Flavivirus, detected in a wide variety of mosquito species.Flaviviruses are important pathogens for humans and animals . Culex flavivirus (CxFV) is an insect-specific virus of the genus To detect Flavivirus in mosquitoes of a tropical region of the Colombian Caribbean.Mansonia (n = 3271/39.5%), Culex (n = 2668/32.26%), Anopheles (n = 840/10.15%), Aedeomyia (n = 411/4.9%), Psorophora (n = 397/4.8%), Coquilletidia (n = 369/4.46%), Uranotaenia (n = 261/3.15%) and Aedes (n = 53/0.6%). All mosquito species were collected in dry tropical forest of the Caribbean area. Universal primers for NS5 gene (958 pb), RT-PCR for flavivirus and sequencing were used for molecular identification of viruses detected.In 2014, an entomological surveillance of arboviruses was conducted in the department of Cordoba area of the Caribbean, Colombia. A total of 8270 mosquitoes were captured as follow: Culex coronator were positive for flavivirus RNA sequence by RT-PCR. The sequences of the PCR amplicons, matched that of the Culex flaviviruses, CxFv COL PM_149 (GenBank: KR014201) and CxFv COL PM_212 (GenBank: KT307717). Phylogenetic analysis of the NS5 protein sequences of the Culex flaviviruses sequences with those of reference sequences available in GenBank indicated viruses of Genotype II, closely related to the Brazilian strain, BR_SJRP_01_ (GenBank: KT726939), from Culex sp. The alignment of Culex flavivirus sequences CxFv COL_ PM 212 and CxFv COL_ PM 149 with sequences of strains detected in different geographical regions grouped the strains in a Latin American clade reported in Brazil, Argentina and Mexico.Two pools belonging to The present work illustrated that CxFV was circulating among vectors of human pathogenic arboviruses in Colombia, but the impact of CxFV on other flaviviruses which are endemic in the study area still remains to be explored.The online version of this article (10.1186/s12985-018-1108-2) contains supplementary material, which is available to authorized users. Culex coronator in Colombia. The strains detected are phylogenetically related to Africa/Caribbean/Latin America genotype 2.Culex flavivirus (CxFV) is an insect-specific virus of the genus Flavivirus detected in mosquitoes. The present report shows the first detection of CxFV in mosquitoes Arboviruses are a serious public health problem in tropical countries, the genus flavivirus included a group of these viruses which some of them are highly pathogenic, and can cause encephalitis or hemorrhagic fevers in humans . In eachCulex pipiens, Culex quinquefasciatus, and other Culex sp. around the world [Flaviviridae, the viruses within this family are transmitted horizontally between vertebrate hosts and hematophagous arthropods. However, certain members of the genus Flavivirus are known to be vertebrate-specific whilst others are insect-specific flaviviruses (ISFV). Transmission of ISFVs in natural mosquito populations is maintained by vertical transmission and in minor role the venereal transmission [Culex flavivirus (CxFV) is an insect-specific virus of the genus Flavivirus. CxFV strains have been detected from mosquitoes he world , 3. CxFVsmission –6.Psychodidae [In Europe, species of ISFV have been identified in field mosquitoes from Italy, Portugal, Spain, the United Kingdom, the Czech Republic and Greece –9. Sequechodidae . Despitechodidae .ISFV do not cause disease in humans or animals, but they are important because they co-infect mosquito vectors and compete with the viruses that are pathogenic to humans and animals .The circulation of ISFVs in nature is of increasing interest due to their influence onarboviruses transmission . In vitrCulex erraticus, Cx. quinquefasciatus and Mansonia titillans, confirming that these ecological conditions are favorable to high viral biodiversity [Colombia, due to its geographical location, has a great biodiversity of reservoirs and arthropods, which have supported the emergence of viruses such as Chikungunya and Zika. These viruses have adapted to the vectors circulating in the tropics, which facilitated their spread, and caused the two most recent major epidemics of arboviruses in the Americas. The Colombian Caribbean also maintains circulation of pathogenic Saint Louis encephalitis virus (SLEV) and West Nile Virus (WNV), transmitted by mosquitoes of iversity –13.The objective of this work was to detect Flaviviruses in mosquitoes of a tropical region of the Colombian Caribbean.From August 2012 to December 2014, an observational, descriptive and prospective study was conducted to identify circulating flaviviruses in different mosquito species of the department of Córdoba, Colombia. The department is located in the Caribbean region between 09° 26′16" and 07° 22′05 “N, and 74° 47’ 43” and 76° 30’01 ‘W. The department of Córdoba has areas with wetlands and tropical dry forest, the area is characterized by rainfall that does not exceed 2000 mm per year, of which, 80% is during the rainy season (May-October). The tropical climate has an average annual temperature of 28°C, with heights between 0 and 40 masl. The department is divided geographically into sub-regions according to the territorial development plan: High, Medium and Low Sinú, and the samplings were carried out in these areas. One of the geographical zones corresponded to the municipality of Montería (8 ° 43’15.80 “N -075 ° 59′27.27” W (Medium Sinú), where the circulation of WNV and SLEV have been reported [2 were used to capture gravid females of different genera. The sampling sites were close to small lagoons and areas of tropical dry forest. During each sampling, the traps were placed between 17:00 and 6:00 h. Manual aspirators were also used. The collected mosquitoes were kept at 6 °C, until being transported to the laboratory of the Biological Research Institute of the Tropic (IIBT), in less than 12 h post-capture. Once in the laboratory the mosquitoes were stored at − 80 °C for molecular analysis.In each of the selected sites, a total of five samplings were set-up. Four Light-CDC traps were used to capture mosquitoes, and 2 BG Centinell ™ traps fed with COCulex genus. They were classified according to their morphology based on specialized taxonomic keys [Male mosquitoes were identified first. It was necessary to clarify Terminalia , due to mic keys –18. To mmic keys . For femmic keys . The ideRNA extraction was performed using the QIAamp Viral RNA Mini kit  following the manufacturer’s instructions. The RNA was extracted from 140 μl of the homogenized mosquitoes sample. For detection of flavivirus reverse transcription RT-PCR with universal primers previously described by Bronzoni et al.  were usehttp://beast.bio.ed.ac.uk/Main_Page, to model sequence evolution, invariants, gamma distribution, size of the chain run (20 million generations), coalescent constant population and uncorrelated molecular clock were chosen for this inference [The amplified fragments were sequenced in both directions in an ABI 3730 XL Automated sequencer. Thirty-six representative sequences belonging to different geographic regions were downloaded from Genbank and aligned using MUSCLE in MEGA v7 . Alignmenference .Mansonia (N = 3271/39.5%), Culex (n = 2668/32.2%), Anopheles (n = 840/10.1%), Aedeomyia (n = 411/4.9%), Psorophora (n = 397/4.8%), Coquilletidia (n = 369 / 4.4%), Uranotaenia (n = 261/3.1%) and Aedes (n = 53/0.6%). The genus of mosquitoes of greater medical importance, distributed monthly is shown in the Fig. Nine thousand eight hundred ninety eight mosquitoes were captured, and 8270 of them were identified and grouped in 334 pools. 41 species were identified and distributed in 8 genera: Culex coronator were positive for flavivirus. These mosquitoes were collected in the municipality of Montería (PM 149 and PM 212). The 958-bp amplicons from the NS5 gene were sequenced, and the sequences were edited and assembled to obtain a fragment of 758 bp for analyses. Sequences were aligned and a 99% similarity was obtained with Culex flavivirus virus sequences from position 8142 to position 8899 of the complete genome of the isolate . Sequences were deposited in the GenBank: CxFv COL_ PM 212 (Genbank accession number: KT307717) and CxFv COL_ PM 149 (Genbank accession number: KR014201).Of the 334 analyzed pools, 2 pools of Culex flaviviruses, CxFv COL_ PM 212 and CxFv COL_ PM 149, with NS5 sequences from strains detected in different geographical regions allowed their grouping in a clade that includes virus strains reported in Brazil, Argentina and Mexico and belonging to the genotype II group of strains of Africa/Caribbean/Latin America  and Culex (32%), these results differ somewhat from those reported by Hoyos et al. [Culex (50%) was the most abundant. The difference between these results is possibly due to the geographic, climatic and environmental characteristics of the selected areas.In the present study, the most abundant mosquito genera were s et al.  in a neaThe limited number of positive samples (pools) in the present study 0.6% (2/334 pools) is similar to reported by Bryant et al 2005 in Vietnam, where only 2.3% of analyzed pools were positives 26/1122) . Ochieng/1122 . OCx. coronator species in Colombia. CxFV was previously reported in Colombia, in the department of Córdoba in Cx. quinquefasciatus (KM031075) and Cx. erraticus (KM031073) species [According to the taxonomic definition of flaviviruses, based on the identity of the conserved region of NS5 gene (> 84%) , the det species . InteresCx. pipiens is the main reservoir. However, other mosquito species, such as Cx. tritaeniorhynchus and Anopheles sinensis [CxFV have a great diversity of reservoirs and their phylogeny could be influenced by these. The phylogenetic tree shows two clades Fig. : the firsinensis , have beCx. quinquefasciatus as the main reservoir [The second phylogenetic group of CxFV have been reported mostly in the Americas and Africa, with eservoir , 29, 30,Cx. coronator in the department of Córdoba, the presence of these viruses in mosquitoes could play an important role from the public health point of view, because it has been shown that previous CxFV infection can increase or block the infection of the mosquito by other pathogenic flaviviruses. CxFV-positive mosquitoes also differ in flight activity, which could decrease contact with arbovirus amplification hosts [The present study reports the first description of CxFV in on hosts , 31–34. Additional file 1:Culex flavivirus aligment Bayesian inference phylogeny of representatives CxFv sequences belonging to different geographic regions strains obtained from the GenBank database. (FAS 29 kb)


B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore’s interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis.This work dramatically improves our understanding of the assembly of the outermost layer of the  Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis. We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.Bacterial spores produced by the  Bacillus anthracis, the causative agent of anthrax, these layers are the cortex, coat, interspace, and exosporium (B. anthracis and the model organism Bacillus subtilis (which lacks the interspace and exosporium) seek to identify the functions of these layers and elucidate the mechanisms directing their assembly.The bacterial endospore, an abundant cell type found in many environments , is highly resistant to diverse stresses and survives under extreme conditions for extended time periods. These properties are important for persistence in the environment, colonization of the human host, and pathogenesis –3. The s–osporium  3, 4). . Bacilluosporium . OngoingB. anthracis spores lacking the exosporium are largely functional . At. AtB. anexsY, encoding a major basal layer structural protein  what directs the cap to form at the forespore? and (ii) what guides exosporium elongation to form a closed shell around the forespore, rather than disconnected sheets in the mother cell cytoplasm? Addressing these questions is essential to a robust explanatory model of the assembly of this distinctive and unique cell layer, both in position , 30.B. subtilis and B. anthracis spores, previous work shows that several proteins with important roles in spore assembly in these two species are conserved evolutionarily and, at least in some cases, functionally , resulted in spores lacking the exosporium . The resulting spores also had an intact coat, but ∼96% lacked the exosporium , and 120 µg of protein was separated via 15% (A) or 10% (B) SDS-PAGE gels. Sporangial proteins were extracted by resuspending cell pellets in Laemmli buffer and were mixed with 0.1 mm glass beads. Mother cell lysis was achieved, and forespore proteins were extracted by bead beating, immediately followed by three rounds of incubation at 100°C for 5 min and 1 min of vortexing. Samples were then centrifuged at 17,000 × g for 5 min, and the supernatant was collected. The concentration of protein extracted was measured using a NanoDrop spectrophotometer, and 120 µg was separated via 15% SDS-PAGE gels (C). Gels were stained with Bio-Safe Coomassie G-250 stain (Bio-Rad). Download FIG S1, PDF file, 2.2 MB.SDS-PAGE of proteins extracted from released spores or sporangia. Washed spores were resuspended in Laemmli buffer, and proteins were extracted by incubating at 100°C for 5 min, followed by 1 min of vortexing. Samples were heated and vortexed 3× and centrifuged at 17,000 × Copyright © 2018 Boone et al.2018Boone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.01166-18.2FIG S2B. anthracis spores. Wild-type spores or spores lacking an exosporium (exsY or cotO) were germinated using a combination of inosine and alanine (A), inosine and serine (B), or alanine alone (C). For spores germinated using inosine and alanine, 100 µl of a stock solution containing 50 mM alanine and 10 mM inosine was used to induce germination in a total volume of 1 ml. This stock solution was either used directly or diluted 1:100 or 1:150 before use. Inosine and serine were used at final concentrations of 1 mM or 0.2 mM. Alanine alone was used at final concentrations of 100 mM or 50 mM. Download FIG S2, EPS file, 1.8 MB.Effect of the exosporium on germination of Copyright © 2018 Boone et al.2018Boone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the cotO into the TJB124 genome at an ectopic site (creating strain TJB130) restored the expression of cotO . Possibly, this failure was due to increased cotO expression. To test the hypothesis that elevated CotO levels cause an exosporium assembly defect, we transformed the wild-type strain with a plasmid expressing cotO from its endogenous promoter. The resulting strain (TJB116) had increased CotO levels during sporulation  and TJB124 (cotO), harvested between 1 and 8 h after the onset of sporulation (T1–T8). Consistent with previous studies (2–T5). At these times, the cap covered less than one-quarter of the two-dimensional circumference of the developing spore and was close (∼35nm) to the forespore membrane (cotO mutant) and 80% (45 of 54) of MGM76 (cotOΩpMGM3) sporangia at these times  is insufficient to complete multiple exosporia. These data suggest a role for CotO in initiating cap assembly at the mother cell pole.If CotO is required for cap formation, overproduction of CotO might result in the assembly of additional caps. To address this hypothesis, we used TEM to search for caps in strain TJB116 . Prior tect caps . Strikin caps (6 . We infe2) in total sporangia . We detected CotO at all time points ..cotY gencotY mutation on the timing of exosporium formation and the location of exosporium initiation, we compared the degree of exosporium assembly at intermediate times in wild-type and mutant sporangia. We also compared the stage of exosporium assembly to the stage of coat assembly as a separate marker of progression through sporulation. In wild-type sporangia, exosporium elongation occurs simultaneously with coat assembly . Instead, we observed what appear to be small patches of exosporium at random locations around the spore , harvested at intermediate time points (T5), were visualized by TEM. Sporangia in the early stages of exosporium formation were specifically selected to determine if the timing of exosporium nucleation affects its localization. Arrows indicate the location of small patches of exosporium-like material, which are located at various points around the developing forespore, suggesting that exosporium nucleation is not limited to the mother cell-proximal pole of the forespore in this strain. Download FIG S3, PDF file, 2.5 MB.Effect of early cap formation on the location of exosporium initiation. Sporangia of strain TJB239  also plays a role in directing elongation of the exosporium around the forespore. To investigate the role of CotO in guiding exosporium elongation, we characterized exosporium assembly in the ore pole , the leaore pole  and C. Iorespore , are preorespore , or appeorespore . Our datB. subtilis, coat assembly begins at the mother cell-proximal pole of the forespore  of wild-type sporangia  of cotE mutant sporangia, and 35% (9 of 26) of cotY mutant sporangia had electron-dense coat material covering the mother cell-proximal pole of the forespore at this stage , CotO-GFP localized to, and eventually surrounded, the forespore (cotE (MUS8189), CotO-GFP accumulated in the mother cell cytoplasm early during sporulation but did not localize to the forespore. No fluorescence was visible in this strain at later time points (T7). We conclude that CotE has a significant role in CotO localization and may impact CotO stability.To address the possibility that CotE directs CotO assembly early in sporulation, we used epifluorescence microscopy to monitor the localization of a CotO-GFP fusion protein in wild-type and orespore . In a stcotE is required for CotO stability, we measured CotO steady-state levels throughout sporulation in wild-type and cotE mutant sporangia using Western blot analysis. In wild-type sporangia, we detected CotO at all time points after T2. In contrast, CotO was not detected after T5 in cotE mutant sporangia . We found that CotE bound to CotO immobilized to the wells of a 96-well plate in a concentration-dependent manner  likely reside between CotE and the forespore membrane and anchor the network to the forespore  at the mother cell-proximal pole, occludes further deposition of coat proteins at this location. We argue that coat proteins are unable to diffuse through the cap and therefore must traffic between the exosporium and the forespore membrane surface, resulting in late coat formation proceeding from the edge of the cap. After interspace widening, however, the cap no longer interferes with coat assembly, and electron-dense layers form where the cap used to be. We suggest that the mechanism of coat assembly may be more flexible than previously thought, changing as a result of the evolution of additional outer structures. In this view, no single model of sporulation is likely to completely describe spore formation in all species.Our second key finding is that, unlike he spore . We founB. subtilis as being important for proper coat assembly, are often conserved in other Bacillaceae , kanamycin (25 μg/ml), or carbenicillin (80 μg/ml). B. anthracis strains were grown in the presence of polymyxin B (60 units/ml), kanamycin (20 μg/ml), erythromycin (10 μg/ml), chloramphenicol (10 μg/ml), or spectinomycin (250 μg/ml). Recombinant DNA techniques were performed as previously described (All strains were cultured in Luria-Bertani (LB) medium. When needed, escribed . PCR proescribed  were pur10.1128/mBio.01166-18.4TABLE S1Table S1, DOCX file, 0.02 MB.Primers, plasmids, and strains. Download Copyright © 2018 Boone et al.2018Boone et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the cotO gene was initially disrupted via Campbell-type single-reciprocal recombination, as previously described  under its native promoter, immediately downstream of the eag1 gene . The resulting plasmid (pBT4653) was introduced into B. anthracis using electroporation, followed by selection with chloramphenicol.To generate strains of B. anthracis spores were generated by growth to exhaustion in Difco sporulation medium (DSM) (600) to determine the initiation of sporulation (T0), defined as the end of exponential-phase growth. Two-milliliter samples were taken every hour during sporulation (T1–T12) and either immediately fixed for analysis by transmission electron microscopy (TEM) (um (DSM) . Spores py (TEM)  or storeThe CotE and CotO proteins were overproduced and nickel-affinity purified, using the QIAexpressionist system, according to the manufacturer’s instructions (Qiagen). Purified CotO was used to generate anti-CotO antibodies in New Zealand White rabbits , which were protein A affinity purified.g for 5 min, and the supernatant was collected.Coat and exosporium proteins were isolated from harvested spores by resuspending in Laemmli buffer, followed by three rounds of heating (100°C for 5 min) and then vortexing (1 min) . SolubleProteins were quantified using a NanoDrop spectrophotometer (Thermo Scientific), 120 μg was separated via 10% or 15% SDS-PAGE gel, and the gel was stained with Bio-Safe Coomassie G-250 stain (Bio-Rad) .For Western blot analysis, 40 μg of extracted proteins was separated by 15% SDS-PAGE gels and transferred to a nitrocellulose membrane (Bio-Rad). Membranes were washed with Tris-buffered saline  and incubated for 30 min with blocking buffer . Membranes were washed with TBS and incubated with anti-CotE  or antil-lysine (Sigma-Aldrich). Slides were washed with PBS, treated with blocking solution (2% BSA in PBS) for 30 min, washed 9× with PBS, and incubated for 1 h with either polyclonal anti-BclA  or anti-CotE  antibodies. Slides were washed 9× with PBS and incubated for 1 h with goat anti-rabbit antibodies conjugated to Alexa Fluor 488 . Slides were washed 9× with PBS, and coverslips were mounted using PermaFluor Mountant mounting medium (Thermo Scientific). Spores were then visualized using a Leica DM IRB fluorescence microscope. Images were collected with a MagnaFire cryo-cooled charge-coupled-device (CCD) camera and processed using the ImageJ software.Washed spores (10 µl) were adhered to the wells of a multiwell microscope slide  coated with poly-in vitro was measured using an enzyme-linked immunosorbent assay (ELISA), as previously described (A405) was measured.The potential for CotE and CotO to interact escribed . CotO wa


Organ failure, including acute kidney injury (AKI), is the third leading cause of death after bleeding and brain injury in trauma patients. We sought to assess the prevalence, the risk factors and the impact of AKI on outcome after trauma.We performed a retrospective analysis of prospectively collected data from a multicenter trauma registry. AKI was defined according to the risk, injury, failure, loss of kidney function and end-stage kidney disease (RIFLE) classification from serum creatinine only. Prehospital and early hospital risk factors for AKI were identified using logistic regression analysis. The predictive models were internally validated using bootstrapping resampling technique.p < 0.001). AKI was independently associated with a twofold increase in ICU mortality.We included 3111 patients in the analysis. The incidence of AKI was 13% including 7% stage R, 3.7% stage I and 2.3% stage F. AKI incidence rose to 42.5% in patients presenting with hemorrhagic shock; 96% of AKI occurred within the 5 first days after trauma. In multivariate analysis, prehospital variables including minimum prehospital mean arterial pressure, maximum prehospital heart rate, secondary transfer to the trauma center and data early collected after hospital admission including injury severity score, renal trauma, blood lactate and hemorrhagic shock were independent risk factors in the models predicting AKI. The model had good discrimination with area under the receiver operating characteristic curve of 0.85 (0.82–0.88) to predict AKI stage I or F and 0.80 (0.77–0.83) to predict AKI of all stages. Rhabdomyolysis severity, assessed by the creatine kinase peak, was an additional independent risk factor for AKI when it was forced into the model (OR 1.041 (1.015–1.069) per step of 1000 U/mL, AKI has an early onset and is independently associated with mortality in trauma patients. Its prevalence varies by a factor 3 according to the severity of injuries and hemorrhage. Prehospital and early hospital risk factors can provide good performance for early prediction of AKI after trauma. Hence, studies aiming to prevent AKI should target patients at high risk of AKI and investigate therapies early in the course of trauma care.The online version of this article (10.1186/s13054-018-2265-9) contains supplementary material, which is available to authorized users. Organ dysfunction remains the third leading cause of death in trauma patients, after hemorrhage and traumatic brain injury . Among oSevere trauma triggers initial AKI risk factors including hemorrhage, rhabdomyolysis, traumatic inflammation and leads to second hits due to emergency surgery or infections that may cause additional renal disorders resulting in renal function impairment. Identifying AKI risk factors after trauma is essential to establish a strategy aiming to prevent AKI and its related complications. Previous studies reporting AKI incidence after trauma focused either on medical history , 5, hemoThe objectives of this study were (1) to report the prevalence of AKI, (2) to describe risk factors associated with AKI and (3) to explore whether AKI is independently associated with mortality in a multicenter cohort of trauma patients whose characteristics and physiological variables are prospectively collected in a research registry.http://www.traumabase.eu/fr_FR). Clinical, biological and anamnestic data on every patient admitted to each of these three centers were prospectively collected in the database. The TraumaBase® group obtained approval for this study, including waived informed consent from the Institutional Review Board . The database was approved by the Advisory Committee for Information Processing in Health Research , and the French National Commission on Computing and Liberty .We conducted a retrospective multicenter observational study in three French regional level-1-designated trauma centers between May 2011 and July 2014. The three trauma centers progressively joined the TraumaBase registry between 2011 and 2012  transport were included in the study. Beaujon, Pitié-Salpêtrière and Bicêtre University Hospitals are three trauma centers in the Paris region. They manage the care of approximately 60% of patients with severe trauma in the Paris region (12 million inhabitants). The Paris emergency medical system and trauma management have been described elsewhere . PatientData collected in the prehospital setting included blood pressure, heart rate, peripheral oxygen saturation, trauma characteristics, Glasgow Coma Scale (GCS) score and vasopressor use. On arrival at the hospital, hemodynamic, respiratory and neurologic variables were recorded. Blood was immediately sampled for blood typing and analysis that usually  included blood gas, lactate, creatine kinase (CK) and coagulation tests. Maximum CK was the highest level of CK recorded during the ICU stay. The clinical severity was assessed using the following severity scores: Simplified Acute Physiology Score II (SAPS II) and Sequential Organ Failure Assessment (SOFA). The severity of traumatic injuries was assessed using the Abbreviated Injury Scale (AIS) 1998 version and the Injury Severity Score (ISS). The trauma-related injury severity score (TRISS) was also determined , 13. ConThe TraumaBase registry included all the patients admitted to the study centers. Data are entered manually by dedicated research technicians. Algorithms for consistency and coherence are integrated into the database structure. A core dataset of 35 variables for which the collection of data is considered mandatory was established prior to data collection in the registry. Completeness of data was monitored and regularly checked in each center. Data monitoring is performed by a central administrator.In the database, hemorrhagic shock is defined by the transfusion of at least four units of packed red blood cells (RBC) within the first 6 h , 15. Tra− 1 per 1.73 m2 in a population of young trauma patients [Renal function was assessed with serum creatinine variations according to the classification of risk, injury, failure, loss, end-stage of kidney disease (RIFLE)  and the patients . We defipatients .Quantitative variables were expressed as mean (SD) or median (25th–75th interquartiles) according to their distribution and categorical variables were expressed as counts (proportions). Prevalence of each AKI severity stage was reported in the whole cohort and in three subpopulations commonly reported in the literature: (1) severely injured patients presenting with an ISS ≥ 16 , (2) trat test for Gaussian variables, chi-square test for proportions and Mann Whitney test for non-normally distributed variables). We selected those factors significantly associated with AKI in the univariate analysis (p < 0.20) with two other clinical relevant factors: age, which is a well-known risk factor for AKI in ICU and direct/secondary transfer to a trauma center as a surrogate of time to specialized care, which is elsewhere reported to be linked to outcome [r > 0.8). If variables were correlated, we chose the most relevant one from a clinical/physiological perspective. Candidate variables were entered into stepwise multivariate logistic regression using a backward selection model. To better assess the performance of the predictive model, bootstrapping  analysis was used to estimate the odd ratios (OR) and 95% confidence intervals (CI). As an internal validation, bootstrapping provides robust estimates with low bias by addressing optimism and overfitting [Risk factors for stage I or F AKI  were evaluated in univariate analysis  .We built ROC curves for various thresholds of variables associated with AKI. Sensitivity, specificity, positive and negative predictive values (PPV and NPV), positive and negative likelihood ratios (PLR and NLR) and calculation of the AUC-ROC were reported for each variable. The best threshold was defined as the value maximizing the Youden index (sensitivity + specificity – 1). To investigate the association between AKI and ICU mortality, the factor AKI was entered into a stepwise logistic regression model with other factors associated with the outcome (death) in the univariate analysis. Missing data were not replaced and only complete cases were included in the multivariate models. Proportions of missing data and characteristics of patients with missing data were reported for each model. The two-sided level of significance was fixed at 5%. Results were analyzed using R open source software 3.1.1 (n = 3111) thereby expecting to provide robust estimates even in the case of severe AKI prevalence as low as 3%.The prevalence of severe AKI (stage I or F) is variable in cohort of trauma patients, with reported percentages ranging from 1 to 26% , 17, 18.There were 3488 trauma patients recorded in our database from June 2011 to July 2014. Data on renal function were available for 3111 patients. Characteristics of the 377 excluded patients are reported in Additional file n = 388) had early AKI, as they reached the creatinine peak within 5 days after trauma, while 4% of patients with AKI (n = 17) had late AKI (8 patients with AKI stage R and 9 patients with AKI stage F) and reached the creatinine peak after 5 days. Renal replacement therapy was used in 49 patients (1.6%).The prevalence of AKI in the overall population was 13% (CI 11.8–14.2) including 7% (CI 6.1–7.9) of patients with stage R, 3.7% (CI 3.0–4.4) of patients with stage I and 2.3% (CI 1.7–2.8) with stage F  and more blood product transfusion Table . Among vp < 0.0001). The CK peak value reached 977 U/L (383–2417) in patients with no AKI while it reached 2800 U/L (1032–7938) in patients with AKI stage R, I or F (p < 0.0001). When adding CK peak to the factors previously included in the multivariate model, it was significantly associated with AKI stage I or F (OR 1.041 (1.015–1.069) per step of 1000 U/mL, p < 0.001) and with AKI of all stages (OR 1.041 (1.010–1.073) per step of 1000 U/mL, p < 0.001) , 0.79 0.75–0.82), 0.75 (0.71–0.79), 0.72 (0.68–0.77) and 0.66 (0.62–0.71), respectively, to predict AKI stage I or F Fig. . The AUC–0.82, 0.p = 0.001) and OR = 1.943 (1.300–2.890) for AKI stage R, I or F (p = 0.001))  for AKI stage I or F , injury severity (ISS), renal trauma and delayed admission. Fourth, rhabdomyolysis severity (CK peak) was an additional independent risk factor for AKI. Fifth, AKI was independently associated with an increased risk of ICU mortality. To our knowledge, this is the largest multicenter cohort of trauma patients in which AKI risk factors were assessed. For the first time, in association with hospital variables collected early on admission, we report prehospital variables to predict the occurrence of AKI after trauma. This is all the more relevant that AKI has an early onset after trauma, thereby calling for its early prediction to direct treatment aiming to prevent AKI.The overall incidence of 13% of AKI is consistent with previous studies, though wide ranges of AKI proportions from 1 to 50% have been reported in trauma patients , 28, 29.The severity of trauma injuries (ISS) was associated with AKI in our study, which is inconsistently reported in other studies in the field of trauma , 5, 17. Patients directly transported to a trauma center were less likely to experience AKI than those who were secondarily admitted to the referral trauma center. The most common reasons for such transfers include injuries requiring specialized care that cannot be provided in the first hospital the patient was admitted to, thereby corresponding to under-triage. Early admission to the referral trauma center likely allows earlier hemorrhage control and injury care, preventing renal aggression. Surprisingly, age was not significantly associated with AKI. This has been reported elsewhere , 17 and Regarding AKI prediction, none of the variables associated with AKI performed satisfactorily to individually discriminate the occurrence of AKI stage I or F. However, by using prehospital and early hospital variables, our logistic regression model performed well to predict AKI stage I or F with an AUC-ROC of 0.85. The model for AKI all stages  performed worse, with an AUC-ROC of 0.80. By comparison, in a well-conducted study, Haines et al. recently used hospital variables to build a model predicting stage 2 or 3 AKI  classification) in trauma patients, with an AUC of 0.81, while their model predicting AKI of all stages had an AUC of 0.77 . Taking Several investigations have emphasized that AKI is independently linked to adverse clinical outcome in ICU patients  or in tr2. Nevertheless, the definition we used was reported to be more accurate to diagnose AKI in ICU patients than creatinine estimated by MDRD [There are several limitations to our study. This is a retrospective study, though data used in the study were prospectively collected and registered in a research database. Since blood lactate and CK were not systematically measured in trauma patients during the study period, missing values led us to exclude patients from the predictive models. However, the number of AKI patients in our sample far exceeded the number of events that is recommended (at least 10 events (10 patients with AKI) per candidate variable) when building a predictive model and therefore is expected to provide robust estimates . Data on by MDRD , especia by MDRD . Last, tAKI has an early onset and is independently associated with mortality in trauma patients. Its prevalence varies by a factor of 3 according to the severity of injuries and hemorrhage. Prehospital and data on risk factors collected early after hospital admission can be effective in the early prediction of AKI after trauma. Hence, studies aiming to prevent AKI should target patients at high risk of AKI and investigate therapies early in the course of trauma care.Additional file 1:French Vittel triage criteria. (DOCX 30 kb)Additional file 2:General characteristics of patients with missing data. (DOCX 23 kb)Additional file 3:Univariate analysis and stepwise logistic regression model including risk factors associated with the occurrence of AKI of all stages . (DOCX 32 kb)Additional file 4:Risk factors associated with the occurrence of AKI stage I or F and with the occurrence of AKI of all stages  in a stepwise logistic regression model including CK peak. (DOCX 20 kb)Additional file 5:Predictive performances of different models and variables for AKI stage I or F and for AKI of all stages . (DOCX 16 kb)Additional file 6:Assessment of AKI stage I or F and AKI of all stages  as risk factors associated with mortality in a stepwise logistic regression model. (DOCX 29 kb)


Neospora caninum, Liverpool isolate (NcLiv), used by our labs over the last few years as a base strain for genetic modification assays, with a Toxoplasma gondii knockout strain. This issue has been made public by our research groups during the retraction of another article (https://doi.org/10.1038/s41598-018-28052-2). Regarding the article herein referred, this issue has compromised a single set of experiments, which are displayed in In the original article, there was an error. We have recently discovered the contamination of a batch of All the other experiments of the originally published article were not affected by this error, since other parasite stocks/isolates were used, and the data is reproducible and sound.T. gondii's GRA24 (type II) using the NcGRA7 promoter, proper localization of the protein in the vacuoles of infected cells, compatible MAPK p38 phosphorylation and induction of differential IL-12 production; with the exception of the identification of biotinylated p38 MAPK by MS.In order to address this issue, we have repeated, with success, the experiments contained in N. caninum, since GRA24-p38 MAPK interactions had been extensively described elsewhere (DOI: 10.1084/jem.20130103).Although unfortunate, this experiment was a mere additional control of the ortholog gene expressed in We truly believe that scientific integrity is the basis of the advancement of knowledge.Results, section N. caninum-Triggered p38 Activation Is Induced by a Distinct Mechanism thanT. gondii's GRA24 Protein. Paragraph two has been removed and the following paragraph has been corrected to:A correction has therefore been made to the N. caninum GRA24-BirA* expressing tachyzoites did not retrieve biotinylated p38 MAPK within its results, we continued to investigate whether the mechanism behind p38 pathway triggered by N. caninum shares common features with those described for TgGRA24, BMDMs were infected for 30 min and 18 h by parental (NcLivΔHPT), TgGRA24+ N. caninum (NcLiv_GRA24) or type II T. gondii (PRU) tachyzoites. As seen in N. caninum tachyzoites would further enhance IL-12 production. For that purpose, cells were treated with p38 inhibitor SB203580 and infected with NcLivΔHPT, NcLiv_GRA24 or PRU tachyzoites. This assay demonstrated that all tested parasites induced similar cytokine production, as inhibition of p38 MAPK induced higher IL-12p40 production in all infected BMDMs, if compared to infected and untreated cells (N. caninum, demonstrating that the mechanisms herein reported—downregulation of IL-12 by activation of the p38 MAPK pathway by Neospora's antigens—are distinct from those previously described for T. gondii (Braun et al., “Although MS experiments with ed cells . These rThe corrected The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.


Rhodococcus peritonitis in PD patients.Galactomannan index (GMI) at a level higher than 0.5 provides high sensitivity and specificity for the diagnosis of fungal peritonitis. Here, we report the false-positive of GMI in peritoneal dialysis (PD) effluent (PDE) due to Rhodococcus colonies were 1.27 and 1.56, which were isolated from case #1 and #2, accordingly.GMI in PDE of case #1 and case #2 were 1.53 and 0.76, respectively, while serum GMI of both cases was less than 0.5. In addition, GMI from the specimens obtained directly from the stationary phase of Rhodococcus spp., especially in endemic areas.High GMI in PDE of PD patients is not specific just for fungal infections but may also be secondary to other infections, such as  Rhodococcus spp. There were 2 patients with rhodococcal infection who had orange stains inside their PD catheters. Both cases were mistakenly diagnosed as fungal peritonitis and resulting in initial treatment failure.Fungal peritonitis is a relatively uncommon but serious complication of peritoneal dialysis (PD) and is associated with a high risk of technique failure and mortality, especially in cases of delayed diagnosis. The galactomannan (GM) test is an easier technique for the detection of fungal cell wall components shedding out of the fungi during fungal growth and death . Indeed,Case #1 was a 71 year-old man with diabetic end-stage renal disease (ESRD) who was performing continuous ambulatory PD (CAPD) at Sunpasitthiprasong Hospital since 2013. On 30 July 2017 (day 0), he presented with acute abdominal pain and cloudy dialysate associated with a normal exit-site. The diagnosis of peritonitis was confirmed with a PDE leukocyte count of 2900 cells/μL of which 91% were neutrophils. The empirical therapy for bacterial peritonitis with intraperitoneal (IP) cefazolin 1 g together with ceftazidime 1 g IP once daily was commenced. Although the dialysate leukocyte gradually decreased, the leukocyte persisted at more than 100/μL with neutrophil predominance. The dialysate from day 0 failed to culture any organism. However, many orange spots were noticed inside the PD catheter on day + 8. GMI in the PDE and the sera were 1.53  and 0.39 , respectively. The provisional diagnosis of fungal peritonitis was made. Intravenous amphotericin B, 0.5 mg/kg/day was promptly started on the following day (day + 9), and the PD catheter was subsequently removed on day + 11. The PD catheter, serum, and drained PDE were submitted to the central microbiology lab for microorganism identification. The patient was transferred to hemodialysis (HD) following PD catheter removal. The patient continued to improve while having intravenous (IV) amphotericin and ceftazidime that were prescribed for a total of 2 weeks.Corynebacterium spp. The primary physician swapped the antibiotics to IV vancomycin, 1 g every 3 days, and IP amikacin, 25 mg loading dose followed by 12 mg once daily for a total duration of 2 weeks. During the follow-up period, salmon-pink colonies were observed inside the PD catheter and GMI in PDE from day + 10 was later reported positive at a level of 0.76 (< 0.5). Superimposed fungal peritonitis was suspected, although IP amphotericin B was added on day + 13, and the PD catheter was removed on day + 16. Amphotericin was later withdrawn after a return of culture report from the central microbiology lab on day + 19 demonstrated Rhodococcus equi. Temporary HD was performed during day + 17 to day + 56. The PD catheter was reinserted on day + 58, and the patient has remained well on CAPD without any subsequent peritonitis.Case #2, a 59 year-old man with a past medical history of diabetes and hypertension, was diagnosed with ESRD from diabetes. He commenced CAPD (4 exchanges/day) in January 2017 at Nakhon Pathom Hospital. While performing CAPD, he was able to maintain adequate dialysis small solute clearance and had never experienced peritonitis. On 16 January 2018 (day 0) the patient presented with cloudy dialysate, abdominal pain, poor appetite, and ultrafiltration failure, associated with a normal exit-site. The PDE cell count was 497 cells/μL with a neutrophil predominance (63%). He was initially diagnosed with CAPD-related peritonitis and received a combination of IP cefazolin and ceftazidime on the same day resulting in partial resolution of the abdominal pain. However, the PDE leukocyte count that was repeated on day + 3 indicated an increased count of 2080 cells/μL (85% neutrophils), while the dialysate culture from day 0 later yielded Rhodococcus spp. and Rhodococcus equi was identified by modified AFB and the biochemistry assay from case #1 and #2, respectively. The GMI in the specimens obtained directly from colonies of Rhodococcus were 1.27 (Case #1) and 1.58 (case #2), which were above the cut-off value (≥ 0.5) using a GM test kit .At the central microbiology lab, the removed catheters and the PDE were submitted for culture in several media. Interestingly, the orange colonies on tryptic soy agar (TSA) and chocolate agar were observed at 72 h, but no colony growth on SDA Fig. . RhodocoRhodococcus, a gram-positive filamentous bacteria which belongs to phylum Actinobacteria, causes disease only in immune-compromised hosts [Rhodococcus is sensitive to several antibiotics [ed hosts . This ored hosts , 7. Fungibiotics , early cibiotics . Thailanibiotics .Rhodococcus does not express GM, it contains a substantial amount of arabinogalactan [AG: a linear galactan of galactofuranose  residues + discrete arabinan side branches which contain arabinofuranoses) and lipoarabinomannan (LAM: lipoglycan core + mannan side chains) of which both are bound to branched long-chain lipids termed mycolic acids. The galactan of R. equi contains a mixture of (1 → 3), (1 → 5), and (1 → 6) linkages [Rhodococcus is on the backbone of AG [The cell envelop of the linkages . On the linkages , 10. Botlinkages , 10. Howne of AG .Rhodococcus is initially sensitive to cephalosporins, it is not recommended in clinical practice because of the potential development of late resistance [Rhodococcus peritonitis due to the unpredictable response of this organism to treatment [Rhodococcus peritonitis with 2 weeks of antibiotics combined with the PD catheter removal. Case #2 was treated with IP vancomycin plus amikacin. Meanwhile, case #1 was successfully treated by IP ceftazidime. The Rhodococcus infections in both of these cases either responded to the empirical treatment or PD catheter removal.Since there are very few case reports of rhodococcal peritonitis, experiences of the preferred treatment are limited. A combination of antibiotics is the mainstay of treatment, especially antibiotics with intracellular activity against the organism. Although sistance . The dursistance . Removalreatment . Our 2 cRhodococcus infection. Thus, various culture media and polymerase chain reaction should be used to confirm the results of high GMI in PDE, especially in endemic areas of rhodococcal infection.In conclusion, false-positive GMI results may occur with


The whole-volume apparent diffusion coefficient (ADC) histogram analysis generated ADCmean, skewness, kurtosis, and entropy values from bilateral parotid glands. Continuous variables were compared using an independent two-sample t test, and categorical variable compared using the Fisher’s test between the two groups. Receiver operating characteristic (ROC) analysis was used to evaluate the diagnostic performance of the indexes. Fisher’s tests demonstrated that some clinical indexes and MR morphology grades differed significantly between patients with SS and patients suspected of having the disease . The parotid entropy value of patients with SS was significantly higher than that of patients suspected of having the disease (P < 0.001). Among MR parameters, entropy combined with kurtosis performed the best in differentiating patients with SS from those suspected of having SS (area under the ROC curve = 0.955). A whole-volume ADC histogram analysis might provide a series of parameters that reflect tissue characteristics.At present, no gold standard for diagnosing Sjögren’s syndrome (SS) is available in clinical practice. The 2002 American–European Consensus Group classification criteria are used to diagnose SS. Clinically, it is challenging to distinguish patients with SS from suspected patients undergoing different therapies. A total of 52 patients with SS and 24 patients suspected of having the disease prospectively underwent 3.0-T magnetic resonance (MR) scanning, including diffusion-weighted imaging ( Imaging the injury to the salivary glands is critical for diagnosing SS.Sjögren’s syndrome (SS) is a chronic autoimmune systematic disease, causing direct damage to exocrine glands. At present, a gold standard diagnostic assay for SS is not available in clinical practice. According to the 2002 American–European Consensus Group (AECG) classification criteria3 and technetium-99m pertechnetate scintigraphy results in exposure to radionuclides5. Ultrasonography and computed tomography (CT) provide qualitative evaluation; however, CT involves the use of ionizing radiation7. Magnetic resonance (MR) imaging (MRI) is increasingly used because it is noninvasive and does not require radiation exposure9. However, MRI, including MR sialography, proved to be insensitive to early injury of the parotid glands11.Although X-ray sialography and sialoscintigraphy have been added to the AECG criteria as imaging modalities, X-ray sialography is invasive13, dynamic contrast enhancement (DCE)14, and intravoxel incoherent motion (IVIM)15. DWI has been the most widely used modality because of its ability to noninvasively quantify water molecular diffusion by calculating apparent diffusion coefficient (ADC) values16. Diagnosing SS using DWI has been investigated by several groups. Regier et al. demonstrated that ADC values increase in early-stage SS and decrease in advanced-stage SS, based on MR sialography grading. They declared that DWI could display functional changes in the parotid gland affected by SS and might be a useful tool for differentiating between the early and advanced disease stages if combined with MR sialography17. Ding et al. reported that when using multiple small regions of interest (ROIs), the signal intensity ratio (SIR) values of the parotid versus spinal cord and parotid ADC values in DWI differed significantly between SS patients and non-SS patients 12. The receiver operating characteristic (ROC) analysis showed that ADC values are less diagnostically effective than SIR values. Multiple small ROIs could better reflect the heterogeneity of the parotid glands, presenting a higher diagnostic value compared with traditional ROI. However, Ding et al. did not conduct a ROC analysis for patients with SS and patients suspected of having SS, probably due to the small sample size (only five patients were suspected of having SS)12. Xu et al. also confirmed the use of ADC values in diagnosing a patient with early-stage SS13.Therefore, various functional MR sequences were applied to evaluate parotid injury in SS, such as diffusion-weighted imaging (DWI)18, or edema and neovascularization in inflammatory diseases20. The histological analysis of early parotid injury typically reveals a loss of normal gland architecture and lymphocytic and plasma cell infiltration22. ADC texture analysis was used in our study to evaluate the disease activity of patients with primary SS using MRI and clinical and laboratory indicators23. To date, whole-volume ADC histogram analysis had never been reported in diagnosis of SS.ADC histogram analysis could provide many parameters reflecting tissue characteristics, for instance, hypoxia, angiogenesis, and cellular proliferation in cancer lesionsHence, his study aimed to explore the use of parotid whole-volume ADC histogram analysis to distinguish patients suspected of having SS from those with the disease.The MR morphology grades of bilateral parotid glands were found to be consistent in all patients and were valued at 0 for patients suspected of having SS. The MR morphological grades were 0, 1, 2, and 3 in 21 (40.4%), 11 (21.2%), 12 (23.1%), and 8 (15.4%) patients with SS, respectively. The MR morphology of the bilateral parotid glands were consistently grade 0 in all volunteers.P = 0.008 and 0.039, respectively). However, no significant differences were observed between patients with SS and patients suspected of having SS (P = 0.625).There were no significant differences of ADC values based on one ROI between bilateral parotid glands. Hence, we calculated average ADC values of bilateral parotid glands as the final one for each patient. Parotid ADC values from volunteers were significantly higher than those of patients with SS or patients suspected of having SS . The parotid entropy values of healthy volunteers were significantly lower than those of patients with SS (P < 0.001), and the entropy values of healthy volunteers were significantly lower than those of patients suspected of having SS (P < 0.001). Scatterplots of entropy in patients with SS, patients suspected of having SS, and healthy volunteers are shown in Fig. Parotid entropy values of patients with SS were significantly higher than those of patients suspected of having SS P < 0.00. The parmean, skewness, and kurtosis values between the two groups. Detailed results are shown in Table mean and skewness values of volunteers were significantly higher than those of patients with SS . The parotid kurtosis values of healthy volunteers were significantly lower than those of patients with SS (P = 0.004). The parotid ADCmean, skewness, and kurtosis values of volunteers were significantly higher than those of patients suspected of having SS .No significant differences were observed in the ADCP = 0.036 and <0.001, respectively).The parotid kurtosis and entropy values of patients with SS of grade 0 were significantly higher than those of patients suspected of having SS , and that parotid entropy correlated positively with MR morphology grade . Parotid skewness and kurtosis showed no significant correlation with the MR morphology grade .The Spearman correlation test showed that the parotid ADC and ADCb = 1000 s/mm2), corresponding ADC maps, and ADC histograms of both groups.Figure P = 0.906). Kurtosis combined with entropy yielded a sensitivity value of 86.5%, a specificity value of 91.7%, an accuracy level of 88.2%, and an AUC value of 0.955 (P < 0.001) (Table P < 0.05).ROC analysis showed that entropy presented the largest area under the ROC curve (AUC) of 0.924 Table . The gooWith a cutoff value of 4.825, parotid kurtosis could differentiate patients with SS of grade 0 from patients suspected of having SS with a sensitivity of 57.1%, a specificity of 83.3%, and an accuracy of 71.1% (AUC = 0.683). With a cutoff value of 6.075, parotid entropy could differentiate patients with SS of grade 0 from patients suspected of having SS with a sensitivity of 85.7%, a specificity of 75.0%, and an accuracy of 80.0% (AUC = 0.839).ADC and all ADC histogram parameters of parotid glands showed excellent intraobserver and interobserver agreements with intraclass correlation coefficients (ICCs) of 0.898–0.986 Table . The intIn this study, the differences in the whole-volume ADC histogram analysis on the bilateral parotid glands were compared between patients with SS and patients suspected of having SS. The study established the role of ADC histogram parameters in differentiating SS in the suspected group for the first time. It could help clinicians make different treatment plans.et al.12 also reported that the ADC value could hardly differentiate patients with SS from non-SS patients (patients suspected of having SS + volunteers). In clinical practice, some patients suspected of SS may be diagnosed with dry mouth or other symptoms related to xerophthalmia and xerostomia; also, some of them have immunity-related complications, such as undifferentiated connective tissue disease or early-stage SS. Differentiation between patients with SS and patients suspected of having SS can prevent overtreatment of patients.Parotid ADC values were compared among patients with SS, patients suspected of having SS, and healthy volunteers. The ADC values of patients with SS were significantly lower than those of healthy volunteers, probably due to damage to the parotid glands. Nevertheless, no significant difference was found between patients with SS and patients suspected of having SS. Ding mean, skewness, kurtosis, and entropy values based on ADC histogram and texture analysis between patients with SS and healthy volunteers. Only the entropy differed significantly between patients with SS and patients suspected of having SS. Kurtosis describes the sharpness and tails of ADC distribution, and skewness characterizes the degree of asymmetry from the normal distribution24. However, our study did not detect significant differences of skewness and kurtosis between patients with SS and patients suspected of having SS. Entropy is a measure of the disordered state of the system in statistical thermodynamics. The ADC entropy reflects the heterogeneity of various tumors, such as those of lung, breast, esophageal and colorectal cancers, which is closely related to tumor malignancy, patient survival, and prognosis28. The ADC entropy can also be used in evaluating inflammatory diseases. For instance, Makanyanga et al. indicated that the entropy could reflect bowel activity in Crohn’s disease through image heterogeneity and complexity19. Sidhu et al. suggested that ADC entropy might partially reflect changes in perfusion difference29. Perfusion changes in the parotid glands of patients with SS have been demonstrated in studies using DCE-MRI14 and IVIM15. The ADC entropy of SS patients was significantly higher than that of patients suspected of having SS and healthy volunteers. It was speculated that the microstructural and perfusion heterogeneity of patients with SS was more pronounced than that of patients suspected of having SS and healthy volunteers.Significant differences were observed in the ADC11. According to the grading system of Makula et al.11, patients with SS who have no morphological changes in the parotid glands were graded as 0. The present study found that both kurtosis and entropy could differentiate patients with grade 0 SS from patients suspected of having SS. Additionally, the diagnostic performance of entropy was relatively better, providing a new way to address the clinical challenge.The parotid glands of patients with SS showed characteristic morphological changes on MRImean correlates negatively with the MR morphology grade, which was in line with a previous study of diffusion kurtosis imaging (DKI)30 in SS. It also showed that parotid entropy correlated positively with the MR morphology grade. The authors speculated that as the MR morphology grade increased, heterogeneity in the microstructure and microperfusion of the parotid glands also increased29.This study found that the parotid ADCTo better distinguish patients with SS from patients suspected of having SS, binary logistic regression modeling of all parameters was performed. The optimal combination of kurtosis and entropy yielded the highest AUC of 0.955. Since traditional X-ray sialography involves an invasive process with radiation, parotid MRI with an ADC texture analysis may serve as a replacement in some clinical settings.15. Secondly, only X-ray sialography was used for the salivary test. Thirdly, only two b values were used in DWI, and the perfusion effect could not be eliminated, affecting measurement of the ADC. Fourthly, bilateral submandibular glands were not taken into consideration. Finally, the study was performed in a very homogenous setting with only one scanner in a single center. However, it is believed that these parameters could be reproduced across different scanners because the diagnostic performance of DWI did not differ significantly between 1.5-T and 3.0-T MR scanners32. Of note, according to this and previous studies, a b value of 1000 s/mm2 was recommended for DWI13.There are some limitations in our study. Firstly, our sample size was small but still larger than previous studiesIn conclusion, the present study confirmed that parotid whole-volume ADC histogram analyses, especially entropy, had great potential in diagnosing SS.2, with one focus defined as an aggregation of ≥50 mononuclear cells), and X-ray sialography (according to Rubin and Holt scores) to establish the diagnosis; and (3) a willingness to undergo parotid MR scanning. Exclusion criteria included a medical history of radiotherapy to head and neck area, infection of hepatitis C virus (HCV), lymphoma, acquired immunodeficiency syndrome (AIDS), sarcoidosis, a history of drug use , and any association with other autoimmune diseases . Patients were also excluded if contraindications to MR examination were present (such as cochlear or cardiac pacemaker implantation).The ethics committee of Nanjing Drum Tower Hospital approved this study. All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent was obtained from all participants. For human participants under the age of 18 years, informed consent must have been obtained from a parent and/or legal guardian. From July 2015 to December 2016, patients exhibiting xerostomia or xerophthalmia were included consecutively and prospectively based on the following criteria: (1) an initial suspected diagnosis of SS according to symptoms; (2) a willingness to undergo laboratory tests (anti-SSA and anti-SSB using enzyme-linked immunosorbent assay), ocular tests , labial gland biopsy of the lower lip  were enrolled. According to the AECG criteria, 52 patients  were diagnosed with SS. The remaining 24 patients  with a suspected diagnosis of SS due to the symptoms of thirst and/or xerophthalmia did not fulfill the AECG criteria, including 8 patients with 0 positive findings, 6 patients with 1, and 10 with 2. All 76 patients had clinical symptoms of xerostomia or xerophthalmia and underwent all the listed examinations, including a serological test, Schirmer’s test, and/or the Rose Bengal test, lip biopsy, sialography, conventional imaging and DWI. Table During the same period, healthy volunteers were enrolled according to the following criteria: (1) no presentation of symptoms, signs, or history of mouth, eye, or salivary gland disease; no radiotherapy history for head or neck area, infection of HCV, AIDS, lymphoma, or sarcoidosis; and no history of drug use ; (2) willingness to undergo serological tests (anti-SSA and anti-SSB) and Schirmer’s test; and (3) willingness to undergo parotid MR examination and a lack of contraindications. A total of 42 healthy volunteers  were enrolled.All participants were scanned head first on a platform of 3.0T MR scanner  by using a 16-channel head&neck coil in a supine position. MR scan covered the skull base to the submandibular glands, thus covering the whole volume of the bilateral parotid glands. The participants were told to refrain from swallowing during the scanning procedure.2; field of view (FOV) = 240 × 240 mm2; slice thickness = 4.25 mm; intersection gap = 0.67 mm; number of signal averages (NSA) = 2; and bandwidth = 287.9 Hz/pixel]; axial T2-weighted (T2W) turbo spin-echo sequence ; axial T2-weighted short tau inversion recovery (STIR) sequence ; and coronal T2-weighted STIR sequence . The DWI was performed with a single-shot turbo spin-echo (TSE) sequence . The diffusion time and duration of the motion probing gradient (MPG) were 39.9 and 23.1 ms, respectively, Single-shot TSE-DWI used a 180° radiofrequency refocusing pulse for each measured echo, explaining the reduction of the susceptibility artifact. The phase-encoding direction was anterior to posterior. Spectral presaturation with inversion recovery fat-suppression was used for the DWI sequence. Three motion-probing gradients along the readout, phase-encoding, and slice-selection directions were adopted. The DWI acquisition period took approximately 3 min and 48 s, and the total scan period lasted approximately 17 min and 47 s. All participants successfully underwent MRI tests without experiencing any adverse effects or discomfort.MR sequences included axial T1-weighted imaging, T2-weighted (T2W) imaging, axial and coronal fat-suppressed T2W imaging, and DWI. The maximum gradient strength was 45 mT/s and slew rate of the MR scanner system was 200 mT/m/s. MR sequences were as follows: axial T1-weighted (T1W) turbo spin-echo sequence [parameters: repetition time (TR) = 400–675 ms; echo time (TE) = 18 ms; in-plane resolution = 0.65 × 0.75 mmThe MR images were transferred to the workstation . Two radiologists , with two and ten years of experience with head and neck radiology respectively, independently performed the measurements and interpretations independently. The radiologists were blinded to all clinical and laboratory records.et al.11, which reads as follows: grade 0, normal homogeneous gland parenchyma; grade 1, fine reticular or small nodular structures, nodule diameters <2 mm; grade 2, medium nodular patterns, nodule diameters between 2 and 5 mm; and grade 3, coarsely nodular, nodule diameters >5 mm. Grade 0 was treated as negative while grades 1–3 were considered as positive for diagnosing SS on MRI. A consensus was reached through discussion when divergences in the results obtained by the two radiologists occurred.Injury degree of bilateral parotid glands was assessed separately with T1W, T2W, and T2-STIR images based on the scale developed by Makula b × ADC). DWI (b = 1000 s/mm2) presenting with the largest slice of parotid glands was adopted, and the ROI was drawn by freehand to cover a unilateral parotid gland as large as possible  while maintaining a distance of 1 mm from the boundary and avoiding the retromandibular vein and external carotid artery within the gland. The ROIs were automatically copied to the ADC maps, and mean ADC value within the ROI was calculated. The mean value for bilateral parotid glands was the obtained.ADC maps were produced via DWI using the monoexponential model: S = S0 × exp (−33. The ROIs were drawn by freehand to cover the parotid gland as large as possible on each DWI slice (b = 1000 s/mm2). The ROIs were automatically copied to the ADC maps. After selecting all ROIs of a unilateral parotid gland , a volume of interest (VOI) was composed  to calculate the following parameters with following formulas, where X indicates the set of all ADC values, N is the number of sampled ADC pixels,X, and P(i) is the frequency of voxels with intensity i divided by N.(i)mean is the mean value of all ADC values within the VOI, ADC(ii)Skewness is histogram asymmetryaround the mean,(iii)Kurtosis is a measurement of histogram sharpness, (iv)Entropy is the distribution of gray levels over the VOI, A whole-volume ADC histogram analysis was performed by using an in-house software  as described in our previous studiesMeasurements made by each radiologist were separately recorded for interobserver agreement analysis. The averaged value of the two measurements was calculated as the final value for each subject. One of the radiologists (Chen Chu) repeated all measurements one month later to perform intraobserver agreement analysis.No obvious artifacts were found on DWI owing to quality control and quality assurance. Measurements were performed in all of the patients because of the lack of too much diffusion distortion. Single slices were not excluded from the whole-volume analysis on any of the occasions because of distortion artifacts.P > 0.05), which were recorded as mean ± standard deviation, while qualitative data were recorded as ratios. Continuous variables were compared using an independent two-sample t test, and categorical variables were compared by Fisher’s test. A ROC analysis was applied to assess the diagnostic performance of parotid ADC histogram parameters. The maximal Youden index (sensitivity + specificity − 1) was calculated to establish cutoff values. A binary logistic regression analysis based on a backward stepwise selection procedure was carried out to identify independent predictors for differentiating patients with SS from patients suspected of having SS. Goodness-of-fit Hosmer–Lemeshow test was used to assess multivariate model calibration, and graphical decile group probability was determined from a calibration plot. AUCs were compared with McNeil test. The Spearman rank correlation was performed to evaluate correlation between MR morphology grade and ADC histogram parameters. Interobserver agreement for MR morphology assessment was evaluated by calculating kappa coefficient. Intraobserver and interobserver agreements for ADC parameters measurement were assessed with ICCs . We performed all statistical analyses with SPSS . A two-tailed P value < 0.05 was treated as statistically significant.Kolmogorov–Smirnov test confirmed the normal distribution of quantitative data (all


Immune-related enterocolitis (irEC) is the most common serious complication from checkpoint inhibitors (CPIs). The current front-line treatment for irEC, high-dose corticosteroids (CS), have significant side effects and prolonged therapy may reduce CPI-anti-tumor activity. Early addition of TNF-α inhibitors such as infliximab (IFX) may expedite symptom resolution and shorten CS duration. Thus, we conducted the first retrospective study, to our knowledge, evaluating symptom resolution in patients with irEC treated with and without IFX.Data were collected from the medical records of patients diagnosed with irEC. The primary endpoint was time to symptom resolution for irEC for cases managed with IFX plus CS (IFX group) versus CS alone (CS group). Duration of CS, overall survival (OS), and time to treatment failure (TTF) were secondary endpoints.p < 0.001), median times to diarrhea resolution  and to steroid titration  were shorter in the IFX group than in the CS group without a negative impact on TTF or OS. Total steroid duration  was numerically lower in the IFX group.Among 75 patients with irEC, 52% received CS alone, and 48% received IFX. Despite higher grade colitis in the IFX group  contains supplementary material, which is available to authorized users. Checkpoint inhibitors (CPIs) have improved overall survival (OS) for patients with various malignancies –5. CurreIrEC extensively affects the gastrointestinal tract. If irEC is not treated promptly, it can lead to bowel perforation, sepsis, and death . AlthougThe recommended front-line treatment for irEC is high-dose corticosteroids (CS)  , 10–12. Adult cancer patients treated with CPIs  between January 1, 2012, and June 30, 2017, were identified by review of the institutional pharmacy database at MD Anderson Cancer Center. Included in our analysis were patients for whom there was adequate documentation of the timing of our primary endpoints in the medical records. Patients who received IFX and CS  as well as those who received CS alone were identified. Their records were then reviewed to identify the indication for those treatments, and patients who received IFX and/or CS for a reason other than irEC were excluded from further analysis. All treatments for irEC were per the treating oncologist’s discretion. Baseline characteristics at the time of irEC diagnosis, including age, sex, co-morbidities, cancer type, and CPI regimen, were collected. Stool studies for infectious cause, endoscopic and radiographic evaluations, immunosuppressant regimens, CS duration, number of IFX doses, and treatment side effects were documented.In this study, irEC was defined as diarrhea requiring immunosuppressants in the setting of CPI therapy and was graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0 based solely on diarrhea. CTCAE classify and grade diarrhea and colitis as two separate entities. In the setting of CPI therapy, diarrhea is a widely accepted surrogate for irEC and is treated as presumed irEC . We gradThe primary endpoint, time to symptom resolution, was defined in two ways: the time from initiation of IFX and/or CS to either (i) complete resolution of diarrhea (absence of diarrhea) or (ii) initiation of steroid titration due to improvement of irEC symptoms as indicated by documentation in the electronic medical record. Although the timing of initiation of steroid titration depends on the physician’s discretion, we considered this co–primary endpoint another surrogate for resolution of colitis symptoms. Among the secondary endpoints was CS treatment duration, defined as the time from initiation of IFX and/or CS to the last dose of CS.To assess the relative impact of these immunosuppressive therapies on CPI clinical activity, we evaluated OS and time to treatment failure (TTF), defined as the time from the initiation of CPI to oncologic therapy change due to disease progression among patients with stage IV melanoma only.Patient characteristics were reported as percentages for categorical variables and as medians for continuous variables. Categorical and continuous variables were compared between groups by Fisher exact test and Kruskal-Wallis test, respectively. The log-rank and Renyi tests were used to compare survival curves between treatment groups. Forest plots based on results from gamma generalized linear models (with log link function) were used to display treatment differences regarding the time to diarrhea resolution and time to steroid titration within subgroups of given variables. For each subgroup, the treatment multiplicative effect and its 95% confidence intervals (CI) were estimated. Safety assessments were descriptive in nature. Statistical analyses were performed using R version 3.3.1. All statistical tests used a significance level of 5%. No adjustments were made for multiple testing.A total of 75 patients with irEC were included in the study Fig. . Of thesClostridium difficile infection was found in three patients in each group. Endoscopic evaluation was performed in 42 (56%) patients, and biopsy-proven colitis was found in 37 (49%). In the 5 patients who underwent endoscopy without a tissue diagnosis of colitis, there was either clinically evident colitis on endoscopy or reports were unavailable. Pathology findings consistent with colitis were seen in 20 (56%) patients in the IFX group and 17 (44%) patients in the CS group. Computerized tomography was performed in 42 (56%) patients, and wall thickening was present in 21 (28%). In the IFX group, one to three doses of IFX were administered; 26 (72%) patients had one dose, eight (22%) patients had two doses, and two (6%) patients had three doses. IFX was administered within a median of 8 days after CS initiation. Every patient had CPI therapy discontinued at the time of diagnosis of irEC. There were 18 patients re-challenged within 6 months of completing steroids and/or infliximab, 12 patients (33%) in the IFX group and 6 patients (15%) in the CS group.The clinical characteristics of the irEC cases are presented in Table p = 0.077). Initial CS therapy alone did not seem to impact the severity of irEC in the IFX group. In fact, the grade of colitis significantly worsened from the onset of diarrhea to the time of IFX administration; grade 3/4 colitis was seen in 86% of the IFX group (by the time of IFX initiation) but only 34% of the CS group (p < 0.001)  than in the CS group (34%) (1) Table .Table 2Gp < 0.001) and to steroid titration  compared with the CS group  than did the CS group  in the IFX group and 12.5 months (95% CI 5.8 months–not reached) in the CS group   and the CS group  than with agents targeting the PD-1/PD-L1 axis  , 10, 11.p = 0.150) without a negative impact on TTF or OS in the patients with metastatic melanoma. Most patients only required one dose of IFX (72%) to achieve symptom resolution. The recommended front-line treatment for grade 3/4 irEC is high-dose CS [In this study, IFX use was associated with a numerically shorter median duration of steroid exposure  due to insufficient enrollment further highlights the relevance of these data. It is important to identify more targeted approaches to treat irEC that mechanistically do not inhibit the anti-tumor immune response. However, until newer approaches for irEC are available, we recommend the early addition of IFX for irEC.Additional file 1:Figure S1. Kaplan-Meier analysis of time to treatment failure in patients with stage IV melanoma. Median TTF was 9.0 months (95% CI 5.6 months–not reached) in the IFX group and 12.5 months (95% CI 5.8 months–not reached) in the CS group. Median follow-up 26 months. (DOCX 48 kb)


Mouse models of Alzheimer’s disease (AD) exhibit marked differences in life expectancy depending on their genotype and sex. The assessment of frailty could provide a measure of healthspan to facilitate comparisons between different AD models. We used a validated mouse frailty index (FI) assessment tool to explore genotype and sex differences in lifespan and healthspan of 3xTg-AD mice and their B6129F2 wild-type (WT) controls. This tool is based on an approach commonly used in people and quantifies frailty by counting the accumulation of age-related health deficits. The number of deficits in an individual divided by the total number measured yields an FI score theoretically between 0 and 1, with higher scores denoting more frailty. Male 3xTg-AD mice aged 300–600 days had higher FI scores (Mean FI = 0.21 ± 0.03) than either male WT (Mean FI = 0.15 ± 0.01) or female 3xTg-AD mice (Mean FI = 0.10 ± 0.01), and the elevated frailty scores were accompanied by parallel increases in mortality. Frailty increased exponentially with age, and higher rates of deficit accumulation elevated mortality risk in all groups of mice. When mice were stratified by FI score, frailty predicted mortality, at least in females. Therefore, the mouse clinical FI provides a valuable tool for evaluating healthspan in mouse models of AD with different lifespans. Alzheimer’s disease (AD) is the most common type of dementia and its prevalence is increasing as the population ages  is a commonly used measure of overall health status and resilience as it is considered a measure of a person’s biological age, as distinct from their chronological age  and B6129SF2 WT mice  were bred at Dalhousie University from parents originally purchased from the Jackson Laboratory . Mice were weaned at 21 days of age, and housed in groups of 2–4 same-sex littermates in plastic cages (18.75 × 28 × 12.5 cm) with a PVC tube (4 cm diameter × 7 cm length) for enrichment, woodchips for bedding and metal wire covers. Animals were maintained on a reversed 12:12 light–dark cycle with lights off at 9:30 am, and ad libitum access to food (Purina #5001) and water. Frailty testing was completed during the dark phase of the light-dark cycle. All experimental procedures were conducted in accordance with the guidelines published by the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Laboratory Animals (#15-099).The 3xTg-AD mice .Four groups of mice were compared in these experiments: female WT, male WT, female 3xTg-AD and male 3xTg-AD. Mice were followed over their lifespan for survival. Date of death was determined as the day a mouse was found dead or determined to be moribund by a veterinarian. Some mice were used for separate experiments and these were included in the longevity analysis as censored data. For some mice the date of death was unknown, and these mice are included in frailty analyses but excluded from survival analyses  and SigmaPlot . P values less than 0.05 are considered significant.Data are shown as mean ± SEM unless otherwise indicated. Kaplan Meier (KM) curves were plotted to show survival probabilities over time in genotype and sex groups. Male and female TG and WT mice were also divided into low and high FI score groups (<0.21 and ≥0.21), and survival for each genotype and sex group was plotted on KM curves. Log rank analyses were used to determine if there were differences between the KM curves. The correlation between FI score, or the natural log of FI score, and age was determined for each group with Pearson correlation statistics. Mean FI scores, age at FI score and lifespans were compared between groups using a 2-way analysis of variance (ANOVA) for genotype and sex. An ANCOVA of sex/genotype group with age as a covariate was used to compare the slopes of ln FI vs. age. Mean FI scores were determined for each genotype and sex group in three different age groups: 0–300 days, 300–600 days and more than 600 days old. A 3-way ANOVA was used to determine the effect of genotype, sex and age on FI scores. Two-way ANOVAs were used to determine the effect of genotype/sex, age and the interaction of these variables on FI scores. Bonferonni p < 0.001). Pairwise comparisons show that male 3xTg-AD mice have lower survival probability than male WTs (p < 0.001), but there was no difference in survival probability between female 3xTg-AD mice and WT mice. In terms of sex differences, there was no significant difference between the curves for WT male and female mice, but male 3xTg-AD mice had shorter lifespan than female 3xTg-AD mice (p < 0.001).KM survival curves were plotted to determine differences in survival probability for male and female 3xTg-AD and WT mice Figure . Log ranF = 16.03, p < 0.001), sex  = 3.79, p < 0.05) and their interaction  = 8.44, p < 0.01). Post hoc analyses showed the same trend as the KM curves, with male 3xTg-AD mice having a significantly lower mean lifespan than male WT, and female 3xTg-AD mice  and maximum lifespans are summarized for each group in Table F,243 = 16The MCFI was scored in 3xTg-AD and WT male and female mice. The data that were used to construct the MCFI, grouped by systems, are presented in Supplementary Table F = 3.163, df = 3,260, p < 0.05). Post hoc pairwise comparisons showed that male 3xTg-AD mice have a significantly higher rate of deficit accumulation (0.035) than female 3xTg-AD (0.031) and female WT mice  = 32.14, p < 0.001), age group  = 6.59, p < 0.001) and the interaction of sex and genotype  = 6.59, p < 0.01). Two way ANOVAs of age group and either sex or genotype showed that, for all groups, mean FI scores increased with increasing age  = 5.19, p < 0.05), but not females. Male 3xTg-AD mice were more frail than WT males at 300–600 days of age. A 2-way sex and age ANOVA showed that male 3xTg-AD mice were more frail than females at all ages  = 23.55, p < 0.001, Figure F = 9.56, p < 0.01, Figure To analyze sex and genotype differences in FI scores over the lifespan, mice were binned into three age groups, and mean FI scores were calculated for each sex/genotype group at each age Figure . There wp < 0.05; F WT: p < 0.01; Figures To determine the correlation of FI score with survival probability, each group of mice was divided into those with high and low FI scores. Days to death post FI score measurement were plotted against survival probability in KM curves Figure . OverallThis study was the first to explore the relationship between genotype, sex and frailty in a mouse model of AD. Male 3xTg-AD mice had higher FI scores than either male WT or female 3xTg-AD mice, and these differences in frailty were accompanied by differences in mortality. Frailty increased exponentially with age in all groups, and higher rates of deficit accumulation corresponded to increased mortality risk. Finally, mortality differences in female 3xTg-AD and WT groups were revealed when groups were stratified by FI score, with higher FI scores predicting higher mortality risk.Male 3xTg-AD mice were shorter lived than male WT mice but this genotype difference in lifespan was not seen in female mice. Additionally, both male 3xTg-AD and WT mice had higher FI scores than the corresponding female groups. Previous work in this AD mouse model has shown this sex difference in mortality risk, with male, but not female, 3xTg-AD mice showing much shorter lifespans than WT controls  also corresponded to greater mortality, with male 3xTg-AD mice showing the highest mortality rate, and female WTs the lowest. This shows, for the first time, the importance and value in measuring not just lifespan but also overall health status in studies of longevity and aging in transgenic mouse models.In addition to sex differences in frailty in the 3xTg-AD mice, we also found that WT males had higher frailty scores than WT females in mice over 300 days of age. This higher baseline frailty in the males may explain why their mortality is generally more impacted by the AD mutations than females  in the male high FI groups reduced the power to detect changes in mortality risk for these mice.The neuropathological changes in brains of AD patients show a gradual increase over age and these changes have been classified into a series of seven stages , to reduce the number of items in the FI and identify latent variables. Typically, this approach is resisted as a matter of principle in constructing an FI. That is because, although it is possible to do, it detracts from the central idea that there are different ways to become frail and each of them is valid for the person or mouse who becomes frail in that way. Indeed, several lines of evidence show that the dimensionality reduction that is achieved by a single variable—the FI—is preferable to a PCA that is likely to yield more variables (Song et al., In summary, this study was the first to use the MCFI in a transgenic AD mouse model. The MCFI showed similar characteristics to FIs measured in other studies in rodents and humans (Whitehead et al., In conclusion, we found significant sex differences in both the lifespan and healthspan of 3xTg-AD mice. Male 3xTg-AD mice were more frail than male WT and female 3xTg-AD mice, and this was accompanied by increased mortality. Frailty increased exponentially with age in all mice, and higher rates of deficit accumulation corresponded to increased mortality. Stratification of female mice by frailty scores showed that higher MCFI scores were related to increased mortality. These results show the importance of considering the overall health status of mice, not just their chronological age. The MCFI is a valuable measure of healthspan for use in AD animal studies.AK wrote the manuscript. SS collected the data. AW organized data and edited the manuscript. EF and NF organized data. SH and RB supervised the project and edited the manuscript.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.


Background: The study was designed to explore any beneficial effect of Ocimum sanctum (Linn) (OS) in experimental pulmonary hypertension (PH) in rats. OS is commonly known as “holy basil” and “Tulsi” and is used in the Indian System of Medicine as antidiabetic, antioxidant, hepatoprotective, adaptogenic, and cardioprotective. Methods: Monocrotaline (MCT) administration caused development of PH in rats after 28 days and rats were observed for 42 days. Treatments  were started from day 29 after the development of PH and continued for 14 days. Parameters to assess the disease development and effectiveness of interventions were echocardiography, right and left ventricular systolic pressures, and right ventricular end diastolic pressure, percentage medial wall thickness (%MWT) of pulmonary artery, oxidative stress markers in lung tissue, NADPH oxidase (Nox-1) protein expression in lung, and mRNA expression of Bcl2 and Bax in right ventricular tissue. Results: OS (200 mg/kg) treatment ameliorated increased lung weight to body weight ratio, right ventricular hypertrophy, increased RVSP, and RVoTD/AoD ratio. Moreover, OS treatment decreases Nox-1 expression and increases expression of Bcl2/Bax ratio caused by MCT. Conclusion: The present study demonstrates that OS has therapeutic ability against MCT-induced PH in rat which are attributed to its antioxidant effect. The effect of OS was comparable with sildenafil. Ocimum sanctum (OS) (Linn)  is one of the medicinal plants widely used in the Indian System of Medicine. Various animal and clinical studies have validated its antioxidant  were calculated . ThereafBoth lobes (right and left) of the lung were separated and weighed excluding the bronchi and trachea, and the ratio of total lung weight to body weight was estimated (TLW/BW). Right middle lobe was preserved in 10% formalin for histopathology, while other lobes were snap frozen in liquid nitrogen and stored at −80 °C for oxidative stress markers and western blotting. Wet weight of left lobe of lung was measured then it was dried at 70 °C in oven for 3 days until the weight becomes constant and the percentage of dry weight to wet weight was calculated .After 48–72 h in formalin, the lung tissue samples get fixed and then processed under routine automated histokinetic processor. Paraffin blocks were prepared, 4–5 µm thick sections were cut and stained for routine hematoxylin and eosin stain. Special histochemical stain (Elastic Van Gieson) was carried out to highlight the elastic lamina. Thirty vessels were randomly selected from each section . The vesg at 4 °C for 15 min . Dilution of 1:10 (w/v) was used for homogenization. Supernatant was taken for oxidative stress markers.Lung tissue was homogenized in 0.1 M phosphate buffer (pH-7.4) and centrifuged at 10,000× TBARS was estimated by the method described by Ohkawa et al. . BrieflyReduced glutathione was estimated by the method described earlier . Briefly2O2 was added and extinction was read at 240 nm at 15 s interval for a total of 60 s by spectrophotometer .Catalase was estimated as described earlier . BrieflySuperoxide dismutase was estimated by the method described earlier . Briefly® west Pico chemiluminescent substrate, Thermo Scientific, Waltham, MA, USA). Then, the blots were exposed to the  chemiluminescence detection system and developed. Expression was normalized with loading control. Identification of band intensity was performed using the Image J Software .Total protein extraction and immunoblotting were performed in RIPA buffer enriched with protease and phosphatase inhibitors. Protein concentration was determined by the Bradford assay. An equal amount (30 µg) of protein was used for the immunoblotting and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, protein was transferred to PVDF membranes . The membranes were then blocked in Tris-buffered saline Tween-20  and 5% non-fat dry milk for 1 h and subsequently washed and incubated with primary antibodies in TBST at 4 °C for overnight. The titer of polyclonal antibody  used was 1:1000. After washing with TBST, the membrane was incubated with goat anti-rabbit IgG-HRP  antibody with 2.5% non-fat dry milk at room temperature for 2 h. After washing with TBS-T, immunoreactions were visualized with a chemiluminescence detection kit . The RPL-32 gene was used as a reference gene. We evaluated and quantified PCR product by agarose gel electrophoresis. Quantification of bands on the agarose gel was performed using Image J Software . Fold-change analysis was based on normalizing to RPL-32 transcript levels in each sample.Total RNA was isolated from RV of rat heart using TRIzol (Sigma). Reverse transcriptase reactions were performed for cDNA synthesis according to the method described earlier . Primersp < 0.05.Results are represented as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test  was done. The level of statistical significance was set at No significant difference in body weight was observed among the groups at the end of the study.p < 0.05) in the MCT administered group, which was significantly decreased by OS (p < 0.05). However, sildenafil did not cause significant change  and the ratio of right ventricular (RV) weight to left ventricle (LV) plus interventricular septum (IVS) weight (Fulton index) (p < 0.001) were increased in MCT administered rats as compared to control (p < 0.001), and OS (p < 0.001) cured  the parameters of RV hypertrophy  and end diastolic pressures (RVeDP) at 42 days A,B. OS ap < 0.001); and at 42 days ] and decrease in PAAT/ET ratio at 28 days  along with systolic notch in pulsed wave doppler images which were typical of pulmonary artery hypertension in MCT administered rats. After 14 days of OS treatment, it ameliorated both the increase in RVoTD/AoD ratio  and decrease in PAAT/ET ratio  caused by MCT (p < 0.001) caused by MCT , while the vessels with more than 100 µm external diameter did not show any significant change in %MWT as compared to control. Sildenafil  treatment for 14 days after 28 days administration of MCT significantly decreased the increase in %MWT of pulmonary artery with external diameter less than 100 µm  of the pulmonary artery with external diameter less than 100µm was found to be increased in MCT administered rats at 42 days . Sildenafil  and OS  treatment significantly reduced lung TBARS levels caused by MCT . OS and sildenafil administration did not cause significant difference in lung reduced GSH level as compared to MCT administered rats.There was a significant decrease in lung reduced GSH levels in MCT group as compared to control at 42 days . OS treatment after 28 days of MCT administration significantly increased the decrease lung catalase level  as compared with control rats. OS and sildenafil treatment did not alleviate the decrease in lung SOD levels caused by MCT .p < 0.001). OS treatment significantly decreased the protein expression of lung Nox-1 caused by MCT administration (p < 0.001). However, sildenafil did not significantly decrease protein expression of Nox-1 caused by MCT (A significant increase in lung Nox-1 protein expression was observed in MCT administered rats as compared to control (d by MCT A.p < 0.001), suggestive of apoptotic changes which was preserved significantly by OS and sildenafil (p < 0.001) administration  and right ventricular hypertrophy (RVH), as evidenced by the RV/LV + IVS (Fulton index) and RV/HW ratios. It also caused vascular smooth muscle hypertrophy of pulmonary arterioles in these rats. These findings established the development of PH, as also reported in earlier studies ,45,46. OIt has been demonstrated that MCT-induced PH causes structural changes in pulmonary arteries which are similar to the characteristic features of human PH in terms of marked medial wall thickening resulting in a dramatic increase in pulmonary arterial resistance ,49. In oIt has been demonstrated that echocardiography is a non-invasive and a key screening tool in the diagnostic algorithm for PH in human and it is also used in rat models . In the It has been reported that oxidative stress plays an important role in the development and progression of PH. Increased oxidative stress has been also reported in the lungs of different rat models of PH . NicotinIt has been demonstrated that PH induced by a single pathological way, such as with MCT or chronic hypoxia, do not fully show the severity of PH observed in clinics with respect to histological and/or hemodynamic parameters . It has In the present study, it has been demonstrated that OS improved major characteristic pathological features of MCT-induced PH in rats, including increased RVSP, right ventricular hypertrophy, echocardiographic parameters (increased RVoTD/AoD and reduced PAAT/ET), altered oxidative stress markers, increased expression of lung Nox-1 protein, proapoptotic changes in right ventricle, and most importantly, increased thickness of distal arterioles (of diameter <100 μm). It has been observed that OS has a certain therapeutic advantage over sildenafil, a commonly used drug for this condition.