The topcoat color of motor vehicles offers vital information while investigating vehicular accidents, especially in instance of hit-and-run, since witnesses seldom perceive and retain the plate details. Differences in color perceptions among individuals with normal vision may lead to confusion in determining the color of the car involved. In this way, witnesses of crash accidents could potentially initiate flawed leads in forensic investigation, and thus affect the administration of justice. In this study, the inter-rater reliability of vehicle color determination by different volunteers was explored. Six individuals observed the topcoat colors of 500 stationary and 500 moving vehicles from five locations, employing a common system of color gradation. The outcome was binary: the vehicle color was either a “match” or “non-match”. This was followed by statistical analysis in terms of the colors’ frequencies and inter-rater reliability, based on which more suitable color descriptions were determined for subsequent comparisons of stationary and moving vehicles. Higher match frequencies and greater inter-rater reliability were observed when color gradations were disregarded. The frequency of correct matches could have been closely related to their relative on-the-road distribution, regardless of the statuses of observed vehicles. It was also found that black and white were associated with a greater number of matches than were intermediate colors, which should be carefully interpreted during forensic investigation to avoid wrong leads. In conclusion, the present study demonstrated the forensic significance of vehicle topcoat color determination, particularly in cases where witness statements are crucial. Multiple coatings of automotive paints are applied to vehicles for both protective and decorative purposes , 2. ThesDuring a trial, investigative officers or forensic scientists are often required to explain the evidential value of questioned paint evidence recovered from a vehicular accident, including its color , 4. SurvInter-rater differences in color determination could be related to categorical differences in observers’ knowledge and perceptions . Color pThis study was conducted in five locations within the boundary of the Health Campus of Universiti Sains Malaysia, where a total of 500 stationary and 500 moving vehicles were randomly sampled and studied. Topcoat colors of vehicles were observed at noon in clear weather by six students (aged between 20 and 22 years) from various universities undergoing industrial attachment training in the forensic science program. In this study, only passenger-type vehicles, such as cars and vans, were included; heavy-duty vehicles such as trucks and buses were excluded.Prior to the survey, the authors conducted an introductory session with the six observers using a standard auto color chart available in forensic laboratories as a guide. This was to assure consistency in color determination, noting the choice of colors available and the color naming system. Buckle et al.’s  chart coIn each location, the six observers simultaneously noted the topcoat color of each car at a distance of approximately two meters. For both stationary and moving vehicles, each observer was given three seconds to write down their observation on a sheet of paper provided. No attempt was made to identify whether a vehicle’s paint system was solid or metallic since this is difficult to determine at a glance. As the survey was conducted within the university, the speed of moving vehicles could not have exceeded 60 km/hr.The observers’ perceptions were tabulated. Subsequently, the extent of agreement between individuals’ observation regarding topcoat color was checked and interpreted. Regarding agreement, the outcome was binary: it was either a “match” or “non-match.” It was a match when all the observers described the same color code, while it was a non-match when even one observer varied in his/her description of the topcoat color.Statistical analysis was conducted using Stata software version 12 . Data cleaning and descriptive analyses were performed to ensure there were no errors.Using the colors listed above , the freInter-rater reliability of color determination for both color descriptions was investigated. Kappa test (κ) statistics were used to assess agreement among the six observers. These values were interpreted as poor agreement 0.00–0.20), fair agreement (0.21–0.40), moderate agreement (0.41–0.60), good agreement (0.61–0.80), and very good agreement (>0.80) [, fair agThe observational data set was further analyzed to determine the colors with a greater possibility of non-matches among the six observers. The frequency and percentage of the matches and non-matches for each color were demonstrated and compared. Colors that could easily be described differently by the six observers were identified.In this study, 214 matches were recorded, which was 72 cases less than the non-matches. This finding indicates that all six observers concurred in the descriptions of only 42.8% of topcoat colors when using the color shades described earlier . Then, bInter-rater reliability (κ) values for each color scored in the two calculations (one that included the variations in shades and the other that considered the basic colors) were computed . By inclUsing basic color descriptions, the number of matches decreased by 12 when vehicles were in motion , but theIn this study, a non-match was scored if there was even a single difference among observers. Therefore, cases involving determination of two or more colors by the six observers were separately recorded as non-matches in their respective color categories. White scored the lowest percentage of non-matches, followed by black and gray. On the contrary, there were colors with only non-matches, such as red-brown, gold-bronze, and yellow-green, where no agreement was achieved among observers. It was also noted that the colors that could lead to differences in color determination were similar, regardless of whether the vehicles were stationary or moving.The majority of non-matches reported in this study were seen as a consequence of the existence of multiple shades of the same basic color. For example, the six observers did not have mutual agreement in determining “white” and “off-white” where 81 cases, accounting for 28.3% were recorded as non-matches. “Dark gray” and “medium gray,” with non-matches recorded in 22 cases (7.7%), and “light gray” and “medium gray,” with non-matches recorded in 21 cases (7.3%), showed similar trends. The higher proportion of matches reported when considering only the basic color description as compared to when shades were included [When shade variations in blue, brown, gray, green, yellow, and white were disregarded, the inter-rater reliability increased considerably. White, which was initially coded separately as “white” and “off-white,” became the most reliable color, replacing black. In the basic color description, only the crossover colors  demonstrated poor inter-reliability (κ<0.60), suggesting differences in agreement on such intermediary shades of colors.Although an introductory session was conducted to calibrate the observers prior to sampling, the variations in color determination could have been due to differences in their ability to discriminate colors as well as personal experiences , 21. A sSince all six individuals in this study made their observations in similar conditions, the use of only basic colors, which ensures good inter-rater reliability, is proposed for describing vehicle topcoat colors during forensic investigations, particularly when recording witness statements. In addition, such basic color-based enquiries would limit the use of jargon, thus conforming to the suggestion that an observer feels more comfortable describing a color in a few words rather than having to rely on a spectrum of colors with broad and hardly discriminable shades . HoweverHigher match frequencies for white, gray, and black could be linked to the on-the-road topcoat color distribution found in an earlier survey . A greatGood inter-rater reliability indicates greater consistency in the estimation of a phenomenon; in this case, matches during color determination. The use of basic colors has been associated with more consistent determination and less confusion as compared to the use of intermediate colors . In thisThis study suggests that witness statements regarding intermediate colors should be carefully interpreted during forensic investigations to avoid following wrong leads. Additionally, exact agreement among the observers which did not occur, particularly in determination of intermediate color, could have been due to personal variations , 19. ThiAccording to Bae et al. , an obsePrevious literature suggests that memory retrieval delays could affect observers’ color perception , 26, espThis study was conducted in optimal rating conditions with adequate illumination at a fixed distance for the young observers to determine topcoat colors. However, further collation of information, including vehicle color perception under different conditions, particularly accounting for environmental factors and observers’ vision and attention toward color determination, is necessary for broader forensic intelligence. However, percentage of agreement among colors with greater number of observers observing on the smaller number of objects could be proposed, perhaps in subsequent studies upon the determination of suitable color system and combination of “non-match” colors identified in the current study.In general, the frequency of matches in topcoat color determination, both for stationary and moving vehicles, could be related to their relative on-the-road distribution. This study indicated that using basic colors without shade variations could lead to better determination of color by an observer, resulting in a greater frequency of matches. The motion of vehicles did not have much effect on the scoring a match, given that the environmental conditions were adequate for an observer to encode the color. This study supports that for forensic intelligence purposes, cases involving descriptions of vehicle topcoat colors shall need greater investigative efforts including a more careful interpretation of witness testimony. It should also be emphasized that individual differences could lead to differences in color perception, especially when involving intermediate colors.A survey to investigate the inter-rater reliability of color determination among observers who visually perceived the topcoat colors of both stationary and moving vehicles indicated that the frequencies of matches, and subsequently inter-rater reliability of color determination among observers, significantly increased when using basic color descriptions, disregarding their shades. White and black had the greatest matches, while intermediate colors like green-blue, yellow-green, red-orange, gold-bronze, and red-brown were considered confusing, and thus require careful interpretation during forensic investigation. Information from this study can prove useful in interpreting witness descriptions of vehicle topcoat colors for more reliable statements.


Enterocytozoon bieneusi and Giardia duodenalis are common human and animal pathogens. Studies have increasingly shown that non-human primates (NHPs) are common hosts of these two zoonotic parasites. However, few studies have explored the genetic diversity and public health potential of these pathogens in laboratory monkeys. In this study, we examined the genetic diversity of the two pathogens in crab-eating macaques (Macaca fascicularis) in a commercial facility in Hainan, China.Enterocytozoon bieneusi and G. duodenalis were detected by PCR analysis in 461/1452 (31.7%) and 469/1452 (32.3%) fecal specimens from the animals, respectively. Significantly higher detection rates of E. bieneusi were detected in males  than in females , in animals with loose stools  than those with normal stool , and in animals of over 3 years of age  than those of 1–3 years . For G. duodenalis, the detection rate in males  was higher than in females but not statistically significant , in monkeys with loose stools  than those with normal stools , and in monkeys of 1–3 years of age  than those over 3 years . Nine E. bieneusi genotypes were detected in this study by DNA sequence analysis of the internal transcribed spacer of the rRNA gene, namely Type IV (236/461), Peru8 (42/461), Pongo2 (27/461), Peru11 (12/461), D (4/461) and PigEbITS7 (1/461) previously seen in NHPs as well as humans, and CM1 (119/461), CM2 (17/461) and CM3 (3/461) that had been only detected in NHPs. DNA sequence analyses of the tpi, gdh and bg loci identified all G. duodenalis specimens as having assemblage B. Altogether, eight (4 known and 4 new), seven (6 known and 1 new) and seven (4 known and 3 new) subtypes were seen at the tpi, gdh and bg loci, leading to the detection of 53 multi-locus genotypes (MLG-B-hn01 to MLG-B-hn53). Most of them were genetically related to those previously seen in common Old-World monkeys.E. bieneusi and assemblage B of G. duodenalis in farmed crab-eating macaques in Hainan, China.Data from this study indicate a common occurrence of zoonotic genotypes of  Giardia duodenalis and Enterocytozoon bieneusi are common human pathogens. At present, there are more than 200 million of annual giardiasis cases in humans, while microsporidiosis is a common cause of diarrhea [G. duodenalis every year [E. bieneusi, the detection rates range between 2–78% in industrialized and developing countries [E. bieneusi also caused an outbreak in France [diarrhea , 2. The diarrhea . In Chinountries –6. Over n France .E. bieneusi and G. duodenalis, which are transmitted in similar fecal-oral routes [Non-human primates (NHPs) are important experimental animals in public health research because of their high genetic similarity to humans . A growil routes , 11. In l routes , 13.E. bieneusi is the most common [E. bieneusi genotypes have been identified [E. bieneusi genotypes are divided into at least 11 groups [E. bieneusi in NHPs can help us understand the zoonotic potential of E. bieneusi in these animals.Of the 17 known human-pathogenic microsporidian species, t common . Based oentified . In phyl1 groups . Among t1 groups . Thus, gE. bieneusi genotypes have been found in NHPs, most of which belong to Group 1 [E. bieneusi genotypes [E. bieneusi.At present, more than 50  Group 1 . Among t Group 1 , 24–30. enotypes , 31. TheG. duodenalis assemblages (A-H) have been identified by genetic analysis of triosephosphate isomerase (tpi), ssrRNA, β-giardin (bg), glutamate dehydrogenase (gdh) and other genes [E. bieneusi, genotyping G. duodenalis also can help us to understand the transmission of this pathogen.Similarly, eight distinct er genes , 32, 33.G. duodenalis [G. duodenalis in NHPs.NHPs are also potential reservoir hosts for zoonotic transmission of odenalis , 12. In odenalis . Among todenalis –38. Althodenalis , 40. Mulodenalis –44. Contodenalis . There aE. bieneusi and G. duodenalis in fecal specimens from commercial crab-eating macaques (Macaca fascicularis) in Hainan. The genetic diversity of the two pathogen species was assessed using sequence analysis of the ITS (E. bieneusi) and using MLG analysis of the tpi, gdh and bg gene . The data generated were used to explore the human-infective potential of these two common gastrointestinal parasites in NHPs.In the present study, we examined the prevalence of 2. The cages were elevated 1 m from the ground so that the feces could fall onto the ground. Every monkey had contact with animals in the neighboring cages. The rooms were cleaned every morning and afternoon to ensure a clean living environment. Feed, fruits  and drinking water were regularly distributed by farm staff to each cage every day.A total of 1452 fecal specimens were collected in April 2016, June 2017, October 2017 and January 2018 from laboratory crab-eating macaques kept on a commercial farm. The farm was founded in 2003 and has been awarded full accreditation from the International Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) since 2008, with over 20,000 animals at the sampling time. All animals were kept in separate cages (1 × 1 × 1 m) after they were born on the farm, with approximately 30 animals kept in each room of ~ 60 mE. bieneusi and G. duodenalis genotypes in these animals. Among the sampled animals, 706 were male, 599 were female and 147 sampled animals had missing information on the sex. The sampled animals belonged to two age groups: 1102 were 1–3 years-old and 350 were adult monkeys older than 3 years. Regarding the latter, as monkeys over 4 years-old were often sold, the oldest animals sampled in the study were 5 years-old. At the time of sampling, 365 monkeys had loose stools, as defined by runny fecal consistency, and 1087 monkeys were apparently normal. The specimens were stored in 2.5% potassium dichromate solution at 4 °C prior to DNA extraction.The sampling plan took into consideration the number, age and sex of animals on the farm, and the needed number of positive specimens to generate data for a meaningful assessment of the distribution and human-infective potential of g for 10 min. Genomic DNA was extracted from the washed fecal material using the FastDNA SPIN Kit for soil  [The stored fecal specimens were washed three times with distilled water by centrifugation at 2000×Ana, CA) . DNA wasEnterocytozoon bieneusi was detected by nested PCR amplification of a 392-bp fragment of the rRNA gene containing the entire ITS sequence [E. bieneusi found in this study were determined by sequencing the PCR products and comparing the sequences obtained from the specimens with the reference sequences from known genotypes. The established genotype nomenclature was used in naming E. bieneusi genotypes identified in this study [sequence . The genis study .Giardia duodenalis was detected by nested PCR amplification of a 530-bp fragment of the tpi gene, a 511-bp fragment of the bg gene and a 599-bp fragment of the gdh gene [G. duodenalis-positive if any of the PCRs generated the expected PCR product. The genotypes of G. duodenalis found in this study were identified by sequencing PCR products from G. duodenalis-positive specimens and comparing sequences obtained with the reference sequences from the known genotypes at each genetic locus.gdh gene –50. The tpi, bg and gdh genes were sequenced in both directions on an ABI 3730 Genetic Analyzer . Nucleotide sequences generated were assembled and edited with software ChromasPro v.1.32 (http://technelysium.com.au/ChromasPro.html), and aligned with reference sequences from GenBank by using ClustalX .The secondary PCR products of the ITS, G. duodenalis, tpi, bg and gdh sequences from specimens with complete data at the three genetic loci were concatenated to form multi-locus sequences. They were compared with the reference sequences MLG1-MLG15 from NHPs, and Sweh001, Sweh059, Sweh074, Sweh107, Sweh136, Sweh158, ECUST5414, ECUST1710, ECUST4064 and ECUST981 from humans [https://www.megasoftware.net) using evolutionary distances calculated by the commonly used general time reversible model. The reliability of clusters formed was assessed by bootstrap analysis using 1000 replicates.To explore genetic diversity within the assemblage B of m humans , 34, 51.E. bieneusi and G. duodenalis detection rates between groups of different sex, age, or fecal consistency were assessed by using the Chi-square test implemented in SPSS Statistics v.20.0 . The difference was considered significant when P < 0.05.Differences in E. bieneusi. Significantly higher detection rates of E. bieneusi were identified in animals with loose stools  than animals with normal stools , in males  than females , and in old animals  than young animals  were positive for G. duodenalis, 362 (24.9%) specimens were positive by tpi PCR, 315 (21.7%) by bg PCR and 240 (16.5%) by gdh PCR. Altogether, 469 (32.3%) specimens were positive for G. duodenalis in at least one PCR. Significantly higher detection rates of G. duodenalis were found in animals with loose stools  than animals with normal stools , and in 1–3 year-old monkeys  than older animals . Nevertheless, detection rates of G. duodenalis were comparable between males  and females , CM1 (119/461), Peru8 (42/461), Pongo2 (27/461), CM2 (17/461), Peru11 (12/461), D (4/461), CM3 (3/461) and PigEbITS7 (1/461).Nine E. bieneusi genotypes were found in animals with loose stools, namely Type IV (74/151), CM1 (40/151), Pongo2 (12/151), Peru8 (11/151), CM2 (8/151), Peru11 (4/151), PigEbITS7 (1/151) and D (1/151). Similarly, eight E. bieneusi genotypes were detected in animals with normal stools, namely Type IV (162/310), CM1 (79/310), Peru8 (31/310), Pongo2 (15/310), CM2 (9/310), Peru11 (8/310), CM3 (3/310) and D (3/310). A similar distribution of E. bieneusi genotypes was also seen between male and female monkeys as well as young and old monkeys , B1 (n = 75), B6 (n = 27) and B2 (n = 17) found in this study were identical to reference sequences JX994245, KC441076, GU564284 and KC441077, respectively. The new subtypes B-hn02 (n = 78), B-hn04 (n = 32), B-hn01 (n = 13) and B-hn03 (n = 12) had one, one, two and one single nucleotide polymorphism (SNP), respectively, compared with the reference sequence MF095053 , B2 (n = 59), B-Egyh8 (n = 58) and B-VANC/91/UBC/67 (n = 5) found in this study were identical to reference sequences KY696837, MG736242, KC441079 and KM190799, respectively. The new subtypes B-hn08 (n = 20), B-hn06 (n = 1) and B-hn07 (n = 1) had four, two, and one SNP, respectively, compared with the reference sequence KY696837 , B-VANC/87/UBC/8 (n = 40), B-VANC/91/UBC/67 (n = 9), BIV (n = 7), B-Afu97 (n = 5) and B-sh03 (n = 2) found in this study were identical to the reference sequences KM190707, KM190714, KM190708, KF679733, HM134210 and JX994233, respectively. The new subtype B-hn05 (n = 15) had three SNPs compared with the reference sequence KM190707 . Among them, MLG-B-hn01 (16.7%) was the most common, followed by MLG-B-hn02, MLG-B-hn03 and MLG-B-hn04, with frequencies of 7.5%, 6.2%, and 5.0%, respectively. In contrast, the frequency of MLG-B-hn05 and MLG-B-hn06 was 4.3%, the frequency of MLG-B-hn07 and MLG-B-hn08 was 3.7%, while the remaining MLGs were each seen in fewer than five specimens . This is higher than the reported detection rates in NHPs in various countries [E. bieneusi in NHPs in China [E. bieneusi in NHPs were performed using wild, captive and zoo animals [Data from this study suggests that crab-eating macaques in Hainan, China are commonly infected with 7% 461/14. This isountries . Similarin China , 56–59. 7% 461/14. This is animals , 55, 57.E. bieneusi genotypes. In this study, all nine E. bieneusi genotypes detected in these animals belong to the zoonotic Group 1  in Hainan, China. This confirms the prevalence of this pathogen in NHPs in various countries [G. duodenalis as well as E. bieneusi in the present study could be attributed to the intensive farming of NHPs in this study, which congregates numerous susceptible individuals in confined areas.Laboratory crab-eating macaques are also apparently common hosts of ountries , 63, 64 ountries , 57, 65.G. duodenalis have been reported in NHPs [G. duodenalis genotype in the crab-eating macaque. This could have been due to the confined nature of animals in the facility, which limits the introduction of new G. duodenalis genotypes. Nevertheless, a high genetic heterogeneity of assemblage B was seen in animals in the laboratory facility, as revealed by subtype analysis at three genetic loci.To date, assemblages A, B and E of  in NHPs , 65–67.  in NHPs , 63–65.  in NHPs , 12, 28.G. duodenalis assemblage B in crab-eating macaques was supported by subtype analysis of specimens. Of the eight subtypes detected at the tpi locus, B-sh01 (JX994245) and B6 (GU564284) have been previously found in humans [gdh locus, B-sh03 (JX994233) and B-VANC/87/UBC/8 (KM190714) have been previously found in humans [bg subtypes, B-Egyh8 (MG736242) has been previously found in humans [G. duodenalis obtained in this study at individual genetic loci had been previously found in humans, supporting the human-pathogenic potential of the assemblage B in crab-eating macaques.The zoonotic potential of  B6 GU5644 have ben humans . Similar B6 GU5644 have ben humans . Likewisn humans . TherefoG. duodenalis [Nevertheless, there appears to be some host segregation within assemblage B of odenalis . In thisodenalis , 51. In E. bieneusi and G. duodenalis subtypes in crab-eating macaques in one commercial laboratory animal facility in Hainan, China. Most of the E. bieneusi genotypes and G. duodenalis assemblage B subtypes are potentially zoonotic. Additional genetic characterizations of these pathogens at other genetic loci, including more conservative ones for G. duodenalis, are needed to better understand the transmission of these pathogens and possible occurrence of host segregation within G. duodenalis assemblage B. Measures should be implemented at the commercial facility to reduce the transmission of enteric parasites.In this study, we have shown a frequent occurrence and high genetic diversity


The aim of this study was to investigate whether uric acid (UA) might exert neuroprotection via activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway and regulating neurotrophic factors in the cerebral cortices after transient focal cerebral ischemia/reperfusion (FCI/R) in rats. UA was intravenously injected through the tail vein (16 mg/kg) 30 min after the onset of reperfusion in rats subjected to middle cerebral artery occlusion for 2 h. Neurological deficit score was performed to analyze neurological function at 24 h after reperfusion. Terminal deoxynucleotidyl transferase-mediated dNTP nick end labeling (TUNEL) staining and hematoxylin and eosin (HE) staining were used to detect histological injury of the cerebral cortex. Malondialdehyde (MDA), the carbonyl groups, and 8-hydroxyl-2′-deoxyguanosine (8-OHdG) levels were employed to evaluate oxidative stress. Nrf2 and its downstream antioxidant protein, heme oxygenase- (HO-) 1,were detected by western blot. Nrf2 DNA-binding activity was observed using an ELISA-based measurement. Expressions of BDNF and NGF were analyzed by immunohistochemistry. Our results showed that UA treatment significantly suppressed FCI/R-induced oxidative stress, accompanied by attenuating neuronal damage, which subsequently decreased the infarct volume and neurological deficit. Further, the treatment of UA activated Nrf2 signaling pathway and upregulated BDNF and NGF expression levels. Interestingly, the aforementioned effects of UA were markedly inhibited by administration of brusatol, an inhibitor of Nrf2. Taken together, the antioxidant and neuroprotective effects afforded by UA treatment involved the modulation of Nrf2-mediated oxidative stress and regulation of BDNF and NGF expression levels. Thus, UA treatment could be of interest to prevent FCI/R injury. The principal therapy for cerebral ischemia is the restoration of cerebral blood flow as early as possible. Early recanalization with recombinant tissue plasminogen activator (rt-PA) has been developed to treat acute ischemic stroke. However, reperfusion after cerebral ischemia may be injurious and increase the risk of brain hemorrhage, promote the development of cerebral edema, and cause more serious damage to the blood-brain barrier. The potential mechanisms responsible for the additional injuries in the ischemic brain caused by reperfusion itself remain unclear . Acute cPhase 2 enzymes have been implicated to be the important means by which neurons protect themselves against intense oxidative stress. The expression of phase 2 enzymes, including heme oxygenase (HO)-1, is regulated by nuclear factor E2-related factor 2 (Nrf2) . ConsideNeurotrophic factors are essential regulators in poststroke recovery –11. BothUric acid (UA) is a major antioxidant in the blood in humans and is responsible for almost two-thirds of all free radical scavenging capacity with a concentration that surpasses other antioxidants by as much as tenfold in plasma . Since Un = 45), vehicle-treated I/R group (n = 45), 16 mg/kg UA-treated I/R group , and UA plus brusatol-treated I/R group .Male Sprague–Dawley (SD) rats (280–300 g) from Pengyue Experimental Animal Breeding Institute of Jinan, China, were maintained in a room  under 12 h light–dark cycle with free access to water and food. All experimental protocols were conducted according to ARRIVE  guidelines. Every effort was made to minimize animal suffering and the number of animals used. The Ethics Committee of Jining Medical University approved all the experimental protocols. All sample analysis was conducted under a strictly blinded manner. 193 rats were used, of which 11 died after transient FCI/R insult and 2 died after left lateral ventricle puncture, and consequently a total of 180 rats were separated randomly into 4 groups which included the sham group (The procedure for transient FCI/R has been described previously . For MCAμl 1% DMSO) [UA diluted with Locke's buffer (vehicle) was injected intravenously through the tail vein (16 mg/kg) 30 min after reperfusion. An equal volume of vehicle was given to sham and model rats. Rats in the UA + Bru group were treated with UA plus brusatol. The dose of UA was chosen based on previous reports of UA treatment in rats induced by transient FCI/R –26. Left2,3,5-Triphenyltetrazolium chloride (TTC) staining technique was employed to test the cerebral infarct volume. TTC stains the noninfarcted region with a deep red pigment, while the infarcted brain area appears white. Infarct volume was calculated with correction of edema in the ischemic hemisphere as previously described .A blinded observer evaluated the neurological status 24 h after FCI/R. A 5-point scale was used as follows , 32: 0, The brains were carefully removed after decapitation. The wet weight was obtained immediately by weighing the ischemic hemispheres. The dry weight was obtained by weighing the samples dried at 60°C for 72 h. The percentage tissue water content was determined as previously described .n = 3/group). The brain coronal sections from bregma at 0.3 to −1.8 mm were cut at 20 μm thickness. The sections were stained for hematoxylin and eosin (HE), terminal deoxynucleotidyl transferase- (TdT-) mediated dNTP nick end labeling (TUNEL), the expression of BDNF and NGF, and 8-hydroxy-deoxyguanosine (8-OHdG). Sections were incubated with primary antibody  and then with the biotinylated secondary antibody. Negative controls performed the same staining procedure with the omission of primary antibody. HE staining was performed according to the standard procedure. TUNEL labeling assay kit  was used to measure DNA fragmentation. The number of HE-, TUNEL-, BDNF-, NGF-, and 8-OHdG-positive cells in the penumbra of ischemic ipsilateral parietal cortex in the ischemic area was quantified under a 400x magnification by a blinded investigator.Rats were transcardially perfused with PBS and 4% paraformaldehyde 24 h after transient FCI/R (n = 4/group) was dissected to extract total protein using a total protein extraction kit . A nuclear-cytosol extraction kit  was used to isolate the nuclear and cytosol fractions. We examined the expression levels of Nrf2 in the cytoplasm and nucleus and HO-1 in the cytoplasm. Each sample contained 50 μg protein. The primary antibodies were anti-HO-1 polyclonal antibody  and anti-Nrf2 polyclonal antibody . Each protein blot was normalized to histone H3 or β-actin to obtain the final result expressed as intensity ratio (% sham-operated control).The ischemic side cerebral cortex  ELISA-based TransAM® Nrf2 kit was used to analyze Nrf2 DNA-binding activity . 10 μg nA commercial OxyBlot kit  was employed to determine the extent of protein oxidation . The proWe analyzed total malondialdehyde (MDA) levels as the indicators of lipid peroxidation with the MDA detection kit  . The absP value < 0.05 was considered to indicate statistical significance.Neurological deficits were analyzed using the Kruskal-Wallis test followed by Dunn's test, and results are expressed as median ± interquartile range. One-way analysis of variance (ANOVA) with subsequent Duncan's test as post hoc analysis was used for statistical analysis of all the other data, and results are reported as mean ± standard error (SE). A P < 0.01). In order to evaluate the role of Nrf2 pathway in UA-induced neuroprotection, the effect of brusatol on cerebral infarct volume was also detected. As observed in the UA + Bru group, a larger cerebral infarct volume was presented compared with the UA-treated I/R group , whereas brusatol attenuated the decrease . More intact cells in UA-treated rats were observed in comparison with model rats owing to the neuroprotection of UA . TUNEL-positive cells were markedly reduced by UA administration . However, a marked increase of TUNEL-positive cells was observed in the UA + Bru group , suggesting brusatol pretreatment attenuated neuroprotection of UA . In addition, cotreatment with UA and brusatol decreased BDNF and NGF expressions comparable to the UA-treated group  decreased cerebral infarct volume, neurological deficit core, and degree of brain edema, consistent with the previous studies –25. The The cellular injury and downstream pathway activation caused by oxidative stress may exacerbate the postischemic brain damage . MDA, prγ-glutamyl cysteine ligase (GCL) and NAD(P)H:quinone oxidoreductase 1 (NQO1) and protein levels of GCLM in astrocyte cultures, whose inductions are governed by Nrf2, suggesting other antioxidants induced by Nrf2, may also contribute to the protective effects of UA [Little is known about the mechanisms on how UA treatment protects neurons against oxidative stress. Evidences demonstrated that Nrf2 is a master regulator of the antioxidative defense responses . Under bts of UA . Thus, tIn addition, our study demonstrated that UA significantly increases the endogenous BDNF and NGF expressions. Several studies indicate that increased levels of neurotrophins have a protective role in experimental stroke studies . The famOur study has several limitations. First, it has been demonstrated that hyperemia at reperfusion can be a sign of worse I/R injury , 52. PriFor the first time, this study demonstrates that UA confers neuroprotection against FCI/R-induced oxidative stress via Nrf2 pathway activation as well as BDNF and NGF expression upregulation. The protective effect of UA was blocked by Nrf2 inhibitor brusatol. Nonetheless, further study deserves to be done in the future to investigate whether other pathways or mechanisms participated in the neuroprotective effects of UA.


FASCIN2 (FSCN2) gene could cause autosomal dominant retinitis pigmentosa. Recent studies have indicated that a missense mutation in mouse Fscn2 gene (R109H) can contribute to the early onset of hearing loss in DBA/2J mice. To explore the function of the gene, Fscn2 was knocked out using TALEN (transcription activator-like effector nucleases) on the C57BL/6J background. Four mouse strains with deletions of 1, 4, 5, and 41 nucleotides in the target region of Fscn2 were developed. F1 heterozygous (+/−Fscn2) mice carrying the same deletion of 41 nucleotides were mated to generate the −/−Fscn2 mice. As a result, the −/−Fscn2 mice showed progressive hearing loss, as measured in the elevation of auditory brainstem-response thresholds. The hearing impairment began at age 3 weeks at high-stimulus frequencies and became most severe at age 24 weeks. Moreover, degeneration of hair cells and loss of stereocilia were remarkable in −/−Fscn2 mice, as revealed by F-actin staining and scanning electron microscopy. Furthermore, compared to the controls, the −/−Fscn2 mice displayed significantly lower electroretinogram amplitudes and thinner retinas at 8, 16, and 24 weeks. These results demonstrate that, in C57BL/6Jmice, Fscn2 is essential for maintaining ear and eye function and that a null mutation of Fscn2 leads to progressive hearing loss and retinal degeneration.Fascin2 (FSCN2) is an actin cross-linking protein that is mainly localized in retinas and in the stereocilia of hair cells. Earlier studies showed that a deletion mutation in human  Age-related hearing loss (presbycusis), one of the most common perceptive diseases among the elderly population, causes both communication disorders and psychological problems.Fscn2) is first identified as a retina-specific transcript localizing in the inner and outer segments of bovine photoreceptor cells thresholds that are elevated by 15-20 dB at age 3 weeks and approach to deafness at about 3 months of age(ahl) in cdh23, a locus named ahl8 recently has been identified as the main contributor to early-onset hearing loss in DBA/2J mice techniques. A total of 354 littermates with ages from 3 to 52 weeks were included in this study, with 185 Fscn2+/+), heterozygous mice (Fscn2+/−), and homozygous mice (Fscn2−/−) were genotyped using a polymerase chain reaction (PCR). The primers .The wild-type mice , and cDNA was synthesized using Random Hexamer primers following the First Strand cDNA Synthesis Protocol . Two sets of primers  spanning the deletion were finally sequenced. GAPDH was used as the internal control.Transcription of Paraffin sections were made following the previous methods were diluted in 0.01 M PBS. The samples were incubated overnight at 4° with primary antibodies. After being washed three times with 0.01 M PBS, the samples were incubated with secondary antibodies  at room temperature for 1 hr and then diluted in the 0.01 M PBS. Counterstaining was completed using Hoechst33342 (3 min at room temperature). Finally, the samples were mounted with VECTASHIELD Mounting Medium H-100  and were observed under immunofluorescent confocal microscopy .Paraffin sections were made as described above. The sections were immersed in 0.5% Triton X-100 for 30 min and blocked with 5% bovine serum albumin for 60 min at room temperature. Primary antibodies . The membrane was immersed in 5% nonfat milk and buffered in Tris-buffered saline containing 0.1% Tween-20 at room temperature for 1.5 hr; it was then incubated at 4° overnight with primary antibody  and β-actin . After being washed three times with the Tris-buffered saline, the membrane was incubated with horseradish peroxidase and conjugated with goat anti-rabbit secondary antibody  at room temperature for 1 hr. Protein expression was detected using SuperSignal West Pico  and was photographed in a chemiluminescence instrument .Western blotting was carried out following the previously described procedures(−/−Fscn2 mice and +/+Fscn2 mice (between 6 and 10 for each group at each of the corresponding time points), following the methods described previously(The mice were anesthetized with 2% tribromoethanol (0.2 mL per 10 g of body weight) and then placed on a heating pad to maintain a temperature of 37°. All operations were carried out in a soundproof and electromagnetic shielding room. We used the IHSS Smart EP 3.30 and USBez Software (Intelligent Hearing Systems) to measure the ABR thresholds and the DPOAE amplitudes at various intervals  in +/+Fscn2 mice and −/−Fscn2 mice (between 6 and 8 in each group for each of the corresponding time points) with reference to the methods described previously, with a focus on their hair cells and stereocilia. After fixation and decalcification, the organs of Corti were carefully microdissected out and subdivided into the base, middle, and apex turns. Then, the tissues were mounted with glycerin on glass slides. The surface preparations were stained for F-actin with Alexa Fluor-488 conjugated with phalloidin  for 1 hr at room temperature to show the hair cell bundles. Then, the samples were mounted with VECTASHIELD Mounting Medium H-100 . After being washed three times in 0.01 M PBS (pH 7.4), the samples were examined with a fluorescent microscope . In each turn of cochlea, counts of hair cells in three continuous views were recorded. Hair cells were counted as present if the hair cell bundles’ V shapes were intact. The percentages of hair cells missing in the three turns of the organs of Corti were calculated and analyzed.The experiments were carried out following the method described.Scanning electron microscopy was carried out following the methods described previously. The Fscn2 null mutation was made using the TALEN technique, with two vectors targeting the first exon of Fscn2  mice carrying the same deletion of 41 nucleotides were mated to generate the −/−Fscn2 mice, which were maintained for 12 generations.The of Fscn2 . The tar−/−Fscn2 mice and the wild-type mice. The weights of −/−Fscn2 mice and wild-type mice are shown in Overall, the mutation doesn’t influence the reproductivity of the mice. The number of pups ranged from 6-9 per litter for the Fscn2 alleles (41 bp) and that the heterozygous +/−Fscn2 mice and homozygous −/−Fscn2 mice carried the mutation in one allele and both alleles, respectively (Fscn2 was then developed to identify the wild-type mice (+/+Fscn2), heterozygous mice (+/−Fscn2), and homozygous mice  of the wild-type mice and homozygous mice (Fascin2 protein was not expressed in the cochleae of the homozygous mice (−/−Fscn2), unlike in the wild-type or heterozygous mice  at all stimulus frequencies . However, the ABR thresholds in the +/+Fscn2 mice did not change until 24 weeks of age. Unlike those of the +/+Fscn2 mice, the ABR thresholds in the −/−Fscn2 mice had already significantly risen at stimulus frequencies of 16 and 32 kHz by age 3 weeks, which indicated an early onset of hearing impairment for high frequencies. Later on, at all time points and all stimulus frequencies, the ABR thresholds in −/−Fscn2 mice were significantly higher than the levels for wild-type mice  at f2 frequency of 17 672 Hz , the amplitudes of DPOAE were measured in 7 672 Hz . These r−/−Fscn2 mice was observed at various ages . OHCs were counted as present if the V shapes of the hair bundles were intact. The hair-cell loss occurred in the basal turns of the cochleae as early as 6 weeks of age (3–4%); it spread to the middle and apical turns at ages 9 and 16 weeks, respectively. More than 80% of OHCs were missing in the basal turns by age 32 weeks, whereas the same number of OHCs was lost in the middle and apical turns at age 40 weeks  flash ERGs were conducted on 24 weeks  and 8B. resented  and 8D. 24 weeks .Fscn2 null-mutation (−/−)Fscn2 mouse model using the C57BL/6J background. Earlier studies showed that the Fscn2 gene was related to retinal pigmentosa in humans and mice(Fscn2 (p.R109H) and progressive hearing loss in DBA/2J mice Hearing impairment in the −/−Fscn2 mice occurred at about 3 weeks of age, which indicated early-onset hearing loss. (2) The hearing impairment was more sensitive to pure tone stimuli at high frequencies (such as 16 and 32 kHz) than to those at a low frequency (8 kHz). This phenomenon corresponded to the loss of OHCs  in several mouse models of age-related hearing loss and Fscn2 (ahl8), the quantitative trait locus on chromosome 5 may also be responsible for severe hearing loss in DBA/2J mice(It souled be mentioned that hearing loss in In summary, the knockout of the Fscn2 gene in C57BL/6J mice causes progressive hearing loss and degeneration of hair cells and retinas. This model will aid in functional studies of Fscn2 to increase understanding of the etiology of FSCN2-associated disorders in humans.


Yes-associated protein, a core regulator of the Hippo-YAP signaling pathway, plays a vital role in inhibiting apoptosis. Thus, several studies and reviews suggest that yes-associated protein is a good target for treating cancer. Unfortunately, more and more evidence demonstrates that this protein is also an essential contributor of p73-mediated apoptosis. This questions the concept that yes-associated protein is always a good target for developing novel anti-cancer drugs. Thus, the aim of this review was to evaluate the clinical relevance of yes-associated protein for cancer pathophysiology. This review also summarized the molecules, processes and drugs, which regulate Hippo-YAP signaling and discusses their effect on apoptosis. In addition, issues are defined, which should be addressed in the future in order to provide a solid basis for targeting the Hippo-YAP signaling pathway in clinical trials. Yes-associated protein (YAP) is a core component of the Hippo signaling pathway in mammals . InitialCyclooxygenase-2 (COX-2) R5  OR YAP [tiab]) AND (apoptosis [tiab] OR autophagy [tiab]) AND (neoplasms [tiab] OR cancer [tiab]). Inclusion criteria: We included all studies which investigated the “mechanism of interaction between YAP and autophagy” or the “mechanisms of interaction between YAP and apoptosis”. Exclusion criteria: We excluded article types, which were reviews or commentaries; literature written not in English and irrelevant literature (literature which does not meet the inclusion criteria). We also added publications, which were cited in the reference list of the included literature or suggested by reviewers. In addition, we searched the


Canonical Wnt signaling is crucial for vascularization of the central nervous system and blood-brain barrier (BBB) formation. BBB formation and modulation are not only important for development, but also relevant for vascular and neurodegenerative diseases. However, there is little understanding of how Wnt signaling contributes to brain angiogenesis and BBB formation. Here we show, using high resolution in vivo imaging and temporal and spatial manipulation of Wnt signaling, different requirements for Wnt signaling during brain angiogenesis and BBB formation. In the absence of Wnt signaling, premature Sphingosine-1-phosphate receptor (S1pr) signaling reduces VE-cadherin and Esama at cell-cell junctions. We suggest that Wnt signaling suppresses S1pr signaling during angiogenesis to enable the dynamic junction formation during anastomosis, whereas later S1pr signaling regulates BBB maturation and VE-cadherin stabilization. Our data provides a link between brain angiogenesis and BBB formation and identifies Wnt signaling as coordinator of the timing and as regulator of anastomosis. Wnt signaling is known to regulate the formation of the blood-brain barrier. Here Hübner et al. dissect the underlying mechanisms using high resolution live imaging in zebrafish, and find that Wnt regulates anastomosis of angiogenic sprouts in the brain by counteracting sphingosine-1-phosphate receptor signaling. During a defined time window of embryonic development, molecular cues from neuronal and perineuronal tissues orchestrate CNS angiogenesis and barriergenesis4. The process of brain angiogenesis is well conserved in vertebrates: After acquiring a pre-sprouting signature , these specified cells migrate out from the resident vessel, with tip cells guiding sprout formation, and invade into the neuronal tissue, where they form cell–cell contacts and anastomose with other sprouts or extra-cerebral vessels in order to establish circulatory loops5. In zebrafish (Danio rerio), hindbrain capillaries  invade the brain parenchyma at around 32 h post fertilization (hpf) from the primordial hindbrain channel (PHBC), extend dorsally and connect ventrally to the basilar artery (BA) or laterally to other CtAs  depends on nutrient and oxygen delivery from blood vessels during the development and homeostasis, but also requires protection from blood-born toxins and pathogens. Endothelial cells (ECs) of CNS blood vessels acquire characteristic properties in order to fulfill the tasks of this blood-brain barrier (BBB), such as expression of a specific subset of junction molecules and nutrient transporters, downregulation of vesicular transport and establishment of cell–cell interactions within the neurovascular unit12. Additionally, Wnt signaling has been shown to be involved in the establishment of BBB characteristics, such as upregulation of tight junction components  or nutrient transporters  and decrease of transcellular transport processes 15. Recently, the Gpr124-Reck complex has been identified as an EC autonomous regulator of Wnt signaling essential for brain vascularization and BBB formation20. Single-cell analysis of brain vascular development through live imaging approaches in zebrafish further revealed that the control of pre-tip cell function by the Gpr124-Reck complex operates within the parental vessels (PHBCs) at pre-invasive stages. This early function of Wnt signaling has so far largely precluded the analysis of Wnt signaling functions at later stages of CNS vascular development, including the invasion process itself and subsequent patterning events.Studies in mice and zebrafish demonstrate β-catenin-dependent Wnt signaling through Wnt7a–Wnt7b to be essential for brain vascularization22. Together with the EC-selective adhesion molecule a (Esama), VE-cadherin is essential for EC–EC recognition and contact formation during intersegmental vessel anastomosis24. Experiments using mice and tissue culture revealed that VE-cadherin is subject to extensive post-transcriptional regulation, including intracellular complex formation and trafficking, membrane localization as well as association in cell–cell junctions. This dynamic regulation of VE-cadherin in adherens junctions is not only crucial for vascular patterning, but also for the function of mature blood vessels, e.g., for maintenance of EC integrity or during leukocyte extravasation26, and can be regulated by Sphingosine-1-phosphate (S1p) signaling28.Vascular endothelial (VE)-cadherin is the major endothelial-specific member of the cadherin protein family and is involved in EC migration, cell–cell contact formation, anastomosis, and barrier formation29 and in zebrafish, two paralogues exist for S1pr3 (S1pr3a and S1pr3b). S1pr1 signaling promotes BBB integrity in mice30. Additionally, postnatal EC-specific knockout of S1pr1 induces loss of VE-cadherin and vascular endothelial growth factor receptor 2 (VEGFR2) from the cell junctions31. To date, the contribution of neither S1pr nor VE-cadherin signaling has been addressed during brain capillary angiogenesis, leaving a missing link between brain EC angiogenesis and BBB formation.ECs express S1p receptor (S1pr) 1, 2, and 312, it is surprisingly dispensable during sprout elongation and migration, although it remains continuously active in the invading sprout. We show that Wnt signaling is regulating brain capillary anastomosis and lumen formation. Interestingly, Wnt signaling is crucial for VE-cadherin and Esama localization at cell–cell junctions in a transcription-independent manner. During active brain capillary angiogenesis, Wnt signaling counteracts S1pr signaling, which enables VE-cadherin-dependent anastomosis and lumen formation. In contrast, when angiogenesis is completed at later stages, S1pr signaling regulates BBB formation. Our data therefore reveal an important functional link between the intertwined processes of brain capillary angiogenesis and BBB formation.In this study, we clarify the distinct requirements for Wnt signaling during brain angiogenesis. Whereas Wnt signaling is essential before sprouting to regulate yet to be defined early function of the future tip cells (pre-tip cells) within the parental vessel14. Studies using zebrafish revealed that this control by Wnt signaling reflects an essential function in defining the pre-tip cell during early, pre-invasive angiogenic events within the parental vessels . As a result, no hindbrain capillaries  are formed in the absence of Wnt signaling. In order to bypass this early requirement, we blocked Wnt signaling pharmacologically using IWR-132 and generated transgenic lines, which allow for temporally and spatially controlled expression of a dominant-negative Tcf transcription factor.Studies in mice have shown that Wnt signaling is essential for hindbrain capillary angiogenesisels Fig. 12. As a Tg(axin2BAC:Venus-Pest)mu288 and Tg(14TCF:loxP-STOP-loxP-dGFP)mu20233. Both reporter lines showed robust fluorophore expression within the migrating CtA sprouts at 34 hpf mu202 embryos starting from 29 hpf  resulted in severe vascular defects and hemorrhages in the brain, but not in the trunk vasculature s898). Validation of Wnt inhibition by heat shock controlled mCherry-dnTcf expression is documented in Supplementary Fig. We therefore generated a transgenic zebrafish line expressing a dominant-negative Tcf transcription factor fused to mCherry (mCherry-dnTcf) under the control of a heat shock inducible promoter (hsp70l). To restrict Wnt inhibition to ECs, we placed a loxP-flanked (floxed) STOP-cassette upstream of the mCherry-dnTcf coding sequence  did not result in differences in filopodia morphology, but instead in high filopodia motility, which resulted in slightly blurry images due to fast filopodia movement y7 embryos. Both, treatment with IWR-1 or heat shock-induced EC-specific mCherry-dnTcf expression, reduced the EC number within CtAs mu240 embryos to illustrate the actin cytoskeleton of ECs  and two paralogues exist in zebrafish, esama and esamb, of which esama is mainly expressed in the vasculature24. To address, whether Wnt signaling inhibition affects protein localization of VE-cadherin or Esama, we performed immunostaining for VE-cadherin, Esama and ZO-1 in 42 hpf-old embryos 37 did not completely restore wild-type VE-cadherin function,  after Wnt signaling inhibition by IWR-1 or EC-specific mCherry-dnTcf expression s843 signal). Surprisingly, we did not find severe changes of the overall ve-cadherin or esama mRNA abundance in CtA ECs at 32 or 42 hpf , but rather slightly increased over time, presumably due to a compensatory mechanism. The mRNA level of esama in CtA ECs was slightly decreased after Wnt signaling inhibition by IWR-1 or mCherry-dnTcf expression both at 32 and 42 hpf ubs3;(UAS:Kaede)rk8 embryos, which were treated with IWR-1 or DMSO  has been shown in vivo and in vitro to increase recruitment of Cadherin protein family members, including VE-cadherin and N-cadherin, to the plasma membrane39. Additionally, S1p receptor 3 (S1pr3) was found to function cooperatively with S1pr1 in promoting adherens junction formation in ECs38, whereas S1p receptor 2 (S1pr2) signaling disrupted EC junctions, thereby increasing vascular permeability40. We confirmed that similar to what is seen in mice30, blocking of S1prs using the pharmacological antagonist VPC23019 (VPC) during barriergenesis (48–72 hpf) resulted in extravasation of red blood cells into the surrounding brain tissue, indicating breakdown of the BBB  signaling, as it is known to regulate EC integrity and behavior by modulating junction protein localization and cell contractilitys1pr1 and s1pr5a as well as very low level expression of s1pr2 and s1pr3b in zebrafish brain capillary ECs  or Rho inhibitor (CT03) alone or together with IWR-1 as described. Single treatment with either Rac1 or Rho inhibitors, did not affect CtA development or lumen formation. Indeed, co-treatment of IWR-1 and Rac1 inhibitor, but not of IWR-1 and Rho inhibitor, robustly restored the lumen formation defects caused by IWR-1 ubs3 fish with Tg(UAS:CA-Rac1)mu211 and analyzed brain capillary lumen formation in 48 hpf-old embryos mu212) could partially rescued lumen formation in IWR-1-treated embryos  should phenocopy Wnt signaling inhibition. Therefore, we mated yos Fig. . Vasculayos Fig.  or ve-cayos Fig. .Hence, Rac1 activation downstream of S1pr1 signaling is required and sufficient to mediate lumen formation defects caused by Wnt signaling inhibition.In summary, we identified a temporal-specific function of Wnt signaling for brain capillary angiogenesis by post-transcriptionally and EC-specifically regulating junction protein localization upstream or at the level of Rac1 activity, but downstream of S1pr1 signaling. We suggest that EC-specific Wnt signaling counteracts S1pr1 signaling during CtA angiogenesis, presumably to prevent premature barrier formation processes, which would impair brain angiogenesis Fig. .15. Treatment of embryos lacking Wnt signaling following IWR-1 treatment or of gpr124 mutants with S1pr antagonists did not restore CtA sprout formation  Wnt signaling is highly active in brain capillary sprouts, but after pre-tip cell formation it is dispensable for brain capillary invasion into the brain parenchyma. Instead, (2) Wnt signaling is required for brain capillary anastomosis. During this process, (3) Wnt signaling influences the post-transcriptional regulation and cell–cell junction localization of the EC-specific adhesion molecules, VE-cadherin, and Esama, by counteracting S1pr1 signaling Fig. .14). Recent reports demonstrate that Gpr124-Reck-mediated Wnt signaling is essential for brain vascularization, as homozygous grp124 or reck mutants are completely devoid of brain capillaries17. We hypothesized that Wnt signaling might be involved in the formation of pre-tip cells within the parental vessel, which most likely occurs at earlier developmental stages12. In line with this, a role of Wnt signaling for EC specification has already been suggested to drive cell fate from the Fli1a-positive precursor cell population into ECs at the expense of primitive erythrocytes33. At later developmental stages, Wnt signaling is required for subspecification of EC populations within the vasculature, which has been shown for hemogenic EC formation46 and lymphatic cell fate specification47.We and others have shown that Wnt signaling is active in brain capillaries during brain angiogenesis Fig. ,11,12,14Here we demonstrate that Wnt signaling is continuously activated in CtAs during all stages of angiogenesis. Surprisingly, Wnt signaling is not involved in the process of CtA sprout invasion and filopodia formation Fig. .24. We detected reduced protein levels of VE-cadherin, Esama, and ZO-1 at the cell–cell junctions following Wnt signaling inhibition  or Esama (by esamaubs19/ubs19) phenocopied CtA lumen formation defects , indicating a compensatory response to the reduced protein levels of VE-cadherin at cell–cell junctions48. We therefore conclude that VE-cadherin and Esama downregulation occurs via a post-transcriptional mechanism, which interferes with their membrane localization. Using a series of inhibitors, we excluded internalization as well as proteasomal and lysosomal degradation as potential mechanisms . In lumenized vessels, activation of S1pr signaling would occur and be most likely enhanced by increased supply of the ligand S1p through the blood circulation41, which would enable functional S1pr signaling for BBB formation and maintenance . We hypothesize that over-activation of Rac1 after Wnt signaling inhibition interferes with efficient cellular junction formation during brain capillary angiogenesis. To date, Rac1 activation is well described to promote EC barrier formation in mature blood vessel44, indicating that Rac1 can act downstream of S1pr signaling in barriergenesis27. However, we demonstrate a different regulation in Wnt-responsive brain capillary ECs during capillary angiogenesis, which is similar to our observation of S1pr signaling regulation . And third, Wnt signaling leads to the induction of BBB components, such as glucose transporter Glut1 and Claudin family members, already during brain angiogenesis in order to subsequently establish a functional BBB after brain angiogenesis has been completed52 and sealing of the BBB is likely to be promoted by S1pr signaling after the onset of blood circulation . The complexity of the signaling network regulating EC behavior during brain angiogenesis and BBB formation is far from being understood. Our study provides a regulatory function of Wnt signaling during brain angiogenesis, which was only unraveled by interfering with Wnt signaling in a temporally and spatially restricted manner in combination with in vivo time-lapse imaging. We were able to identify a post-transcriptional level of regulation, which is essential for cell–cell junction formation and the modulation of EC barrier establishment or tightness. Therefore, interfering with Wnt or S1p signaling during treatment of infectious and neurodegenerative diseases as well as anti-cancer therapy remains a major clinical obstacle as adverse effects and cross-reactions are not well predictable55. The FDA approved sphingosine analog and agonist of S1pr1 and S1pr3–5 called Fingolimod (FTY720/Gilenya) has been reported to cause side effects of increased of vascular permeability in the lung and cardio-vascular complications. Moreover, cases with neuroencephalitis were noted when the drug was applied at higher doses. Therefore, the evaluation of the potential clinical impact for CNS development and pathology of these and future substances requires a greater understanding of signaling cues regulating EC properties and behavior.During brain neo-vascularization, we and others report divers functions of Wnt signaling, which regulate many steps of brain capillary angiogenesis and BBB formation and cross-communicate with other signaling cuesDanio rerio) husbandry and embryo maintenance were carried out under standard conditions at 28.5 °C56. Embryonic developmental stages were determined according to ref. 57.Zebrafish (Tg(14TCF:loxP-STOP-loxP-dGFP)mu20233, Tg(axin2BAC:Venus-Pest)mu28833, Tg(fli1a:dsRed)um1358, Tg(fli1a:lifeact-GFP)mu24059, Tg(fli1a:nGFP)y760, Tgubs361, Tg(gata1:dsRed)sd262, Tg(kdrl:cre)s89863, Tg(kdrl:GFP)s84364, Tg(kdrl:mKate2-CAAX)ubs1623, Tg(kdrl:Hras-mCherry)s89665, Tg(kdrl:tagBFP)mu29366, Tg(UAS:Kaede)rk867, Tg(UAS:VE-cadherinΔC-EGFP)ubs1223 and Tg(ve-cadBAC:ve-cadTS)uq11bh (referred as VE-cadTS)36. Mutants used in this study were esamaubs1924, gpr124s98412 and ve-cadherinubs835.Transgenic lines used in this study were Tg(UAS:CA-Rac1)mu211 and Tg(UAS:DN-Rac1)mu212 lines were generated by injection of plasmids, which were a gift from Gage Crump50, and according to standard transgenesis protocol68. Both constructs harbor α-crystallin:cerulean, inducing blue fluorescence in the lenses at 48 hpf. To overexpress CA-Rac1 and DN-Rac1 in the vasculature the respective fish were mated to Tgubs3;(kdrl:Hras-mCherry)s896. Please note, that in the case of DN-Rac1 overexpression, all embryos were presorted for established circulation in PHBC and BA prior to analysis by confocal microscopy as we most likely obtained multiple integrations of the construct and thereby varying phenotypes in the offspring embryos.The Tg(hsp70l:loxP-STOP-loxP-mCherry-dntcf)mu201 line was generated by fusing the coding sequences of mCherry69 in frame with dntcf70 downstream of a heat shock inducible promoter (hsp70l69). A floxed STOP cassette  was inserted upstream of mCherry by AflII-SpeI digest. Using the gateway system, hsp70l:loxP-STOP-loxP-mCherry-dntcf was cloned into the destination vector pTol2Dest71.The Tg(hsp70l:loxP-STOP-loxP-mCherry)mu279 line was generated by removing the dntcf sequence from Tol2-hsp70l:loxP-STOP-loxP-mCherry-dntcf-Tol2 using AfeI-SnaBI digest followed by Klenow blunting and religation.The 68.For both lines, standard transgenesis was performedcre mRNA (200 pg/nL) were injected at single cell stage. Therefore the cre coding sequence69 was cloned into pCS2+ and transcribed into mRNA by using the Sp6 mMessage mMachine kit (Ambion) following NotI-digest as previously described33.For ubiquitous removal of the floxed STOP cassette by Cre recombinase, 2 nL of s1pr1, s1pr2, s1pr3a, and s1pr5a were amplified from 24 hpf-cDNA and cloned into pCS2+ using the following primers  as described33. Thereafter, 2 nL of 75 ng/µL or 150 ng/µL s1pr1 mRNA, 10 ng/µL s1pr2 mRNA, 150 ng/µL s1pr3a or 150 ng/µL s1pr5a mRNA were injected at single cell stage as indicated for each experiment. For agonist experiments, 75 ng/µL s1pr1 mRNA was additionally supplemented with 200 µM Sphingosine-1-phospate .For overexpression of S1pr’s, the coding sequences of s1pr1, annealed template oligonucleotides were transcribed into gRNAs using MEGAshortscript T7 Kit (Ambion):s1pr1 5′- AAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAACTCTCTGAAGAACCATTGCGTTATAGTGAGTCGTATTACGC-3′ (s1pr1 target sequence: 5′-TCTCTGAAGAACCATTGCGT-3′). A total of 2 nL of 500 ng/µL gRNA and 300 ng/µL nls-zCas9-nls mRNA72 or only nls-zCas9-nls mRNA as control were injected at single cell stage. Efficacy of the CRISPR-targeting was accessed by PCR using s1pr1 fwd 5′-TCTATACTGCCAACATCCTG-3′ and rev 5′-CTGATGAGCATGAAGACCC-3′ mu201) were mated with Tg(kdrl:cre)s898. The resulting embryos carrying both transgenes were used for analysis and in the case of heat shock inducible transgenes were labeled by iEC .For vascular-specific removal of floxed STOP-cassettes, transgenic fish (e.g. Tg(hsp70l:loxP-STOP-loxP-mCherry-dntcf)mu201 and Tg(hsp70l:loxP-STOP-loxP-mCherry)mu279 lines, embryos were incubated at 10 hpf and 15 hpf for 1 h at 39 °C and analyzed at 24 hpf.In order to induce gene expression by heat shock, 26 hpf-old dechorionated embryos were incubated 40 min at 39 °C in pre-warmed E3 medium and analyzed at indicated developmental stages. For validation of the 33, 100 µM Chloroquine (Sigma-Aldrich)74, 10 µM CYM5541 (Sigma-Aldrich), 10 µM CYM5542 (Tocris Bioscience), 80 µM Dynasore (Sigma-Aldrich)75, 20 mM Hydroxyurea 33, 20 µM IWR-1 (Sigma-Aldrich)33, 50 µM JTE013 , 10 µM MG132 (Sigma-Aldrich)76, 100 µM NSC23766 , 5 µg/mL Rho Activator II (Cytoskeleton)59, 2 µg/mL Rho inhibitor CT03 (Cytoskeleton)59, 100 µM TY52156  and 50 µM VPC23019 . Tg(hsp70l:loxP-STOP-loxP-mCherry-dntcf)mu201 and Tg(hsp70l:loxP-STOP-loxP-mCherry)mu279 embryos were incubated directly after heat shock from 27 to 30 hpf with 12.5 µM and from 30 to 48 hpf with 25 µM VPC.For pharmacological treatments, dechorionated embryos were incubated from 5.5 to 24 hpf , from 29 to 48 hpf or from 26 to 32 hpf in E3 medium supplemented with 150 µM Aphidicolin To analyze blood vessel perfusion, 2 nL 40 kDa FITC-Dextran 40  were injected into the blood stream of anesthetized embryos at 48 hpf.77. Additionally, primary and secondary antibody dilutions were supplemented 1:1 with Pierce™ Immunostain Enhancer (Thermo Fisher Scientific). Antibodies used in this study: 1:400 rabbit anti-zf-VE-cadherin77, 1:1000 mouse anti–hZO-1 , 1:400 rabbit anti-Esama23, 1:200 rabbit anti-dnTcf , 1:2000 goat anti-rabbit IgG Alexa Fluor 594 , 1:2000 goat anti-mouse IgG Alexa Fluor 647 , 1:2000 goat anti-mouse IgG Alexa Fluor 488 .Immunohistochemistry was carried out as previously described79. Briefly: MeOH-fixed embryos were rehydrated, digested with Proteinase K  for 12 min and refixed in 4% PFA. After washing with PBS containing 0.3% (v/v) Tween and pre-hybridization at 65 °C, embryos were incubated hybridization buffer containing digoxigenin (DIG)-labeled antisense probes at 65 °C for >12 h. Probe removal, blocking, and primary antibody incubation was performed according to the published protocol. Primary antibodies were POD-coupled anti-DIG  and anti-GFP , which was used for counter-staining GFP in transgenic embryos. After removal of the antibody solution, embryos were incubated for 60 min with TSA Plus Cy3 solution , washed three times with PBS-Tween to remove excess staining solution and subjected to 1:500 goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen) for 60 min. At last, embryos were washed with PBS-Tween >5 h and sequentially taken up in 75% Glycerol for confocal microscopy.Fluorescent in situ hybrisization (FISH) was adapted from refs. ve-cadherin80 and esama81 were generated from plasmid templates using T3 or Sp6 polymerase and DIG-labelled UTP  and purified by precipitation81.The antisense probes for Tgubs3;(UAS:kaede)rk8 embryos was performed using a Zeiss LSM 710 confocal microscope  after embryo anesthesia with a low dose of tricaine. Embryos were mounted laterally in 1% low-melting agarose in glass-bottom Petri dishes . Photoconversion of Kaede fluorescent protein was performed by scanning the selected region of interest with a 405 nm diode laser . After photoconversion, embryos were manually removed from the agarose and placed in fresh egg water  additionally supplemented with N-Phenylthiourea  and covered with aluminum foil to protect from the light before trypsin treatment. After several washes in HBSS  without Ca2+/Mg2+, zebrafish embryos dissociation was performed at 28.5 °C during 30 min using 2 mL of trypsin-LE select (Gibco). Dissociation was stopped by added 200 µL of FBS solution. After centrifugation, pellet was resuspended with HBSS with Ca2+/Mg2+ +5% FBS and filtered. Photoswitched ECs were isolated on FACS Aria II. Cells were immediately centrifuged, frozen by liquid nitrogen and stored at −80 °C.Photoswitching of http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reaper version 13–100 was employed to trim reads after a quality drop below a mean of Q20 in a window of 10 nucleotides82. Only reads between 30 and 150 nucleotides were cleared for further analyses. Trimmed and filtered reads were aligned versus the Zebrafish genome version DanRer10 (GRCz10.87) using STAR 2.4.0a with the parameter “—outFilterMismatchNoverLmax 0.1” to increase the maximum ratio of mismatches to mapped length to 10%83. The number of reads aligning to genes was counted with featureCounts 1.4.5-p1 tool from the Subread package84. Only reads mapping at least partially inside exons were admitted and aggregated per gene. Reads overlapping multiple genes or aligning to multiple regions were excluded. The Ensemble annotation was enriched with UniProt data (release 06.06.2014) based on Ensembl gene identifiers ).For RNA-seq, RNA was isolated from FACS sorted zebrafish CtAs ECs cells using the miRNeasy micro Kit (Qiagen) combined with on-column DNase digestion  to avoid contamination by genomic DNA. RNA and library preparation integrity were verified with a BioAnalyzer 2100 (Agilent) or LabChip Gx Touch 24 (Perkin Elmer). RNA amount was adjusted on number of isolated cells by FACS and ~250–500 pg total RNA was used as input for SMART-Seq® v4 Ultra® Low Input RNA Kit (Takara Clontech) for cDNA pre-amplification. Obtained full-length cDNA was checked on LabChip and fragmented by Ultrasonication by E220 machine (Covaris). Final Library Preparation was performed by Low Input Library Prep Kit v2 (Takara Clontech). Sequencing was performed on the NextSeq500 instrument (Illumina) using v2 chemistry, resulting in minimum of 30M reads per library with 1 × 75 bp single end setup. The resulting raw reads were assessed for quality, adapter content, and duplication rates with FastQC , and cDNA was generated using the SuperScript II reverse transcriptase kit (Invitrogen). Real-time quantitative PCR (RT-qPCR) was conducted using Power SYBR Green (Applied Biosystems) and the following primers CreERT286 mice were interbred. The resulting offspring were injected at P1 and P2 with 50 µg of 4-OHT and samples processed at P3.For immunostaining or gene expression analysis of wild-type primary endothelial cells, C57BL/6J pups were used. To inactivate S1pr1 in the postnatal endothelium, 34. In brief, brains were collected from mice at postnatal day 3 (P3) or adults (>8 weeks old) in ice-cold Dulbecco’s Modified Eagle’s Medium  supplemented with 1% penicillin/streptomycin  and L-Glutamine , hereafter termed dissection media. Brains were transferred to a Petri dish, where olfactory bulbs and cerebellum were removed. The hemispheres were mechanically disrupted to small pieces and further digested for 10 min at 37 °C in 0.5 mg/mL collagenase  dissolved in dissection media . Next, the cell suspension was mixed with an equal volume of DMEM supplemented with 20% fetal bovine serum , filtered through a 70 μm nylon cell strainer  and centrifuged 5 min at 300 g. After centrifugation, the cell pellet was resuspended in dissection media supplemented with 0.5 mg/mL heparin  and incubated for 40 min with CD31  -bound sheep anti-rat IgG magnetic Dynabeads  on a rotary wheel at room temperature. After incubation, microvascular fragments bound to magnetic beads were retrieved using a DynaMag-2 magnet , washed 5 times with heparin-supplemented dissection media and finally resuspended in TrypLE Select  in order to dissociate them from the magnetic beads. After 12 min incubation at 37 °C, unbound beads were removed with the magnet and the suspension containing the microvascular fragments was mixed with EGM2-supplemented EBM2 media . After centrifugation  the microvascular fragments were resuspended in EGM2–EBM2 media supplemented with puromycin  and plated on collagen-I-coated μ-Slide 8-well ibiTreat cell culture chambers  or 24-well cell culture plates. Hereafter, cells were kept in culture at 37 °C and 5% CO2. Twenty hours later, wells were washed three times with PBS to remove dead and unattached cells and fresh EGM2–EBM2 media supplemented with puromycin (5 μg/mL) was added. The next day, pharmacologic treatment started with drugs diluted in EGM2–EBM2 media.Mouse brain primary microvascular fragment isolation and culture was performed as describedIWR-1 (Sigma-Aldrich) and VPC23019  were dissolved in DMSO to a 10 mM concentration. Inhibition of Wnt signaling was achieved after 48 h treatment with 10 or 20 μM IWR-1 for endothelial cells from young pups or adult animals, respectively. Alternatively, 10 μM IWR-1-treated cells from young pups were exposed in parallel to 2 μM VPC to assess the effect of S1pr1 signaling blocking upon Wnt inhibition. Vehicle (DMSO) treated was used in all experiments as control.After inhibitor treatment, cells growing in 8-well ibiTreat cell culture chambers were fixed in 4% paraformaldehyde  for 15 min at room temperature, washed three times with PBS and permeabilized with 0.2% Triton X-100  in PBS for 15 min. After washing three times with PBS, cells were blocked by incubation with 2% normal donkey serum  in PBS in a humidified chamber for 30 min. Primary antibodies  were diluted in blocking buffer and incubated with the cells in a humidified chamber overnight at 4 °C. Wells were then washed three times with PBS and secondary antibodies  together with DAPI  were diluted in blocking buffer and incubated at room temperature for 2 h. After secondary antibody incubation, cells were washed as described above and covered with PBS until imaging.β-mercaptoethanol  and total RNA was isolated using the RNeasy Plus Micro Kit  following the manufacturer’s instructions. Whole RNA was assessed in terms of quantity and quality using Bioanalyzer 2000 (Agilent), reverse transcribed and converted to complementary DNA (cDNA) using the iScript cDNA synthesis kit . The following FAM-conjugated TaqMan gene expression probes  were used: VE-cadherin , Axin2 (Mm01265780_m1), Lef1 (Mm01310389_m1), and Ccnd1 (Mm00432360_m1). VIC-conjugated Actb (4352341E) TaqMan probe was used to normalize gene expression. Quantitative PCR (qPCR) reactions were performed on a CFX96 Touch Real-Time PCR Detection System (BioRad) using the Sso Advanced Universal Probes Supermix . All relative gene expression analyses were performed using the 2−ΔΔCt method in a minimum of four animals per group with triplicate reactions for each gene evaluated.After inhibitor treatment, cells growing in 24-well cell culture plates were collected in 350 μL RLT Plus Buffer  supplemented with 1% 59. For time-lapse analysis, the agarose was additionally supplemented with IWR-1 or DMSO and a stable temperature of 28.5 °C was maintained using a heating chamber. Assembly of confocal stacks and time-lapse movies was conducted using Imaris 8/9 software (Bitplane). Quantification of signal intensity and volume was done using the Imaris surface-rendering algorithm.Confocal microscopy was performed using LSM780 and LSM880 microscopes . For filopodia analysis in zebrafish at high resolution, images were acquired with the LSM880 Airyscan module. For life imaging of VE-cadherinTS embryos, the Online Fingerprinting mode of the LSM880 microscope was utilized. In general, PFA-fixed or living zebrafish embryos were embedded in 0.3% agarose, which was dissolved in E3 medium and additionally supplemented with N-Phenylthiourea  and Tricaine  for living embryos as previously describedp-values were calculated using unpaired two-tailed Student’s t-test or one-way ANOVA for single and multiple comparisons as indicated for each experiment.For all quantifications, statistical analysis was performed using Prism6 software (GraphPad). Graphs show mean ± standard deviation (SD) or standard error of the mean (SEM) and All animal experiments were performed in compliance with the relevant laws and institutional guidelines, were approved by local animal ethics committees and were conducted at the University of Münster and the MPI for Molecular Biomedicine with permissions granted by the Landesamt für Natur, Umwelt und Verbraucherschutz (LANUV) of North Rhine-Westphalia.Supplementary informationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6Supplementary Movie 7Supplementary Movie 8Supplementary Movie 9Supplementary Movie 10Supplementary Movie 11Supplementary Movie 12


Serological screening for human T-cell lymphotropic virus type 1 (HTLV-1) is usually performed using enzyme-linked immunosorbent assay (ELISA), particle agglutination, or chemiluminescence assay kits. Due to an antigen matrix improvement entailing the use of new HTLV antigens and changes in the format of HTLV screening tests, as well as newly introduced chemiluminescence assays (CLIAs), a systematic evaluation of the accuracy of currently available commercial tests is warranted. Serological screening for human T-cell lymphotropic virus type 1 (HTLV-1) is usually performed using enzyme-linked immunosorbent assay (ELISA), particle agglutination, or chemiluminescence assay kits. Due to an antigen matrix improvement entailing the use of new HTLV antigens and changes in the format of HTLV screening tests, as well as newly introduced chemiluminescence assays (CLIAs), a systematic evaluation of the accuracy of currently available commercial tests is warranted. We aimed to assess the performance of commercially available screening tests for HTLV infection diagnosis. A diagnostic accuracy study was conducted on a panel of 397 plasma samples: 200 HTLV-negative plasma samples, 170 HTLV-positive plasma samples, and 27 plasma samples indeterminate by Western blotting (WB). WB-indeterminate samples  were assessed by PCR, and the results were used to compare agreement among the commercially available ELISA screening tests. For performance analysis, WB-indeterminate samples were excluded, resulting in a final study panel of 370 samples. Three ELISA kits  and one CLIA kit (Architect rHTLV-1/2) were evaluated. All screening tests demonstrated 100% sensitivity. Concerning the HTLV-negative samples, the SYM Solution and Gold ELISA kits had specificity values of >99.5%, while the Architect rHTLV-1/2 test presented 98.1% specificity, followed by Murex, which had a specificity of 92.0%. Regarding the 27 samples with WB-indeterminate results, after PCR confirmation, all ELISA kits showed 100% sensitivity but low specificity. Accuracy findings were corroborated by the use of Cohen's kappa value, which evidenced slight and fair agreement between PCR analysis and ELISAs for HTLV infection diagnosis. Based on the data, we believe that all evaluated tests can be safely used for HTLV infection screening. Human T-cell lymphotropic virus type 1 (HTLV-1) and type 2 (HTLV-2) were identified in 1980 and 1982, respectively , 4. It h5–8–HTLV-2 infection is endemic in native Amerindian populations in both North and South America, certain tribes of Pygmies in Africa, and intravenous drug users (IDUs) in urban areas around the world , 16. In Brazil, a country of 200 million inhabitants, has a population of 800,000 who potentially harbor HTLV-1, representing one of the largest areas of endemicity for the virus and its associated diseases anywhere in the world . The virAchieving an accurate diagnosis of HTLV infection is a complex task. Serological screening for HTLV-1 is usually performed using enzyme-linked immunosorbent assay (ELISA), particle agglutination testing, or chemiluminescence assay (CLIA) kits. The Brazilian Ministry of Health recommends the use of ELISA or particle agglutination tests as a screening protocol. Western blotting (WB), or immunoblotting, is used for confirmation, and PCR is employed in the case of inconclusive confirmatory test results . Among t21–In light of this scenario, we endeavored to conduct a systematic evaluation of the performance of commercial screening test kits for HTLV infection diagnosis. Statistical tools were used to obtain a robust assessment of the performance of each serological test by determining the following diagnostic test parameters: sensitivity (the probability of a test being positive in the presence of infection) and specificity (the probability of a test being negative in the absence of infection).The present research protocol was approved by the Institutional Research Board (IRB) of the Bahiana School of Medicine and Public Health (EBMSP) in Salvador, Bahia, Brazil (protocol no. 464.286). All procedures were performed in accordance with the principles established in the Declaration of Helsinki and its subsequent revisions.The present diagnostic accuracy study was carried out between February 2015 and December 2017 using anonymous plasma samples obtained from the biorepository of the Integrated and Multidisciplinary HTLV Center (CHTLV) at EBMSP. CHTLV is an outpatient clinic, open to the public, that provides interdisciplinary care and services, including general medical treatment, laboratory diagnosis, psychological counseling, and physical therapy. All included plasma samples had previously been screened for antibodies against HTLV-1/2 using an enzyme-linked immunosorbent assay , and reactive samples were retested by Western blotting . Test results were interpreted according to the stringent criteria indicated by the manufacturer and in accordance with the guidelines established by the Brazilian Ministry of Health , and 27 WB-indeterminate plasma samples. Briefly, HTLV-negative samples were defined as those lacking reactivity to HTLV-specific proteins; HTLV-1-positive samples were defined as those reactive to Gag (p19 with or without p24) and two Env proteins (GD21 and rgp46-I); HTLV-2-positive samples were those reactive to Gag (p24 with or without p19) and two Env proteins (GD21 and rgp46-II); HTLV-seropositive samples were those reactive to Gag (p19 and p24) and Env (GD21); and samples were considered indeterminate when no HTLV-specific bands were detected; i.e., the criteria for HTLV-1, HTLV-2, or HTLV were not satisfied. Indeterminate samples were assessed by PCR analysis, and the results obtained were used to compare the agreement with ELISA screening test results. For performance analysis, the WB-indeterminate samples were excluded, forming a final study panel of 370 samples .Alternatively, 217 plasma samples  were also assessed by a chemiluminescence assay .Three HTLV-1/2-specific enzyme-linked immunosorbent assay kits, all commercially available in Brazil, were employed in this study: Murex HTLV-1/2 , anti-HTLV-1/2 SYM Solution , and Gold ELISA HTLV-1/2 . Cutoff values, as well as gray zones, were calculated for each test as follows: by adding 0.2 to the mean for the negative-control replicates for Murex HTLV-1/2, by adding 0.18 to the mean for the negative-control replicates for anti-HTLV-1/2 SYM Solution, and by adding 0.25 to the mean for the negative-control replicate for Gold ELISA HTLV-1/2. For data normalization, all results were expressed by plotting values in an indexed format, calculated as the ratio between a given sample's optical density (OD) and the cutoff OD values for each assay. Under this index, referred to as a reactivity index (RI), all results with an RI of <1.00 were considered negative. When a sample's RI value was 1.0% ± 10%, the result was considered indeterminate , and the results for these samples were deemed inconclusive.Peripheral blood mononuclear cells (PBMC) from 27 patients with WB-indeterminate results were obtained from EDTA blood samples under density gradient centrifugation; DNA was extracted using a spin column kit . DNA samples were submitted to nested PCR using HTLV-1 long terminal repeat (LTR) 5′ region-specific primers as described previously , outer pt test, was used. When the assumed homogeneity was not confirmed, Wilcoxon's signed-rank test was used. All analyses were two-tailed, and P values under 5% were considered significant (P < 0.05). Enzyme-linked immunosorbent assay performance was computed using a dichotomous approach, and the performance characteristics of the various assays were compared in terms of sensitivity, specificity, accuracy, likelihood ratio (LR), and diagnostic odds ratio (DOR). Additionally, receiver operating characteristic (ROC) curves were constructed, and the areas under these curves were used as a global measure of test performance. Confidence intervals (CI) were employed at a confidence level of 95%. The strength of agreement between the results of the screening commercial tests and the PCR results was assessed by the use of Cohen's kappa coefficient (κ) . Descriptive statistics are presented as the geometric means ± standard deviations. To test data normality, the Shapiro-Wilk test, followed by Student's ient (κ) , which aient (κ)  has beenient (κ) .Using plasma from 170 HTLV-positive individuals, the performance characteristics of the ELISAs and CLIA were assessed, as shown in As all test kits demonstrated 100% sensitivity, no statistically significant differences were detected. Regarding the HTLV-1/2-negative samples, SYM Solution and Gold ELISA presented specificity values of >99%. Architect rHTLV-1/2 showed a specificity of 98.1%, followed by Murex, which showed a specificity of 92.0%. The differences in specificity and RI between the SYM Solution and Gold ELISA kits were not statistically significant. With respect to the HTLV-negative samples, the maximum RI value was obtained using Murex (RI = 0.30) . When RIAnalysis of the diagnostic accuracy of the three commercial ELISAs with respect to the 27 WB-indeterminate samples, considering PCR amplification as the gold standard for HTLV infection diagnosis, revealed that 8 samples were negative 29.6%) and 19 were positive (70.4%) for HTLV-1 , with al.6% and 1The present study found a high diagnostic value for each of the four different commercially available HTLV screening tests evaluated for the detection of anti-HTLV antibodies in Brazil. In fact, AUC values greater than 99% demonstrate convincing evidence of the optimal discriminative power of these kits regarding HTLV-positive and HTLV-negative samples. Both Gold ELISA and Architect rHTLV-1/2 presented AUC values of 100%. Furthermore, the Murex, SYM Solution, and Architect rHTLV-1/2 assays did not show inconclusive results (gray zone) in HTLV antibody screening procedures. The Gold ELISA yielded a low number of inconclusive results, as only 1 out of 170 HTLV-positive samples tested using this kit produced an RI value that fell in the gray zone.All tests displayed 100% sensitivity in detecting HTLV-positive samples. RI values were higher than 14 for the ELISAs and above 90 for the Architect rHTLV-1/2 kit, which corroborates previous reports . RegardiDue to the high number of samples (4.3%) misdiagnosed by the Murex test, its accuracy was significantly lower than that of the other kits. Gold ELISA, SYM Solution, and Architect rHTLV-1/2 were all found to be 99% accurate, suggesting that these kits can be safely employed for HTLV infection screening. Although the Murex test was less accurate, it nonetheless returned values above 95%, indicating suitability for the diagnosis of HTLV infection; however, the proportion of samples requiring WB confirmation was greater, which increases the cost of performing a diagnosis. In fact, 8% of the HTLV-negative samples assayed with Murex yielded false-positive results, with a specificity of 92%. It is interesting to note that this test's performance has improved over time, as studies performed in 2007 and 2009 described its sensitivity and specificity to be 98.2% and 42.6%, respectively , 33. AnoOf note, the sensitivity, specificity, and accuracy values associated with diagnostic tests are unsatisfactory in terms of influencing clinical decisions . A diagnIt is important to note that, concerning the Architect rHTLV-1/2 test, our findings are in agreement with those reported by other studies. In fact, identical values of sensitivity (100%) and specificity (>99%) have been described for samples from both blood donors and hospitalized patients . Similar42–The results presented herein indicate that all evaluated kits can safely be used for HTLV infection screening. However, it is important to note that the high sensitivity offered by these kits may lead to false-positive results, which could increase the cost of testing as a result of WB confirmation requirements. From the perspective of large diagnostic centers and blood banks, proper screening method selection can substantially reduce the costs associated with confirmatory testing. In an effort to reduce costs and ensure a correct diagnosis, a new diagnostic protocol for HTLV infection diagnosis was proposed by Costa et al. , who sugDespite the scarcity of studies evaluating the diagnostic performance of screening tests in diagnosing HTLV infection by employing LR, DOR, and AUC as performance parameters, we evaluated three ELISAs and one CLIA for HTLV infection screening. Based on the present findings, we conclude that all of the commercially available 3rd-generation kits employed herein presented sensitivity and specificity values higher than those found in previous studies. Among the ELISAs evaluated, the Gold ELISA HTLV-1/2 kit offered the best performance parameters, while Architect rHTLV-1/2 demonstrated the highest performance of all the assays considered. The high sensitivity values produced by screening tests could lead to high proportions of false-positive results. Thus, we reinforce our previous suggestion and urge the consideration of a new protocol employing molecular biology or line immune assay (INNO-LIA HTLV I/II Ab) techniques as a first choice for confirmatory testing in place of WB.


Interestingly, in an ovariectomized mouse model, RANK conditional knock-out mice exhibit resistance to ovariectomy-induced bone loss relative to the sham-operated mice. This study reveals that RANKL forward signaling in BMSCs functions as a negative regulator in osteoblast differentiation and bone formation  and is decreased during osteogenic differentiation. RANK silencing significantly promotes, while overexpression suppresses, the osteoblast differentiation of BMSCs in vitro. Mice with a conditional knock-out of RANK in MSCs  to inhibit RANKL reverse signaling but not forward signaling. Bone formation is disrupted in RANKLP29A mice compared with wild-type mice after recombinant RANKL is administered. At last, the authors show that targeting RANKL reverse signaling prevents decreased bone formation by compensating for the shortage of coupling signals. The results suggest that RANKL reverse signaling is involved in the bone formation as a potential pharmacological target.RANKL belongs to the tumor necrosis factor family and its bidirectional signaling has been indicated.r 2 Fig. . In vivoThese two studies convincingly demonstrate the functions of RANKL-RANK forward and reverse signaling in the regulation of osteoblast differentiation and bone formation. Chen et al. show that RANKL binds to RANK and inhibits osteoblastogenesis, while Yuki et al. demonstrate that vesicular RANK from maturing osteoclasts promotes osteoblastogenesis for the bone formation through RANKL reverse signaling. Specifically, RANKL forward signaling activates NF-κB for degradation of β-catenin. In RANKL reverse signaling, a proline-rich motif in the RANKL cytoplasmic tail interacts with Src homology 3 domains and activates PI3K.In summary, these two reports provide evidence that RANKL-RANK forward and reverse signaling regulates osteoblast differentiation and bone formation. The forward signaling inhibits osteogenic differentiation and the reverse signaling promotes osteoblast differentiation for bone formation Fig. . Not onl