzed multiple information domains  using a collection of statistical and machine learning models with feature selectors. We compared sensitivity, specificity and area under the receiver operating characteristic (AUROC) of our approach with the STONE score, using cross-validation.Thirty eight thousand five hundred and ninety-seven distinct adult patients were admitted to critical care between 2001 and 2012, of which 217 were diagnosed with KS, and 7446 with acute pain (non-KS). The multi-domain approach using logistic regression yielded an AUROC of 0.86 and a sensitivity/specificity of 0.81/0.82 in cross-validation. Increase in performance was obtained by fitting a super-learner, at the price of lower interpretability. We discussed in detail comorbidity and lab marker variables independently associated with KS .Although external validation is warranted, DACA-KS could be integrated into electronic health systems; the algorithm has the potential used as an effective tool to help nurses and healthcare personnel during triage or clinicians making a diagnosis, streamlining patients’ management in acute care. Kidney stone (KS) disease prevalence has increased in the United States from 5.2%  in 1994 to 8.8%  in 2012 [The emergency department (ED) is a common place where patient with KS are evaluated and diagnosed. During the past two decades, a significant increase in ED visits with stone-related symptoms has been observed , with ovA cross-sectional analysis of the 2007–2010 National Health and Nutrition Examination Survey (NHANES) dataset suggests that obesity, diabetes, and gout all have a significant positive association with kidney stone history . In 2014In this study, we tested a big data approach, merging demographic, vital signs, clinical, and laboratory information, to infer and validate a ‘multi-domain’ personalized diagnostic score for KS. We utilized a large, single-center database of patients admitted to ED and other intensive/acute care units in a large tertiary care hospital . We analyzed the information domains individually , together, and compared our approach with the STONE score. A number of statistical and machine learning models were fit and compared to optimize performance. Using this multi-domain integration approach our goal was to significantly improve the sensitivity and specificity of KS diagnosis in acute settings.The study population comprised individuals admitted to critical care units at the Beth Israel Deaconess Medical Center in Boston, Massachusetts, United States, between 2001 and 2012. Data are stored electronically in the Medical Information Mart for Intensive Care (MIMIC-III) database, which is available to the public upon request, upon Collaborative Institutional Training Initiative (CITI) training, and license agreement for full download and research . MIMIC-IThis is a secondary data analysis. We used the MIMIC-III ver. 1.4, released on September 2nd, 2016. Our study included patients aged 18 years and older, divided into four groups based on the ICD-9 diagnoses during hospitalization: (a) KS cases ; (b) patients diagnosed with genitourinary diseases (GUD) except KS , e.g. patients with nephritis, nephrotic syndrome, nephrosis; (c) patients admitted to acute care with other conditions (OTH) who did not have any KS or GUD diagnosed (any ICD-9 code not including 580–599) to represent a general patient population; (d) patients admitted with acute localized pain (ALP) of abdominal (ICD-9 code: 789.0), back (ICD-9 code: 724.2), flank, or groin (identified through patients’ electronic chart record). In addition to ICD-9 codes, we also examined recorded charted events on ALP from the dataset. Patients with both KS and GUD codes were put into the KS group. Each patient was associated to a covariate vector of demographic info, vital signs, clinical diagnoses, procedures, medicaments, and laboratory tests performed during hospitalization.Descriptive analysis was used to assess demographic characteristics , vital signs , laboratory tests (e.g. creatinine), and distribution of ICD-9 diagnoses at admission and during hospitalization. We also calculated the Charlson Comorbidity Index (CCI) using Deyo’s algorithm , and thep-values were adjusted using False Discover Rate (FDR) correction [Due to a low frequency of KS, we included only ICD-9 diagnostic codes that were occurred in less than 5 counts of the KS group, and lab tests that performed in at least 50% of the KS formers. Missing values were imputed via population median/mode. Univariate analysis was conducted to assess differences between KS and GUD/OTH/ALP groups on demographics, ICD-9 diagnoses, and lab tests, using Student’s t-test, Wilcoxon rank test, or chi-square test, where appropriate. Significance rrection .In order to infer a KS diagnostic score, we fitted a collection of multivariable logistic regression models with the GUD, OTH or ALP as negative examples, using different input covariate domains. Specifically, we evaluated seven models: (a) demographic variables and vital signs  (b) CCI, plus demographic variables; (c) eGFR alone; (d) ICD-9 diagnosis , plus demographic variables; (e) laboratory tests (top-25 as selected by the univariate filter), plus demographic variables; (f) ICD-9 diagnosis and laboratory tests (top-50 as selected by the univariate filter), plus all other variables included in models (a) to (e); (g) stepwise (forward-backward) selection of model (f); (h) STONE model. Note that ICD-9 codes used to define the GUD were not used as input covariates to any of the models, except for the STONE model where hematuria (ICD-9 code 599.7) is a covariate. Also, the duration of pain to presentation in the STONE score could not be precisely ascertained from our data; we used ICD-9 codes in the 338 s family plus codes 780.96 and 789.0, excluding chronic pain entries, using a weight of 2 . In addition to ICD-9 codes, we also used charted events to identify pain events. For nausea/vomiting we used ICD-9787.0 codes. In a sensitivity analysis, we also evaluated the contribution of GUD codes to overall performance of models (d) to (g).Model comparison, evaluation, and selection were carried out using a 10-fold cross-validation framework , comparidecision tree by means of the C4.5 algorithms [LogitBoost algorithm in conjunction to logistic regression [random forest (optimizing number of trees up to 1000) [super learner stacking all the above methods plus a single-rule linear model, internally optimized via 5-fold cross-validation [In addition to logistic regression, we also fit a number of machine learning techniques on the full variable set as in model (f). In details: (i) a gorithms ; (ii) Logression ; (iii) ato 1000) ; (iv) a lidation . Given tlidation . The uniThe performance and discriminative ability of models was assessed using sensitivity (true positive rate), specificity (true negative rate), and the area under the receiver operating characteristic (AUROC), which is the expectation that a uniformly drawn random positive case is ranked before a uniformly drawn random negative  . The optThere were 38,597 distinct adult patients (> 18-year-old) in the MIMIC-III database admitted to critical care units between June 2001 to October 2012 , of which 217 were diagnosed with KS, 14,391 with GUD, 23,931 as OTH who did not have any GUD nor KS, and 7446 as ALP with abdominal, back, flank, groin pain.p <  0.05). Most sample population were admitted through emergency or urgent (84.2%). The distribution of race was similar between KS and GUD, but comparing to OTH and ALP, KS had a higher proportion of white (76.5% vs. 71.1% and 72.7%) and black African American . The median eGFR in KS was 65.3, lower than in OTH  and ALP , but higher than GUD . The median (IQR) STONE score in KS formers was 4, higher than in GUD  or in OTH , but not different from ALP . Figure p-values < 0.05).Table Figure p = 0.051, and vs. 1.1% in OTH, p < 0.0001, and vs. 1.5% in ALP, p < 0.0001); 98.6% of KS formers had experienced pain while 53.1% of GUD and 57.6% of OTH had pain events (both p < 0.0001); 0.92% of KS formers had vomiting and 0.46% had nausea recorded, and the percentages of vomiting and nausea in KS were slightly higher than in other three groups. Hydronephrosis (variable from STONE-PLUS) was also positively associated with KS .When looking at the STONE variables, we found that hematuria was positively associated with KS , 83 variables remained. For laboratory tests, a total of 754 entries were found, further condensed to 637 by manual inspection of physicians, and reduced to 69 after frequency filtering. The frequencies of missing values of these included lab tests ranges from 0 to 45%, 66.0% and 45.2% in GUD, OTH and ALP respectively, with the majority of them have less than 50% of missing.p-value below 0.1 (up to the top-25). Overall, 7 ICD-9 were differentially distributed between KS and GUD at the 5% FDR level, while 25 of them were found different between KS and OTH or ALP at the same significance level. Out of the 69 lab tests performed in more than half of KS patients, 43, 50, and 25 showed a significant (5% FDR level) mean or distribution location shift between KS vs. GUD, KSF vs. OTH, and KSF vs. ALP, respectively. The top-25 lab tests rank is shown in Table Table p < 0.05) than these two. Following cross-validated AUROC ranking, the second best-performing models were those with top-ranked ICD-9 codes (d), laboratory tests (e), CCI (b), eGFR (c), and demographics alone (a).In order to derive a multi-domain diagnostic model of KS diagnosis, we fitted different logistic models on selected covariate input domains, as specified in the Methods section, and compared against the STONE. Table Notably, models using top-ranked ICD-9 diagnostic codes showed high sensitivity and moderate specificity, while models using top lab tests showed moderate sensitivity and high specificity, while both high sensitivity and high specificity were achieved in the multi-domain models. The STONE model (h) yielded relatively low AUROC .p < 0.0001 w.r.t. models with non GUD-specific ICD-9 codes) with sensitivity of 77% and specificity of 87%; for KS vs. OTH, model (g) achieved AUROC of 98% (p < 0.0001), with sensitivity of 88% and specificity of 98%; for KS vs. ALP, model (g) achieved AUROC of 87% (p < 0.0001), with sensitivity of 81% and specificity of 82%. Model (f) had very similar performance (not shown). However, these GUD variables were measured concurrently with KS, so we did not include them in our final prediction model, but it could be used as input if these GUD variables happened in one’s history to improve the predictivity and performance of the models.When we added the ICD-9 code for hematuria and other GUD codes to the set of input variables for models (f) and (g), performance increased significantly: For KS vs. GUD, model (g) achieved AUROC of 88% (p < 0.0001). The variables selected by the LogitBoost were concordant with the variables selected from stepwise logistic regression model (g), although the LogitBoost tended to select a few more. The decision tree showed a peculiar behavior as compared to the logistic regression, with increased sensitivity at higher specificity but then lower plateau. The random forest showed higher  AUROC and sensitivity/specificity (significant below the 0.0001 level with respect to the logistic regression and decision tree) and the super learner was comparable to the random forest. In fact, the highest weight of the super learner was that of the random forest, followed by the decision tree, a single rule, and the LogitBoost. The bottom panels of Fig. When we applied the machine learning techniques, using the same cross-validation settings, for the comparison between KS and GUD or OTH, we did not observe a substantial increase in performance indices with the usage of the LogitBoost selector in alternative to the stepwise, but an increased performance was observed for KS vs. ALP , because in conjunction with optimal performance, it included fewer variables than model (f) (15 variables for each comparison vs. 50 variables in model (f)). Table In this large sample of individuals admitted to acute care between 2000 and 2012, we aimed to infer a multi-domain, personalized, diagnostic algorithm risk assessment for KS disease. With a robust model collection and selection framework, under cross-validation settings, we demonstrated that the integrated model improves both specificity and sensitivity as compared to a single domain model. Also, it includes more extensive parameters compared to the STONE score. The STONE score utilizes presentations of KS-related symptoms  and two demographic predictors (gender and race). In our sample population, only a small proportion of (KS) patients had hematuria and nausea/vomiting present or recorded. Our study evaluated thousands of potential predictors among the different domains, comparing relative proportions and shifts in distributions between KS formers and the GUD, OTH and ALP groups, our model can make personalized prediction for each individual based on his/her parameters from different domains. The features used in our final models are usually routinely tested in critical care unit, or tested at admission, therefore, all information to implement our model should be available in an ICU setting, and can be easily adapted to different clinical settings by adding or removing features. We report a series of novel findings in KS that are significantly different than GUD, OTH and ALP populations and which could aid in the triage of patients when they present to the ED or are admitted/transferred into critical care. A number of these variables are worth of discussion in detail.In our study cohort, we found that KS peaked at the 7th decade of age; with variation of prevalence at different age groups between both genders, overall, we found a higher prevalence of females in this cohort. KS prevalence was the highest in non-Hispanic whites, similarly to other studies . Lower rIn our population, KS formers had the highest prevalence of obesity when compared to the GUD/OTH/ALP groups, and our final multivariate model suggested that patients with obesity are two times more likely to be diagnosed with KS comparing with GUD or ALP patients.We found that KS, OTH and ALP were a healthier cohort with lower CCI and higher eGFR when compared to the GUD. Previous studies have demonstrated that KS formers have higher risk of developing chronic kidney diseases , 26; in The most common diagnosis associated with ED visits was hypertension, and its prevalence was higher in patients with KS comparing to GUD and ALP. Disorders of fluids, electrolytes, and acid-base balance was also frequently found in KS and GUD, but not in the diagnosis in the OTH/ALP group. A meta-analysis found that increasing water intake was associated with significantly reduced risk of kidney stones and it was dose dependent for each increase of 500 ml of water . For KS We found that purpura and other hemorrhagic conditions were higher in the KS population when compared to the OTH/ALP population but there was no significant difference when compared to the GUD group.The distribution of serum lipase and creatinine kinase MB isoenzyme were significantly lower in KS as compared to the GUD and OTH/ALP groups. Renal handling of lipase involves removal of lipase from serum by glomerular filtration of lipase with nearly complete absorption of free oxalate in the bowel lumen . DisordeA set of neurologic findings in our study demonstrated that migraine headaches were higher in KS and OTH compared to GUD/ALP. Sleep disorder, neurotic disorder, and depression disorder were also higher in KS patients. Migraine headache medications such as Topamax promote an (RTA)-like phenomenon . Sleep dPerhaps the most important finding and among high morbidity and mortality conditions, septicemia and candidiasis were found to have a high correlation with KS formers only. Reyner et al.  reported2 variables, and we would have needed to use more efficient libraries, with parallel or cloud computing), therefore the model assumed a linear relationship. Ensemble methods, i.e. the random forest and the super learner, achieved almost perfect performance, but the result was not confirmed with the SMOTE class rebalancing, and this warrants further external validation using the TRIPOD protocol [There are several limitations of our study. First, we analyzed a sample from a single site, without external validation; the characteristics of patients in the KS, GUD, OTH and ALP are different and there may be a selection bias which we did not adjust for. In addition, many potentially useful lab tests were dropped because of low frequency in the KS group; other relevant lab predictors for KS may be found outside those routinely measured in people being triaged at the ED based on admission’s symptoms. Second, there was a high-class imbalance, for which the power of the study can be affected, as well as the derivation of a diagnostic model, even though we tried to address in part this issue using the SMOTE technique. Third, when using logistic regression, we did not consider interactions among variables (considering only two-ways interactions would have produced nprotocol . Even thDespite these limitations, our study provided a compact and high-performance diagnostic model for diagnosis of KS.DACA-KS could be integrated into electronic health systems; the algorithm has the potential used as an effective tool to help nurses and healthcare personnel during triage or clinicians making a diagnosis, streamlining patients’ management in acute care. As we enter the era of precision medicine, we envision a family of DACA- models for many other conditions in addition to KS, derived in the same way from big integrated biomedical data bases.


Vibrio vulnificus is a Gram-negative bacterium and an opportunistic pathogen that can cause septicemia or necrotizing fasciitis. Here, we report the draft genome sequences of 42 environmental V. vulnificus strains collected from the northern Gulf of Mexico. These data will allow for more robust comparisons between clinical and environmental strains. Vibrio vulnificus is a Gram-negative bacterium and an opportunistic pathogen that can cause septicemia or necrotizing fasciitis. Here, we report the draft genome sequences of 42 environmental V. vulnificus strains collected from the northern Gulf of Mexico. These data will allow for more robust comparisons between clinical and environmental strains. Vibrio vulnificus is a Gram-negative marine bacterium with a single polar flagellum (vcg)  . Whole-gum (vcg) , and theum (vcg) , 7. Howeum (vcg) . To addrV. vulnificus strains were isolated previously from seven locations in the Texas segment of the NGOM between August 2006 and July 2007 (V. vulnificus with matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry , as described previously  were calculated by QUAST version 4.1 (default options)   to deteroptions) .V. vulnificus genomes, including the overall genome size (bp), number of contigs, N50 value, and G+C content (%). The availability of these genomes will augment future comparative genomic analyses focused on the differentiation of clinical and environmental strains. Additionally, the availability of these genomes will advance the understanding of the environmental V. vulnificus reservoir.PRJNA475262.These whole-genome shotgun projects have been deposited at GenBank under the accession numbers listed in


Machine learning (ML) has become a crucial component of early drug discovery. This research area has been fueled by two main factors. The first is the fast-growing availability of relevant experimental data. Examples of such datasets are those containing the bioactivities of molecules of known chemical structure against a non-molecular target , binding affinities of such molecules against a molecular target  or the X-ray crystal structures of a molecular target. This factor has been boosted by the development of community resources, such as ChEMBL , PubChemk-nearest neighbors (k-NN), convolutional neural network (CNN), or recurrent neural network (RNN), either alone or integrated with dimensionality reduction techniques such as GA-based feature selection (FS) and principal component analysis (PCA).This Special Issue showcases five studies investigating the application of ML for molecular modelling in drug design. These studies have been carried out by 21 academic and industry researchers from around the World. ML techniques include support vector machines (SVM), random forest (RF), 50) data. The QSAR classification models were also built, this time using nuclear magnetic resonance (NMR) data as features. Models were built with k-NN, RF, and SVM algorithms. The authors concluded that the developed models were sufficiently predictive to permit the identification of new inhibitors of this non-molecular target.The first of these papers by Cruz et al.  investigChen et al.  aimed atDetection of mutagenicity during early stages of drug discovery is important to reduce the likelihood of developing drugs with harmful side effects. Norinder et al.  applied Bjerrum and Sattarov  demonstrMachine learning has been used to generate diverse ligand-based predictive models in these four contributions so far ,9,10,11


A critical PUFA, arachidonic acid (ARA), is metabolized to a diverse set of lipids signaling molecules through cyclooxygenase (COX), lipoxygenase (LOX), cytochrome P450 epoxygenase, or cytochrome P450 hydroxylase; however, the majority of ARA is metabolized into anti-inflammatory epoxides via cytochrome P450 enzymes. These short-lived epoxide lipids are rapidly metabolized or inactivated by the soluble epoxide hydrolase (sEH) into diol-containing products. sEH inhibition or knockout has been a practical approach to study the biology of the epoxide lipids, and has been shown to effectively treat inflammatory conditions in the preclinical models including gastrointestinal ulcers and colitis by shifting oxylipins to epoxide profiles, inhibiting inflammatory cell infiltration and activation, and enhancing epithelial cell defense via increased mucin production, thus providing further evidence for the role of sEH as a pro-inflammatory protein. Non-steroidal anti-inflammatory drugs (NSAIDs) with COX-inhibitor activity are among the most commonly used analgesics and have demonstrated applications in the management of cardiovascular disease and intriguingly cancer. Major side effects of NSAIDs however are gastrointestinal ulcers which frequently precludes their long-term application. In this review, we hope to bridge the gap between NSAID toxicity and sEH-mediated metabolic pathways to focus on the role of epoxy fatty acid metabolic pathway of PUFAs in NSAIDS-ulcer formation and healing as well as inflammation-related carcinogenesis. Specifically we address the potential application of sEH inhibition to enhance ulcer healing at the site of inflammation via their activity on altered lipid signaling, mitochondrial function, and diminished reactive oxygen species, and further discuss the significance of dual COX and sEH inhibitor in anti-inflammation and carcinogenesis.Polyunsaturated fatty acids (PUFAs) including epoxide-modified ω-3 and ω-6 fatty acids are made  On the other hand, the inflammatory process itself can cause significant harm to the host , lipoxygenase (LOX), and cytochrome P450 enzymes (including epoxygenase and hydroxylase) , which rapidly converts EETs to dihydroxyeicosatrienoic acids (DHETs) . The eporeviewed . sEH inhreviewed , 2) prevreviewed , and 3) reviewed .via COX enzymes, result in a diverse family of structures that modulate many functions including vascular tone, platelet aggregation, and inflammation (Prostaglandins (PGs), produced through oxygenation of ARA ammation . The NSAammation . Long-teammation .This review aims to highlight a potential strategy combining sEH and COX inhibition for chemoprevention and inflammatory conditions while also mitigating the adverse side effects of single agent sEH inhibitors and NSAIDs.The 20-carbon ω-6 PUFA ARA is a lipid signaling molecule that resides among phospholipids and is a key substrate for a variety of downstream cell signaling mediators known as eicosanoids. ARA is broadly metabolized into one of three main pathways: 1) by COX into prostaglandins; 2) by 5-LOX into leukotrienes; and 3) by cytochrome P450 (CYP 450) epoxygenase and hydroxylase to form epoxyeicosanoids and hydroxyeicosatetraenoic acid. The EETs are signaling molecules formed within various types of cells by the metabolism of ARA . SeveralEETs can act as both autocrine and paracrine mediators of inflammation, angiogenesis, apoptosis, fibrinolysis, mitogenesis, vasodilation, and bronchodilation . The mecThe catabolism of EETs can occur through several pathways including the main pathway of epoxide hydrolysis by sEH into DHETs, but can also occur through β-oxidation , fatty aAcetylsalicylic acid (aspirin) was the first NSAID synthesized and developed for commercial use in 1897 by Felix Hoffman, and it became widely used for its analgesic and anti-inflammatory effects . It tookNSAIDs are among the most commonly administered drugs worldwide, as they are available by prescription and over-the-counter. Excitingly, they are also among the most promising agents for prevention of cardiovascular diseases and cancer including colorectal cancer (CRC) . One of In contrast to the adverse ulcer-causing effects, several clinical trials and epidemiological and animal studies have clearly demonstrated that aspirin or NSAIDs actually decrease the risk and mortality of CRC and prevent colon adenoma formation . To ackn4 hydrolase, cholesterol 5,6-oxide hydrolase, and epoxide hydrolase 3 , sEH, leukotriene Arolase 3 . The majrolase 3 . The 60-rolase 3 .In the early 1990s, concerns about the potential adverse effects of a chronic loss of sEH have centered around the clearance of xenobiotics, particularly the potential loss of function to clear epoxide-containing carcinogens. These concerns have been well studied and demonstrated that these xenobiotics are not the substrates of sEH . StudiessEH shows high tissue expression in liver, intestine, kidneys, brain, and vasculature, with lower expression in spleen, lung, and testes . The subtrans-3-phenylglycidol and chalcone oxide molecules, and while these inhibitors are selective and potent, they are unstable with only temporary effects on sEH . DCU is the basis for several urea-based inhibitors with increased water solubility, and which bind to the active site of sEH via the urea subgroup with hydrogen bonds and salt bridges cyclohexyl oxy]benzoic acid (t-AUCB) is another highly promising sEH inhibitor. Thus far, sEH inhibitors have been demonstrated to be safe, with very few side effects. The effective dosages show very little toxicity.The most recent and well-studied sEH inhibitors are 12-(3-adamantyl-ureido)-dodecanoic acid (AUDA) compounds. These AUDA compounds have been modified to bind the catalytic site of sEH Chronic inflammation has been demonstrated to be among the most important factors leading to carcinogenesis. Obesity, inflammatory bowel disease (IBD), and chronic colitis and pancreatitis are some specific examples of inflammatory conditions that are known risk factors for cancer with shared mechanisms involving altered epoxide fatty acid metabolites and sEH , a low-affinity nuclear hormone receptor involved in fatty acid metabolism, and inhibition of the NF-κB pathway . It is lThe underlying mechanisms of NSAID-induced GI ulcers are not fully understood; however, they likely involve endoplasmic reticulum (ER)/mitochondrial stress and mucinous epithelia-microvascular barrier breakdown . The effNSAID-induced GI ulcers can be counter-balanced by epoxides through direct administration of EETs or indirectly by increasing EETs through sEH inhibition . sEH inhOne of the most common conditions leading to pancreatic tumorigenesis is the inflammatory condition chronic pancreatitis. In the development of chronic pancreatitis aberrant ARA metabolism partially mediated by sEH acts to promote inflammation with a decrease of the anti-inflammatory EETs. Inhibition of sEH has been demonstrated as a successful strategy in murine models to inhibit chronic pancreatitis and the development of pancreatic intraepithelial neoplasms (PanIN) using trans-4-{4-[3-(4-chloro-3-trifluoromethyl-phenyl)-ureido]-cyclohexyloxy}-pyridine-2-carboxylic acid methylamide (t-CUPM) in KRAS-mutant mice . t-CUPM 4 pathway cyclohexyloxy]benzoic acid (t-AUCB)  and COX2 , but not COX-1 , and is effective at suppressing inflammation -ureido]-propyl}-pyrazol-1-yl)-benzenesulfonamide (PTUPB), based on the structures of celecoxib and sEH inhibitor (t-AUCB) , to circ(t-AUCB) . Studies(t-AUCB) . PTUPB iammation .t-AUCB alone and in combination (via the dual inhibitor PTUPB is particularly intriguing for chemoprevention in the colorectum, especially in patients with IBD. This strategy would provide an environment enriched with EETs, with the chemopreventive benefits of COX-2 inhibition while minimizing side effects including GI tract ulceration, as outlined in PTUPB’s effects are more potent than celecoxib or bination . PTUPB hbination . In mousbination . Thus, aThe decades of studies of ARA metabolism and the sEH point to the potential of sEH inhibition and subsequent enrichment of EETs as an effective strategy for therapies in inflammatory conditions. This is particularly promising in colon and pancreas where chronic inflammatory conditions are well-documented risk factors for carcinogenesis, and therefore sEH inhibition may be ideal as a chemopreventive agent alone or in combination with other strategies. These effects have been robustly studied in mouse models, which show significant reductions in inflammation and cancer development with little to no side effects. Additionally, studies have shown that sEH inhibition can dramatically reduce the side effects of NSAIDs when given together. Thus, we believe that sEH should be further investigated in clinical trials for inflammatory conditions and inflammation-induced cancers.One such strategy of sEH treatment involves their co-administration with NSAIDs to further modulate ARA metabolism. NSAIDs including aspirin and celecoxib are among the most widely administered medications worldwide. While they are promising for chemoprevention and in cardiovascular diseases, they also cause significant morbidity through their side effects of gastroenteropathies. These NSAID-related ulcers and bleeding are potentially life-threatening; thus, their use in chemoprevention is not practical. This emphasizes the necessity for a novel approach to harness the beneficial aspects of NSAIDs without the side effects, and one such approach may be through the dual inhibition of COX and sEH. PTUPB is one such agent, although single agent NSAIDs combined with sEH inhibitors may also be effective. This approach has the potential to revolutionize cancer treatment and prevention, and also has broad implications for the millions of people using NSAIDs chronically for pain and rheumatologic diseases.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This study was supported by NIH R01 DK10776, CA172431, and CA164041 to G-YY.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The frequency of occurrence of extrapulmonary tuberculosis (EPTB) has been increasing globally over the last two decades. In Morocco, EPTB cases account for 46% of the patients reported with a new episode of tuberculosis (TB). Lymph node TB (LNTB) is the most common form of EPTB. In line with the guidelines of the National TB Program, the diagnosis is mainly based on clinical evidence, including histopathology.This study aimed to evaluate the yield of histopathology testing in the diagnosis of LNTB.This cross-sectional, prospective study was conducted among patients with cervical lymph node who were enrolled in the study from November 2016 to May 2017 in three regions of Morocco. We compared the outcomes of histopathological testing with those of bacteriology. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of histopathology testing were calculated. Culture and Xpert tests were used as the gold standard Laboratoty Testing.A total of 262 patients were enrolled in this study. The Se, Sp, PPV, and NPV of histopathology testing were 95.6% (129/135), 64.6% (82/127), 74.1% (129/174), and 93.2% (82/88), respectively, in the presence of granuloma with or without caseous necrosis and were 84.4% (114/135), 74.8% (95/127), 78.1% (114/146), and 81.9% (95/116), respectively, in the presence of granuloma with caseous necrosis. The granuloma with caseous necrosis was associated with increased PPV and Sp of histopathology testing .The presence of the granuloma with caseous necrosis in the histopathological examination had significantly improved the yield of histopathology testing for the diagnosis of LNTB. The findings recommend to maintain histopathology testing in establishing the LNTB diagnosis and to explore other techniques to improve it. Tuberculosis (TB) continues to challenge the international community. The global burden is estimated by the World Health Organization (WHO) at 10.4 million incident cases in 2016 . HoweverIn Morocco, TB remains a major problem of public health. The WHO estimates for the year 2016 indicate that approximately 36,000 people were affected with a new episode of TB, with an estimated incidence of 103 per 100,000 population . The freAccording to the NTP guidelines of Morocco, the diagnosis of LNTB should be based on clinical arguments, including histopathological evidence. In fact, the diagnosis of 90% of notified LNTB cases is established through histopathology testing; bacteriological evidence contributes rarely to setting the diagnosis of LNTB . HistopaMycobacterium tuberculosis (MBT) within the tissues because of an anaerobiosis environment. Therefore, the identification of MBT in biopsy fragments, through bacteriological techniques such as a culture or polymerase chain reaction (PCR), is not easy because of the paucibacillary nature of biopsies, and these examinations cannot provide optimal sensitivity [The histopathology is highly sensitive but not very specific for TB diagnosis, in general. Therefore, using histopathology alone may result in a false diagnosis of EPTB. In addition, EPTB is characterized by a low presence of sitivity . This stWe conducted a cross-sectional, prospective study from November 2016 to May 2017 in otorhinolaryngology outpatient departments belonging to 4 university hospitals in 3 regions of Morocco . These regions have a high notified incidence of TB and appropriate laboratory facilities for histopathology and well-developed TB laboratory capacities. The study compares the outcomes of histopathological tests with those of bacteriological examinations using a culture and Xpert tests as the gold standard. The Xpert test was combined with the culture to strengthen the bacteriological gold standard, as its sensitivity (Se) and specificity (Sp), using lymph node biopsies, were, respectively, 96.6% and 88.9% [The study population consisted of all consenting patients with cervical lymph nodes, irrespective of their age and gender, for whom a biopsy was indicated to carry out histopathological testing to establish a diagnosis, including that of TB. All patients for whom the biopsy was contraindicated were excluded.In 2015, approximately 2000 new cases of LNTB were notified in 3 regions. Using an expected proportion of the bacteriological confirmation with histopathological characteristics suggesting TB of 68% [Each lymph node biopsy carried out was examined by a histopathology test and by a culture on the Lowenstein-Jensen (LJ) solid medium and Xpert testing to provide the bacteriological evidence associated with TB. A bacteriological examination was considered positive when a culture or Xpert test or both were positive in a lymph node biopsy. A bacteriological examination was considered as negative when a culture and Xpert test were both negative in a lymph node biopsy. A histopathology test was considered positive when its outcome shows a presence of inflammatory granuloma with or without caseous necrosis. The presence of caseous necrosis in the granuloma was considered as suggestive of TB, whereas its absence as less suggestive, without ruling out the possibility of TB diagnosis. A histopathology test was considered negative when it shows no granuloma with or without nonspecific inflammatory lesions.The data to describe the population study were collected using a questionnaire filled by consenting patients, which included the demographic data , the socioeconomic data , and the clinical characteristics . The results of histopathological and bacteriological tests were collected in the laboratories involved in the study using specific results sheets. All the staff involved in the process of data collection were trained to use the case definitions and collect the relevant information, in line with the study protocol, to standardize the data collection process.Informed consent was obtained for each study participant, and all data obtained during the study were treated confidentially. The study was approved by the National Ethical Committee established by the Ministry of Health of Morocco.P value is less than .05.The data were coded, verified, and analyzed using Epi Info version 7.2.1.0 . We calculated the Se, Sp, positive predictive value (PPV), and negative predictive value (NPV) for each of the histopathological outcomes, namely (1) the granuloma with or without caseous necrosis and (2) the granuloma with caseous necrosis. To calculate these indicators, the outcomes of the histopathological tests were compared with the results of the gold standard (the culture and Xpert test). A chi-square test was used for the comparison of proportions. A chi-square test was considered statistically significant when the The study enrolled 262 patients with cervical lymph nodes from the following locations: 41.2% (108/262) in Casablanca, 35.1% (92/262) in Fez, and 23.7% (62/262) in Rabat. Their mean age was 25 years. Among the study participants, 151 were females , with a sex ratio  of 0.72. A total of 223  patients were living in urban areas, 26  were previously treated for TB, 6  had diabetes, and 2  were HIV positive .Among the 262 enrolled patients, 174 (66.4%) had a positive histopathology test (granuloma with and without caseous necrosis), among whom 146  had granuloma with caseous necrosis. Xpert testing was positive for 124 study participants  and culture, for 27 .The Se, Sp, PPV, and NPV were 95.6% (129/135), 64.6% (82/127), 74.1% (129/174), and 93.2% (82/88), respectively, for the outcomes of histopathological tests, showing granuloma with or without caseous necrosis, whereas the Se, Sp, PPV, and NPV were 84.4% (114/135), 74.8% (95/127), 78.1% (114/146), and 81.9% (95/116), respectively, for the granuloma with caseous necrosis . The PPVEPTB has much less visibility than pulmonary TB and receives less attention because of its low infectiousness ,9. HowevSignificant variations in reporting notified EPTB cases have been observed among WHO regions and countries ,3. They In the context of Morocco, undertaking biopsy and histopathological testing is fully integrated in the clinical practices to establish the diagnosis of LNTB; the diagnosis of 90% of LNTB cases is based on histopathology evidence, whereas bacteriology testing is rarely used to confirm TB diagnosis  because Our study has assessed the performances of histopathology against the established bacteriological gold standard. In general, the results of the study are comparable with those of other similar studies. In our study, the culture made on lymph node biopsies was positive in 10.3% (27/262) cases. According to the various studies, the culture positivity varied from 10% to 69%; it was 10.8% in Tunisia . Other sSeveral studies reported Xpert testing on extrapulmonary biopsies with a positive outcome for TB of 11.5% , 33%, anThe granuloma with caseous necrosis was observed in 83.9% 146/174) of the patients whose histopathology tests show a granuloma, irrespective of the caseous necrosis status. This proportion was more important than what was reported in the study by Lisbet et al (42.5%) . Althoug4 of the With regard to the bacteriological gold standard used in our study, the outcomes of histopathology in favor of LNTB diagnosis were significantly better in the presence of granuloma with caseous necrosis than in the presence of granuloma with or without caseous necrosis.Our study findings report that the Se, Sp, PPV, and NPV were 84.4% (114/135), 74.8% (95/127), 78.1% (114/146), and 81.9% (95/116), respectively, when the histopathological testing showed a granuloma with caseous necrosis, whereas these indicators were 95.6% (129/135), 66.4% (174/262), 74.1% (129/174), and 93.2% (82/88), respectively, when it showed a granuloma with or without caseous necrosis. These results were comparable with a study using a culture alone as the gold standard; this study carried out in Ethiopia reported an Se, an Sp, a PPV, and an NPV of 92%, 88%, 77%, and 97%, respectively, for LNTB when histopathology testing identifies a granuloma with or without caseous necrosis . The Sp Our study reported a 78% PPV in the presence of a granuloma with caseous necrosis; this indicates that 22% of patients might be considered as having LNTB whereas their culture and/or Xpert test are negative. The presence of caseous necrosis may represent a more comfortable argument for the diagnosis of LNTB than the situation when it is absent; however, it is still not compelling evidence that allows clinicians to set the diagnosis of LNTB with confidence because a significant proportion of the patients might not have TB (22% in our study).The NPV exceeded 90% in the presence of granuloma with or without caseous necrosis and 83% in the presence of granuloma with caseous necrosis. Therefore, the proportion of false-negative cases would be 10% to 17%.In conclusion, the presence of granuloma with caseous necrosis in a histopathological examination had significantly improved the yield of histopathology for the diagnosis of LNTB. The evaluation of clinical practices, including the histopathological examination in the diagnosis of LNTB in Morocco, indicates a better yield of histopathology when the histopathological testing shows a granuloma with caseous necrosis. Therefore, it is recommended to maintain the histopathology testing to establish the diagnosis of LNTB, explore other diagnosis techniques, and duplicate this study in other country settings.


The results indicate that the main function of catalysts is to promote the conversion of alcohols into hydrocarbons by reducing energy barriers. Catalysed by the composite molecular sieve, the content of alcohols in the pyrolysis products decreased from more than 30% to less than 10%, the content of hydrocarbons increased from 20% to nearly 60%, while all the adverse components remained at a low level, which indicates that the catalytic pyrolysis products are of high quality. The great deoxidation effect of composite molecular sieves is not only due to the expansion of the range of organic matter during re-pyrolysis, but also the increasing of the residence time of pyrolysis products inside the structure for the external mesoporous structure.In order to improve the quality of catalysis products of algae, composite molecular sieve catalyst was prepared by digestion and crystallization of HZSM-5 to reduce the oxygen content of the catalytic products. According to the analysis of the pyrolysis products, the best preparation conditions were chosen of tetra propylammonium hydroxide (TPAOH) solution 2.0 mol l Therefore, measures should be taken to improve the quality of biological oils. One of the common methods is to use catalysts, such as molecular sieve, metals and metal oxides. Among them, metal oxide has large pore diameter, strong water stability and certain deoxidation performance, which is helpful to improve the stability of biological oil; alkali metals mainly include sodium salt, potassium salt, calcium salt and their oxides; microporous molecular sieve refers to molecular sieve with pore diameter less than 2 nm, which has good deoxidation and aromatization properties. HZSM-5 –14, one cid site ,13. Howecid site , the yiecid site . The mescid site . Howevercid site , while ncid site . Hydrothcid site  can incrcid site  can be acid site ,21 and scid site . HoweverIn this study, a hierarchical micro-mesoporous composite molecular sieve HZSM-5/MCM-41 with external mesoporous and internal microporous properties was developed through digestion and reassembly of molecular sieve HZSM-5, so as to meet re-pyrolysis requirements of pyrolysis steam in a wider range and improve the quality of pyrolysis products. With the support of pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) , this st2.2.1.2/Al2O3 ratio: 26 [The algae used in this study was spirulina, the selected molecular sieve catalyst was HZSM-5(SiOatio: 26 ), and thatio: 26 ,23. PyroTetra propylammonium hydroxide (TPAOH) was dissolved into 50 ml ultrapure water with a certain mass fraction to prepare TPAOH solution, and then 10 g HZSM-5 was added, stirred and heated in a water bath at 40°C for 1 h. Next cetyltrimethylammonium bromide (CTAB) solution of a certain concentration was mixed with HZSM-5 solution and heated in the water bath at 26°C for 1 h. Then, it was poured into the reactor for 24 h at a certain temperature for resolution. After adjusting the pH of the solution to 8.50 [2.2.−1 [−1. Products were identified by comparison with the NIST MS library database, and the analysis results were compared with the references to increase the reliability [−1. Pyrolysis of biomass raw materials, catalytic pyrolysis by HZSM-5 and composite molecular sieve HZSM-5/MCM-41 prepared under various preparation conditions were conducted, respectively. The analysis of organic composition was performed on a 7890A/5975C GC/MS analyser (Agilent Technologies).According to relevant studies, the temperature of pyrolysis by Pyroprobe 5200 pyrolyser   of organic pyrolytic products are calculated using a semi-quantitative method based on the area percentages of the chromatograph peaks and defined as follows [Pi is the peak area of a specific identified product, and Ptotal is the total peak area under a certain condition.In the present work, the relative content  [−1) of TPAOH solution is more effective for HZSM-5 treatment than the low concentration base solution (less than 1.0 mol l−1). Moreover, the modified catalyst does not require NH4NO3-ion exchange. The 2.0 mol l−1 TPAOH modified HZSM-5 led to produce the highest aromatic selectivity and catalytic activity from CFP of algae.The contents of alcohols and hydrocarbons in direct pyrolysis and catalytic pyrolysis products are shown in −1)  of inorg3.6.a is the SEM figure of molecular sieve HZSM-5. It can be seen from the figure that the HZSM-5 is of regular cubic shape with smooth surface. Figure 7b shows the SEM figure of HZSM-5/MCM-41 composite molecular sieve HZSM-5/MCM-41. It can be seen that the surface becomes fragmented and irregular, and many small particles are generated to adhere to the surface of crystal nucleus, namely, the synthetic MCM-41. As can be seen from the figure, compared with the molecular sieve of HZSM-5/MCM-41, the surface pore diameter of the composite molecular sieve HZSM-5/MCM-41 of HZSM-5 is significantly increased, and at the same time, it can maintain a regular overall structure without excessive collapse of the skeleton, which may be the reason for its better catalytic deoxidation effect.In order to study the effect of digestion and crystallization on the microstructure of HZSM-5, SEM characterization of catalyst samples was performed. Figure 74.−1, CTAB concentration 10 wt%, crystallization temperature 110°C, digestion–crystallization time 24–24 h, the external mesoporous structure of the composite molecular sieve can be sufficiently formed and the internal microporous structure would not be excessively damaged. The main components in the direct pyrolysis products of algae are alcohols, followed by N-containing compounds and hydrocarbons, while the adverse components aldehydes, ketones and acids have low contents, which indicate the bio-oils are of high quality. Compared with direct pyrolysis, catalytic pyrolysis by HZSM-5 can effectively reduce the oxygen content of pyrolysis products, mainly by reducing the content of alcohols. However, the adverse components do not change significantly due to their low content, and the content of N-containing compounds is hardly affected. After catalytic pyrolysis by HZSM-5/MCM-41, the alcohols decrease significantly again, and the content of adverse components also decrease, but the content of nitrogen-containing organic matter is not affected. After analysing the specific substances, it is found that HZSM-5 had great hydrodeoxidation performance on micromolecular alcohols, but the catalytic effect was limited by its pore size. The external mesoporous structure of HZSM-5/MCM-41 not only widens the reaction range of hydrodeoxidation reaction, but also increases the residence time of pyrolysis products in the internal structure, making deoxidation hydrogenation reaction more complete. Through the analysis of the products, it is found that the alcohols are mainly converted into hydrocarbons by hydrogenation, deoxidation and dehydration under the catalysis. Finally, high nitrogen content is the problem of algae pyrolytic oil. The composite molecular sieve has no effect on nitrogen content although its deoxidation effect is obvious. How to reduce the nitrogen content in the pyrolysis products of algae and its combination with composite molecular sieves to improve the quality of algae pyrolysis products can be one of the later research directions.By investigating the preparation conditions of HZSM-5/MCM-41, it is determined that under the condition of TPAOH concentration 2.0 mol l


Retention in gas–liquid chromatography is mainly governed by the extent of intermolecular interactions between the solute and the stationary phase. While molecular descriptors of computational origin are commonly used to encode the effect of the solute structure in quantitative structure–retention relationship (QSRR) approaches, characterisation of stationary phases is historically based on empirical scales, the McReynolds system of phase constants being one of the most popular. In this work, poly(siloxane) stationary phases, which occupy a dominant position in modern gas–liquid chromatography, were characterised by theoretical molecular descriptors. With this aim, the first five McReynolds constants of 29 columns were modelled by multilinear regression (MLR) coupled with genetic algorithm (GA) variable selection applied to the molecular descriptors provided by software Dragon. The generalisation ability of the established GA-MLR models, evaluated by both external prediction and repeated calibration/evaluation splitting, was better than that reported in analogous studies regarding nonpolymeric (molecular) stationary phases. Principal component analysis on the significant molecular descriptors allowed to classify the poly(siloxanes) according to their chemical composition and partitioning properties. Development of QSRR-based models combining molecular descriptors of both solutes and stationary phases, which will be applied to transfer retention data among different columns, is in progress. Quantitative structure–retention relationship (QSRR) method is a specialised branch of quantitative structure–activity(property) relationship (QSA(P)R) approach aimed at relating the retention of the analytes in separation chromatographic systems to their molecular structure ,2,3. MulRetention in gas chromatography (GC) is mainly governed by the extent of the intermolecular interactions between the solutes and the stationary phase, since the gaseous mobile phase is not involved in the partition mechanism under the typical experimental conditions of analytical separations . In thisWhile theoretical characterisation of solutes is a consolidated practice in QSRR modelling, description and classification of the GC stationary phases is historically based on empirical approaches enduring until recently . One of Poly(siloxane) stationary phases  occupy aThe dataset investigated in this work consists of 29 poly(siloxane) stationary phases belonging to poly(methylphenylsiloxane), poly(methyltrifluoropropylsiloxane) and poly subgroups displayed in Poly(dimethylsiloxane) (column OV-1) is a nonpolar and low-selectivity phase that can be regarded as the basic structure of the stationary phases here investigated. Substitution of methyl groups with phenyl, trifluoropropyl and cyanoalkyl groups in variable concentration permits extending the selectivity of the poly(siloxanes) over a wide range , which m3 to 106) [r > 0.85). After this preliminary variable selection, 177 molecular descriptors were identified and stored for further analysis.In previous analogous investigations ,24 aimed to 106) , and the to 106) . However2loo-cv. Therefore, the maximum number of descriptors to be selected by GA was set to six. Some hundreds of GA-MLR runs with different starting chromosome populations were performed for each of the five responses and the descriptors selected at least one time were collected together in a same data set that was subjected to a final GA-MLR analysis. The models with the highest Q2loo-cv values for each of the five different responses were finally chosen. These are presented in A specific QSRR model was established for each of the five McReynolds constants by multilinear regression (MLR) coupled with genetic algorithm (GA) variable selection ,29. Befo2 and Q2  and both scores (columns) and loadings (variables) were plotted in the plane of the first two principal components . Based o2 groups, is dipolar and strongly electron attracting. It is therefore able to display dipole−dipole, dipole-induced dipole and charge-transfer interactions. Moreover, the unshared electron pair on the nitrile nitrogen can promote hydrogen-bonding interactions with H-donor solutes. It follows that differentiation of the columns in the subspace of the significant PCs extracted from the QSRR descriptors should reflect the ability of the related McReynolds solute to interact with the various poly(siloxane) stationary phases.r = 0.79, The number of rotatable bonds (RBN) is the most influent molecular descriptor regardless of the QSRR response, according to the higher b’ value of this variable in all the generated models . The conAbout the ability of the selected molecular descriptors for the classification of the poly(siloxane) stationary phases, r > 0.85), only 177 molecular descriptors were retained for further analyses.Starting geometries of the poly(siloxanes) were drawn by means of the MacroModel 7.1 molecular modelling program package  assuming2loo−cv). To avoid the loss of highly predictive models, a predefined number of the best chromosomes  are passed unchanged to the next generation. In this work, the initial population consists of 100 chromosomes and evolution is carried out until no further improvement of Q2loo−cv of the best model occurs after five cycles.A specific QSRR model was established for each of the five McReynolds constants, X, Y, Z, U and S, by multilinear regression (MLR) combined with genetic algorithm (GA) selection to identify a small subset of significant variables within the 177 molecular descriptors provided by Dragon. MLR is the most convenient multivariate tool for QSRR modelling, because of simple statistical bases and easy interpretation of the resulting models: the retention time or a related parameter is expressed as a linear combination of molecular descriptors and the related coefficients are determined by ordinary least squares regression . However2 and SDEC, respectively). The predictive ability of the QSRR models was quantified by the coefficient of determination and the standard deviation of the error evaluated on a predesigned external set , Q2 being computed according to Todeschini et al. [The descriptive performance of each MLR model was evaluated by usual statistical parameters : the coei et al. . The prei et al. . In thisPrincipal component analysis (PCA) was used to help interpretation of the QSRR models and attempt unsupervised classification of the stationary phases based on their molecular structure. PCA  allows tIn the present study, poly(siloxane) stationary phases, the most widely used in liquid−gas chromatography and historically classified by empirical polarity/selectivity scales, were characterised using theoretical molecular descriptors. In spite of the polymeric nature of these stationary phases, the selected molecular descriptors allowed for prediction of the McReynolds constants with acceptable accuracy and are useful to classify the columns according to their partitioning properties. Further work is in progress to evaluate the performance of QSRR−based predictive models combining molecular descriptors of both stationary phases and solutes.


Vibrio vulnificus, an opportunistic human pathogen, identified a set of genes induced upon exposure to NO. Among them, VvhmpA (V. vulnificus hmpA), encoding a multidomain NO dioxygenase, was the most greatly induced upon exposure to NO and was thus further characterized. Absorption spectra demonstrated that VvHmpA is a heme protein in which the heme iron can exist in either reduced, NO-bound, or oxidized state. Biochemical studies revealed that VvHmpA is a flavohemoglobin containing equimolar amounts of heme and FAD as cofactors. The KM and kcat values of VvHmpA for NO at 37°C, the temperature encountered by V. vulnificus in the host, were greater than those at 30°C, indicating that VvHmpA detoxifies high levels of NO effectively during infection. Compared with the wild type, the VvhmpA mutant exhibited a lower NO-decomposition activity and impaired growth in the presence of NO in vitro. Also, the cytotoxicity and survival of the VvhmpA mutant infecting the NO-producing murine macrophage cells were lower than those of the wild type. Furthermore, the mouse lethality of the VvhmpA mutant was reduced compared to that of the parental wild type. The combined results revealed that VvHmpA is a potent virulence factor that is induced upon exposure to NO and important for the survival and pathogenesis of V. vulnificus during infection.Nitric oxide (NO) and its derivatives are important effectors of host innate immunity, disrupting cellular function of infecting pathogens. Transcriptome analysis of  Under ie (N2O3) . Furthere (N2O3)  that acte (N2O3) . RNS cane (N2O3) . Therefoe (N2O3) .c nitrite reductase, S-nitrosoglutathione reductase, and peroxynitrite reductase  heme reacts with NO to yield NO3– and ferric-deoxy (Fe3+) heme, in which both atoms of O2 are incorporated into the NO3– (2 to the ferric heme to regenerate the ferrous heme and the resulting FAD+ is then reduced back to FADH2 by utilizing the reducing power of cellular NAD(P)H H .V. vulnificus, is a causative agent of life-threatening septicemia and necrotizing fasciitis in individuals with predisposing conditions, such as liver damage and kidney failure , which is the most preferentially expressed in V. vulnificus cells exposed to NO. The biochemical and kinetic properties of V. vulnificus HmpA (VvHmpA), the product of VvhmpA, were verified experimentally. Construction of the isogenic VvhmpA mutant and evaluation of its phenotypes provided evidence that VvHmpA could contribute to the survival and thereby the pathogenesis of V. vulnificus during infection.The opportunistic human pathogen,  failure . It is rV. vulnificus MO6-24/O (wild type), VvhmpA mutant, and VvhmpA-complemented strain were grown aerobically in Luria-Bertani (LB) medium supplemented with 2.0% (w/v) NaCl (LBS) at 30°C, and their growth was monitored spectrophotometrically at 600 nm (A600). The RAW 264.7 murine macrophage cells were obtained from Korean Cell Line Bank  and grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and appropriate antibiotics [100 units ml–1 penicillin G and 100 μg ml–1 streptomycin ] in air supplemented with 5% CO2 at 37°C.The strains and plasmids used in this study are listed in V. vulnificus MO6-24/O, grown aerobically to A600 of 0.5 in M9 minimal media supplemented with 0.4% (w/v) glucose (M9G) and exposed to different types of NO donors for 10 min. As NO donors, excess amounts of either NO-releasing poly(lactic-co-glycolic acid)-polyethylenimine nanoparticles (NO/PPNPs) (releasing NO with t1/2 = 24 h at 37°C) or Spermine NONOate  were used . The expression level of each gene was defined using the number of fragments per kilobase of transcript per million mapped reads (FPKM)  in the V. vulnificus exposed to NO/PPNPs.The cDNA libraries were constructed using a TruSeq Stranded mRNA Sample Prep kit . Strand-specific paired-end 100-bp sequences were read from each cDNA library using HiSeq 2500 (Illumina) as described previously . The raws (FPKM) . QuantilTM cDNA synthesis kit , and real-time PCR amplification of the cDNA was performed by using the Chromo 4 real-time PCR detection system (Bio-Rad) with pairs of specific primers  using the PCR-mediated linker-scanning mutation method as described previously  to result in pDY1703 (6-tagged VvHmpA was expressed in E. coli BL21 (DE3) containing pDY1703 and purified by using affinity chromatography (Qiagen) according to the manufacturer’s procedure, and used to raise rabbit anti-VvHmpA polyclonal antibody   .V. vulnificus MO6-24/O grown to A600 of 0.5 in M9G was exposed either to Spermine NONOate as described above or to M9G (negative control) and then harvested to isolate total cellular proteins. To detect VvHmpA, the total cellular proteins (20 μg) were resolved on SDS-PAGE under reducing conditions and immunoblotted using the rabbit anti-VvHmpA antibody as described previously  and used to detect V. vulnificus DnaK   .VvhmpA ORF was amplified by PCR using a pair of primers, HMPA05-F and -R  (Invitrogen) to result in pSH1701 (6-tagged VvHmpA was expressed in E. coli BL21 (DE3) containing pSH1701 and purified by using a HiTrap Q anion-exchange column  and then a HiLoad Superdex 16/60 200 column  as described previously  was mixed with sodium hydrosulfite powder  under anaerobic conditions  (VvHmpA (10 μM) was incubated under anaerobic conditions with 40 μM Diethylamine NONOate sodium salt hydrate (Sigma) (releasing NO with t1/2 = 2 min at 37°C) for 10 min to obtain the NO-bound VvHmpA, or under aerobic conditions for 20 min to obtain the oxidized VvHmpA. The UV–vis absorption spectra of the VvHmpA proteins were recorded by using Shimadzu UV-1800 UV/VIS spectrophotometer  at room temperature.To obtain the reduced nditions . Immediaake, MI) . The redVvHmpA with heme in vitro, 10 μM of the purified VvHmpA was gradually mixed with stoichiometric excess of hemin (1 mM in 0.01 M NaOH), and then incubated for 40 min at 4°C . Similarly, 10 μM of the purified VvHmpA was incubated with 80 μM of FAD for an hour at room temperature, and then residual FAD was removed from the resulting reconstituted VvHmpA using Ultracell-10K centricon  were delivered via separate syringes to 980 μl of the reaction buffer (50 mM potassium phosphate (pH 7.8) with 100 μM EDTA, 1 μM FAD and 100 μM NADH), that is pre-incubated at either 37 or 30°C  was administered to the resuspended V. vulnificus cells to achieve 2 μM NO at a final concentration, and their NO-decomposition activities were determined by measuring the residual NO using the ISO-NOP electrode.The VvHmpA on the survival of V. vulnificus under nitrosative stress, equal numbers (approximately 107 cells ml–1) of the V. vulnificus strains were used to inoculate the M9G containing 0.15 mg ml–1 NO/PPNPs at a final concentration. The resulting cultures were further incubated aerobically with shaking and the viable cells were counted at time intervals.To examine the effect of –1E. coli O111:B4 lipopolysaccharide (Sigma) and 1 mM L-arginine (Sigma) to induce NO production , which is a known NO synthase inhibitor  of 1. To determine the survival of the V. vulnificus strains directly affected by the NO from the RAW 264.7 cells, the culture dishes were washed two times to remove bacteria non-adherent to the macrophages as described previously (V. vulnificus strains was determined by measuring the lactate dehydrogenase (LDH) activity released into the supernatant as described previously  female mice  (n = 5 for each infection). The infection experiments were performed three times with the mice  to ensure reproducibility and percentages of the mice survival were recorded for 24 h. Statistical significance was determined by the log-rank test. All manipulations for mouse lethality assay were approved by the Animal Care and Use Committee at Seoul National University.The 1 under accession number PRJNA5134632.Averages and standard deviations (S.D.) were calculated from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 7.0 (GraphPad Software). All raw transcriptome data were deposited in Sequence Read Archive (SRA)V. vulnificus MO6-24/O cells exposed to NO/PPNPs  upon exposure to NO; 320 genes were upregulated and 231 genes were downregulated , NnrS (putative heme- and copper-containing transmembrane protein), and NrfD (cytochrome c nitrite reductase subunit). The other two genes are predicted to encode transcriptional regulators that control the expression of the genes involved in nitrosative stress defense: NorR (NO reductase transcription regulator) and NsrR .The NO/PPNPs  were harV. vulnificus exposed to NO/PPNPs was reevaluated by using quantitative real-time PCR (qRT-PCR) analyses, further confirming that NO exposure induced transcription of the genes , Salmonella enterica serovar Typhimurium Hmp (StHmp), and Vibrio cholerae HmpA (VcHmpA)  , respectygenases  , indicating that the NO-decomposition activity of the VvhmpA mutant was significantly impaired  compared with that of mice infected with the wild type. At 24 h post-infection, percentages of mice that survived after challenge with the VvhmpA mutant or the wild type were 66.6 and 33.3%, respectively, indicating that the deletion of VvhmpA attenuated the virulence of V. vulnificus in mice. Taken together, the combined results suggest that VvHmpA is important for NO decomposition, survival adjacent to NO-producing immune cells, and thereby the pathogenesis of V. vulnificus.The importance of V. vulnificus identified 15 more genes that are induced upon exposure to NO and potentially involved in nitrosative stress defense H to the ferric heme via FAD  to regenerate ferrous heme, resulting in less efficient NO decomposition (V. vulnificus seems to adopt the multidomain VvHmpA to efficiently overcome nitrosative stress during infection.In order to establish infection successfully, pathogens need to overcome nitrosative stresses that originate from the host immune system . In addi defense . Among tng sites . As well via FAD . This enposition . TherefoEcHmp proposed two possible reaction mechanisms to initiate NO decomposition, namely dioxygenation and nitrosylation (2 binding to the ferrous heme to result in ferrous-oxy heme, most probably under conditions where O2 is not limiting. The ferrous-oxy heme reacts with NO to form a transient Fe-ONOO– intermediate which releases NO3– as described above. In contrast, nitrosylation involves initial reaction of the ferrous heme with NO to form a ferrous-nitrosyl heme, converting transiently to a ferric-nitrosyl heme which in turn reacts with O2 to release NO3– under O2 limiting conditions. Regardless of which mechanistic pathway predominates, they both result in rapid enzymatic turnover of NO to NO3–. Although the exact mechanisms of VvHmpA detoxifying NO have not been yet clarified, the similarities found in the amino acid sequences and absorption spectra of EcHmp and VvHmpA (VvHmpA also could convert NO to NO3– through one of the two pathways depending on O2 availability. Supporting this postulation, the kinetic values of VvHmpA for NO decomposition under aerobic conditions (EcHmp, in which the kcat and KM values are 10-670 s–1 and 0.25 μM for NO, respectively (Biochemical and kinetic analyses of sylation . Dioxyged VvHmpA , 4A led nditions  are alsoectively .KM and kcat values of VvHmpA for NO at 37°C, the temperature to which V. vulnificus is inevitably exposed in the host, are greater than those at 30°C (VvHmpA expressed in the host may be more optimized for acting on the large amounts of NO and decomposing them rapidly to a safe level during infection, before the increased nitrosative stress impairs cellular components of V. vulnificus. It should be noted, however, that VvHmpA may not be the sole enzyme that protects V. vulnificus from NO as the VvhmpA mutation reduced the growth and survival of the pathogen significantly but not completely (V. vulnificus transcriptome upon exposure to NO showed upregulation of the genes encoding a number of NO detoxifying enzymes in addition to NO dioxygenase (VvHmpA could provide an evolutionary advantage for V. vulnificus to survive in the host rather than in nature, where lower concentration of NO may be occasionally encountered. Supporting this hypothesis, loss of VvHmpA led V. vulnificus to show significantly reduced survival adjacent to the NO-producing macrophage cells (After infection, pathogenic bacteria probably encounter increased levels of NO due to the expression of iNOS in cells of the immune system and must overcome the nitrosative stress for successful pathogenesis . The NO  at 30°C . This kimpletely , 7A. Indxygenase  (Poole, xygenase . Neverthge cells , cytotoxge cells , and posge cells .V. cholerae, Salmonella Typhimurium and Staphylococcus aureus were responsible for NO detoxification and thus required for their survival under nitrosative stress and successful pathogenesis (VvHmpA is a flavohemoglobin containing equimolar amounts of heme and FAD as cofactors. Kinetic properties of VvHmpA with greater KM and kcat values at 37°C than 30°C indicated that VvHmpA is optimized to act on and decompose large amounts of NO more effectively in the host. Reduced NO-decomposition activity and growth rate of the VvhmpA mutant in the presence of NO, along with the attenuated virulence observed both in the cell culture and mouse models, indicated that VvHmpA contributes to the survival of V. vulnificus under nitrosative stress, and might play an important role in the pathogenesis of the pathogen. Therefore, VvHmpA could be a promising target for development of new antibacterial agents.It has been reported that the Hmp proteins of miscellaneous pathogenic bacteria including ogenesis . Howeverhttps://www.ncbi.nlm.nih.gov/sra/?term=PRJNA513463.The datasets generated for this study can be found in the Sequence Read Archive – PRJNA513463; The animal study was reviewed and approved by Animal Care and Use Committee at Seoul National University.DK, EN, and SC designed the research. DK, EN, SK, JK, YJ, and JC performed the research. DK, EN, IB, N-CH, and SC wrote the manuscript. All authors made major contributions to the acquisition, analysis, and interpretation of the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.