A 3-year-old neutered male domestic shorthair cat developed pancytopenia 6 months after starting phenobarbital for treatment of recurrent seizures. The cat was switched from phenobarbital to levetiracetam and complete resolution of the pancytopenia was documented within 10 weeks, consistent with phenobarbital-induced pancytopenia.While phenobarbital is frequently used as the first-line treatment for seizures in cats, phenobarbital-induced feline pancytopenia has not been documented in the veterinary literature before. Based on this case, regular monitoring of the complete blood count in cats receiving long-term phenobarbital treatment should be considered. In cases of persistent or severe haematological abnormalities, further investigations are required and treatment discontinuation may be needed in the absence of other causes of pancytopenia. A complete blood count (IDEXX Laboratories) documented a poorly regenerative anaemia ,2 mature neutropenia (0.13 × 109/l [RI 2.62–15.17 × 109/l]) and thrombocytopenia (44 × 109/l [RI 155–641 × 109/l]). The cat was receiving phenobarbital  (3 mg/kg PO q12h) owing to a 6-month history of generalised tonic–clonic seizures suspected to be due to idiopathic epilepsy. Baseline investigations prior to starting phenobarbital treatment had been performed 6 months previously, including complete blood count, biochemistry, electrolytes, thyroxine, feline leukaemia virus (FeLV), feline immunodeficiency virus (FIV), coronavirus and Toxoplasma gondii serological testing, all of which were normal. The phenobarbital serum levels at the time of referral to our hospital were 25.9 mg/l (therapeutic RI 10–30 mg/l) and the most recent seizure episode was 2 months ago. No toxin or other drug exposure was reported and there was no travel history outside of the UK.A 3-year-old neutered male domestic shorthair cat was referred to our hospital with a 1-week history of hyporexia and lethargy. The primary care veterinary surgeon had performed a physical examination that revealed pale mucous membranes, an increased rectal temperature of 39.6°C 1 and pale mucous membranes. Repeat complete blood count (Langford Vets Diagnostic Laboratories) documented a worsening regenerative anaemia  thrombocytopenia  and a persistent mature neutropenia (0.58 × 109/l [RI 3–13.4 × 109/l])  for the management of seizures. Treatment with doxycycline (10 mg/kg PO q24h) was initiated while waiting for outstanding results and discontinued after 7 days upon the receipt of negative feline Haemoplasma PCR testing. Furthermore, broad-spectrum antibiotic cover with amoxycillin/clavulanic acid (20 mg/kg PO q8h) was started in the light of the pyrexia and risk of sepsis associated with severe neutropenia. Further investigations including advanced imaging of the thorax and abdomen and bone marrow biopsy were offered but declined by the owner owing to financial constraints. Repeat complete blood count (Langford Vets Diagnostic Laboratories) 5 days after presentation to our hospital documented static regenerative anaemia , worsened mature neutropenia (0.16 × 109/l [RI 3–13.4 × 109/l]) and improved thrombocytopenia   109/l]) ; day 1. per hpf) ; day 5. 9/l [RI < 50.0 × 109/l]), improved mature neutropenia (0.43 × 109/l [RI 2.62–15.17 × 109/l]) and improved thrombocytopenia  (9/l [<50.0 × 109/l]), normal neutrophil count (4.45 × 109/l [2.62–15.17 × 109/l]) and improved thrombocytopenia  (9/l (RI < 50.0 × 109/l), neutrophil count 2.93 × 109/l (RI 2.62–15.17 × 109/l] and platelet count 140 × 109/l (RI 155–641 × 109/l) and an estimate of 10 platelets per hpf  documented a normal haematocrit  ; day 14.per hpf) ; day 42. per hpf ; day 80.3 Previously reported causes of pancytopenia in cats include infectious agents, immune-mediated diseases, myelofibrosis, neoplasia, and toxin-induced and drug-induced pancytopenia.4 The drugs that have been previously associated with pancytopenia in cats are griseofulvin,5 albendazole,6 azathioprine7 and doxorubicin.8 To our knowledge, phenobarbital has not previously been reported to cause pancytopenia in cats, although it has been previously documented in dogs.913 We report a cat on long-term phenobarbital treatment that developed marked pancytopenia, which resolved after phenobarbital discontinuation.Pancytopenia is defined as the combination of anaemia, neutropenia and thrombocytopenia, and is indicative of bone marrow suppression.1418 The drug-related adverse effects can be divided into type A and type B.19 Type A includes the adverse effects that can be explained by the known pharmacological properties of the agent and therefore are usually dose dependent and predictable. Type B adverse reactions are independent from the known mechanism of action of the drug and the drug dose and are therefore unpredictable.19 Type A adverse effects of phenobarbital reported in cats include sedation, pelvic limb ataxia, paraparesis, polyphagia, polydipsia, weight loss, increased alanine transaminase and alkaline phosphatase activities, and behavioural changes.20 Type B adverse effects reported in cats include dermatitis, pruritus, stomatitis, hypersensitivity,21 pseudolymphoma,22 fever,23 coagulopathies, leukopenia and thrombocytopenia.15 According to the existing literature, haematological abnormalities were mild, transient and did not cause a treatment discontinuation.20 In our case, the severe pancytopenia and associated hyporexia and fever required hospitalisation, phenobarbital discontinuation and broad-spectrum antibiotic coverage. In one recent study regarding the follow-up of cats on phenobarbital treatment, the adverse effects tended to be more severe at the beginning of treatment and at higher doses, although haematological adverse effects were uncommon and the timing of these was not specifically described.20 In dogs, the time to onset of pancytopenia has been reported to be most common within the first 3 months after initiation of phenobarbital treatment.19 In our case report, the cat developed pancytopenia after 6 months of treatment and in spite of the fact that the phenobarbital dose was within the recommended range, suggesting that not only short-term, but also long-term, regular monitoring of the complete blood count is recommended.Phenobarbital is a barbiturate commonly recommended as a first-line drug for cats with recurrent seizures.24 although both myelofibrosis and bone marrow necrosis have been documented in dogs receiving phenobarbital treatment.25 The proposed mechanisms extrapolated from human medicine are either an immune-mediated destruction of the precursor cells in the bone marrow or direct toxic effects of the drug on the bone marrow.26 Phenobarbital has been reported to induce cytochrome P450 enzymes in dogs, which can enhance the drug metabolism of phenobarbital itself.28 Interestingly, phenobarbital does not induce these enzymes in cats,12 which could theoretically lead to higher plasma concentrations and therefore more likely adverse drug effects. However, this is not reflected in the frequency of reported cases in the literature of adverse effects associated with phenobarbital in cats and the cat reported here had a measurably normal serum phenobarbital concentration.The exact mechanism of phenobarbital-induced pancytopenia is not well clarified,15 Despite that, the results suggested levetiracetam to be the next most effective treatment after phenobarbital.15In our case report, levetiracetam was selected to replace the phenobarbital monotherapy treatment. A recent systematic review in feline antiepileptic drugs found overall weak evidence to support the safety and efficacy of different anticonvulsant options.29 although the safety of such a technique has not been demonstrated in cats. However, bone marrow aspiration was declined on financial grounds in this cat. Nevertheless, the resolution of the cat’s pancytopenia following discontinuation of phenobarbital, in the absence of other treatment modalities for pancytopenia, indicates that phenobarbital was the cause. Furthermore, the cat was presumed to have idiopathic epilepsy based on the clinical response to anticonvulsants and the unremarkable blood testing but advanced brain imaging, cerebrospinal fluid analysis and liver function testing would, ideally, have been performed to rule out underlying diseases. Nonetheless, this had no influence on the fact that the phenobarbital appears to have caused the pancytopenia in this case.A few limitations apply to this case report, mainly owing to cost constraints. First, bone marrow biopsy and advanced thoracic and abdominal imaging would be required to fully rule out other causes of pancytopenia. A novel bone marrow aspiration technique via a costochondral rib using local anaesthesia alone has been proposed in one case report of a dog and may be useful in patients too unstable to undergo general anaesthetic,To our knowledge, this is the first report of suspected phenobarbital-induced pancytopenia in a cat. Considering that this possible adverse effect may be life-threatening, regular ongoing monitoring of the complete blood count of all cats treated with phenobarbital should be considered. If any haematological abnormalities identified are persistent or severe, further investigations are required and phenobarbital discontinuation may be needed in the absence of other causes of pancytopenia.


It is unknown whether low serum levels of salivary and pancreatic amylases are associated with the high combustion of carbohydrates or lipids for energy. Elevated blood ketones and a low respiratory quotient (RQ) can reflect the preferential combustion of lipids relative to carbohydrates. Therefore, using the data from our previous study, we investigated if low levels of serum amylases were associated with a high serum ketone level and low RQ in 60 healthy non-obese young women aged 20–39 years old.Serum ketones [3-hydroxybutyric acid (3-HBA) and acetoacetic acid (AA)] were inversely correlated with RQs, but not body mass index (BMI) or glycated haemoglobin (HbA1c) levels. Logistic regression analysis showed that high levels of serum ketones (3-HBA ≥ 24 μmol/L and AA ≥ 17 μmol/L) and a low RQ (< 0.766) were significantly associated with low serum salivary (< 60 U/L) and pancreatic (< 29 U/L) amylase levels, respectively. These associations were not altered by further adjustments for age, BMI, HbA1c, and estimated glomerular filtration rate. These results confirm the high combustion of lipids for energy in individuals with low serum amylase levels, suggesting a close relationship between circulating amylases and internal energy production. Although many clinical studies have shown that low serum amylase is significantly associated with diabetes and obesity [We previously showed an association between low serum amylase and positive ketonuria assessed by dipstick urinalysis in a general population of Japanese adults who underwent a health-screening check-up. These results suggested a lower availability of carbohydrates for energy production in individuals with low serum amylase levels . Howeverabolized , 8. In oabolized , we foun obesity , 11, und obesity , we aime obesity .2), and had no history of metabolic disorders including diabetes and dyslipidemia.We reanalysed the data from our previous study  that con1c), ketones (3-HBA and AA), and amylase , were measured using standard methods by SRL, Inc., a Japanese clinical laboratory test company. HbA1c (Japan Diabetes Society) was converted to HbA1c  [Anthropometric and laboratory measurements were obtained in the morning following an overnight fast. Biochemical parameters, including glycated haemoglobin (HbAProgram) . RQs at Program) , 14. eGFProgram) .\documenBecause the distributions of serum ketones and RQs were expected to be skewed, the correlations of these were tested by Spearman’s rank correlation. Since the median levels of 3-HBA, AA, serum salivary amylase, and serum pancreatic amylase were 24 µmol/L, 17 µmol/L, 60 U/L, and 29 U/L, respectively, high levels of serum 3-HBA and AA and low levels of serum salivary and pancreatic amylases were determined as ≥ 24 µmol/L, ≥ 17 µmol/L, < 60 U/L, and < 29 U/L, respectively, in this study. The proportion of serum salivary amylase in total  serum amylase was also considered. Low proportion of serum salivary amylase was determined as < 66%, which was the mean of proportion of serum salivary amylase.The RQ is usually divided into tertile in terms of the combustion of the three macronutrients  [The characteristics of the participants were reported in our previous study , which iAlthough data is not shown, serum ketones were significantly correlated with RQ  but not BMI  or HbA1c . In addition, no significant correlation was observed between RQ and BMI  or between RQ and HbA1c .Logistic regression analysis showed that low serum salivary amylase (< 60 U/L) was significantly associated with high levels of serum 3-HBA ≥ 24 μmol/L) and AA ≥ 17 μmol/L), but not with a low RQ (< 0.766) (Table 7 μmol/L,4 μmol/L In the current study, we found significant associations between low serum salivary amylase and high serum ketones, and between low serum pancreatic amylase and low RQs in healthy subjects. These associations were independent of relevant confounders including age, BMI, HbA1c, and eGFR. However, the proportion of salivary to total amylase was unlikely to relate with high serum ketones and low RQ, although the proportion of salivary amylase was correlated with the level of blood glucose at early time point after starch loading in our previous study .Because subjects in this study are healthy non-obese young female non-smokers, current results indicate fundamental relationship among serum salivary and pancreatic amylase and metabolic indices such as blood ketones and RQ. As both high serum ketones and low RQs reflect the high combustion of lipids compared with carbohydrates –6, our cNotably, in our previous study , no signIt has been shown that reduced rates of fat oxidation, namely, high RQ, may contribute to the predisposition to obesity or weight gain –18. HoweAlthough high levels of ketones often reflect a deficiency in insulin secretion in diabetic patients –4, no suAlthough current study indicates that fatty acids may be predominantly used as an energy source in individuals with low serum amylase, it is unclear if a diet rich in lipids is suitable for individuals with low serum amylase. Taken together, our studies suggest that a close relationship may exist between serum amylase and the specific type of macronutrient combustion used for energy.While increases in serum amylase occur because of leakage from salivary glands and the pancreas, the clinical relevance of this remains unknown. Circulating amylases might just be a marker of leaks or damage, albeit several investigators have suggested a feedback system between serum amylase and insulin action , 11, 21.In conclusion, the current results obtained from the reanalysis of our previous study confirm the high combustion of lipids for energy in individuals with low serum amylases, suggesting a close relationship between circulating amylases and internal energy production.First, although low serum amylases may be caused by insulin resistance and reduced insulin secretion , 21, insAdditional file 1: Figure S1. Distributions of serum amylases. A, serum salivary amylase; B, serum pancreatic amylase; C, serum total amylase.Additional file 2: Figure S2. Potential underlying mechanism between serum amylases and metabolic indices. *Baseline individual levels of serum amylases, which are genetically determined in most cases.


Vaccinium spp.) fruit. Until now, breeding selection was mostly oriented on the amelioration of agronomic traits, such as flowering time, chilling requirement, or plant structure. Up until now, however, the storage effect on fruit quality has not been extensively studied, mostly because objective and handy phenotyping tools to evaluate quality traits were not available. In this study we are proposing a novel phenotyping protocol to support breeding selection and quality control within the entire blueberry production chain. Volatile organic compounds (VOCs) and texture traits, were measured by Proton Transfer Reaction- Time of Flight- Mass Spectrometry (PTR-ToF-MS) and a texture analyzer respectively, taking into consideration the influence of prolonged storage. The exploitation of the genetic variability existing within the investigated blueberry germplasm collection  allowed the identification of the best performing cultivars, based on texture and VOCs variability, to be used as superior parental lines for future breeding programs. The comprehensive characterization of blueberry aroma allowed the identification of a wide array of spectrometric features, mostly related to aldehydes, alcohols, terpenoids, and esters, that can be used as putative biomarkers to rapidly evaluate the blueberry aroma variations related to genetic differences and storability. In addition, this study revealed a lack of straightforward relationship between harvest and postharvest quality features, that might be genotype-dependent.Improved fruit quality and prolonged storage capability are key breeding traits for blueberry ( Vaccinium spp.) production, for both processed and fresh market, has increased over the last decade making blueberry becoming the second most important soft fruit species after strawberry . Nonetheless, this progression mostly concerns certain production areas, situated for the most part in Peru, Mexico, Spain, Poland, South Africa, and China  synthesized by fruit during ripening . As mostIn order to satisfy consumer demands more effort and attention need to be devoted, from a scientific and practical background, to improve and optimize blueberry quality upon delivery to the consumers. The key attributes of quality may vary with context and depend on the intended use of the product and the available or affordable technology . DefininWhile taste traits (sweetness and sourness) are relatively well explained by the sugar content and titratable acidity, the prediction of aroma and texture traits seems more uncertain  because In this study, a wide blueberry germplasm collection, including southern and northern highbush, hybrids and rabbiteyes, was employed and assessed for both texture and aroma traits applying advanced phenotyping strategies preliminarily developed in Vaccinium accessions  to produce a 20 μM stock solution for fluorescently-labeled universal primer and locus-specific reverse primer and a 10μM stock solution of locus-specific forward primer with universal tail . Primer mix here designated Pmixlocus (three primers were mixed for each specific locus). Each Multiplex included equimolar amounts of the Pmixlocus.Multiplexed reactions (MVA and MVB) were carried out in 10ul reactions using Type-it™ Microsatellite PCR kit (Qiagen) according to the manufacturer instructions. Thermal cycling conditions for MVA were as follow: initial 3 min denaturation step at 94°C, eight touch-down cycles comprising a 30 s denaturation at 94°C followed by 90 s of annealing starting at 64°C and decreasing, 0.5°C per cycle, down to 60°C and 60 s of extension at 72°C. Subsequently, 25 more identical cycles were conducted with and annealing temperature of 60°C followed by a final 30 min extension step at 60°C. Thermal conditions for MVB changed only for the annealing temperature, starting at 60°C and decreasing 0.5°C per cycle, down to 60°C. Subsequently, 25 more identical cycles were conducted with an annealing temperature of 56°C followed by a final 30 min extension step at 60°C. Products from multiplexed reactions were checked on agarose gels and then diluted 1:50 prior to analysis and separated by electrophoresis on an ABI3730XL genetic analyzer (Applied Biosystems).Data generated were then acquired and analysed using the GENEMAPPER (Applied Biosystems) software and checked visually. Final allele sizes were thus obtained per locus and per blueberry accession after removing the length of each universal tail sequence.https://www.r-project.org).To investigate the genetic relationships between blueberry genotypes, the microsatellite dataset was analyzed using the Poppr package  in R  implemented in the Adegenet package ver. 2.0.1 . Prior cV.corymbosum) and hexaploid cultivars . The ‘‘bruvo.boot’’ command  with bootstrap support of 1000 replication was used to produce a neighbor joining tree with the ‘‘njs’’ algorithm from the ape package ver. 5.0 . All data were analyzed by TaxtExpertII software .Texture assessment was performed for each cultivar on 20 homogenous fruit at harvest and after storage. Texture was determined by a texture analyser , which profiled a mechanical force displacement using a 5 kg loading cell and a cylindrical flat head probe with a diameter of 4 mm entering into the berry flesh from the sagittal side  coupled to PTR-ToF-MS.Three biological replicates of 1 g of powdered frozen sample, each obtained by five fruit, were inserted into 20 ml glass vials equipped with PTFE/silicone septa  and mixed with 1 ml of deionized water, 400 mg of sodium chloride, 5 mg of ascorbic acid, and 5 mg of citric acid . MeasureThe analysis of PTR-ToF–MS spectra proceeded as described in The array of masses detected with PTR-ToF-MS was reduced by applying noise and correlation coefficient thresholds. The first removed peaks were not significantly different from blank samples; the latter excluded peaks with over 99% correlation, which correspond for the most part to isotopes of monoisotopic masses .2(QiPH/QiH), where QiH is the value of the i-th quality parameter measured at harvest, and QiPH is the value of the same parameter measured after cold storage. Positive SI values indicate a quality trait enhancement, while negative values highlight a loss of the quality trait during storage.For all quality parameters, both texture and volatiles, a storage index (SI) was computed using the formula proposed by R.3.4.1. R Foundation for Statistical Computing, Vienna, Austria) internal statistical functions and the external packages “mixOmics”, “FactoMineR”, and “ggplot2” were used for the multivariate statistical methods  employed in this work.MVA and MVB multiplexed reactions failed for two blueberry accessions [‘Azur’ (#4) and ‘Southern Belle’ (#43)] out of 46 samples and thus these accessions were not included in the genetic analysis. The obtained 44 molecular profiles were further used to identify K-clusters by successive K-means, resulting in the definition of four clusters Table S3Vaccinium germplasm collection may lead to a high phenotypic variance that can be enhanced and altered by prolonged cold storage. Phenotypic variance for texture qualities and for their alteration during cold storage (Vaccinium genotypes are predominantly related to the first component (PC1) that is highly correlated to the deformation forces, both maximum and minimum. In accordance with the results presented in The broad genetic variability revealed in our  storage Table 2,Vaccinium cultivars  between the storage index values of the deformation strain and deformation  computed on each texture parameter. As for the PCA based on the whole texture database Figure 2Four distinct clusters were statistically distinguished at harvest based exclusively on texture , acetaldehyde (m/z 45.031), C6-aldehydes , monoterpenes (m/z 137.13), benzoic compounds (m/z 107.087), butyrolactone (m/z 87.04), and sulfuric compounds . In agreement with m/z 75.043; 89.055; 103.07; 117.09; 131.107; 145.124) was very low.VOC profile, assessed at harvest, was characterised by mass peaks tentatively identified (t.i.) as methanol , m/z 99.08 , m/z 101.096 , m/z 133.103 , and m/z 137.134 . In contrast, fruit storage significantly enhanced the content of several alcohols  and esters . Ethanol (m/z 47.043) was one of the compounds that increased most (SI: + 6.68), followed by acetaldehyde , and by masses related with ester compounds in particular m/z 131.107 , m/z 117.092 , or m/z 89.055 .VOC profile changed significantly during storage; however, any additional mass peaks were not revealed solely after cold storage. Aldehydes and terpenes decreased during storage, as confirmed by the masses Vaccinium genotypes, especially at harvest, are mostly related to the second component (PC2). In contrast with the outcome of texture assessment  taken into consideration in this study. These results confirmed that the VOC profile of “Rabbiteye” blueberry cultivars is distinguishable from other Vaccinium species, particularly from V. corymbosum, for the most part due to a higher content of esters and alcohols  or ‘Northland’ (#31), are considerably different after storage. These differences may be the consequence of both ripening and senescence processes.m/z 33.033, Vaccinium germplasm is remarkably high at both harvest and storage assessments, without any well-defined trend due to fruit conservation. Several cultivars, such as ‘Compact’ (#13), ‘Centra Blue’ (#10), and ‘Jubilee’ (#24), showed very low concentration of methanol at both stages, while others, such as ‘Azur’ (#4), ‘Jersey’ (#22), and ‘Star’ (#44), are characterized by a severe increase due to storage, independently from the concentration assessed at harvest. On the contrary, several cultivars with high methanol content at harvest, such as ‘Toro’ (#46), ‘Liberty’ (#26), and ‘Top Hat’ (#45), significantly reduced their content after storage.Methanol , ‘Centurion’ (#11), and ‘Sky Blue’ (#42)], presented an extremely low concentration of acetaldehyde at harvest. Variation during storage is related to genotypes: results of this experiment showed cultivars with a low increase, such as ‘Centra Blue’ (#10), ‘Liberty’ (#26), and ‘Aurora’ (#3), and others with a more predominant increase, such as ‘Jersey’ (#22), ‘Star’ (#44), and ‘Safir’ (#40). Cultivars with a high acetaldehyde production had also a high content of methanol and ethanol, but no statistically significant positive correlation was found. Nevertheless, a significant positive correlation is obtained linking SI values of ethanol and acetaldehyde  is another ester compound measured at high concentration after storage, but only in a limited number of accessions (m/z 89.055 are positively correlated with the content of ethanol and methanol as evidenced in the cultivars ‘Jersey’ (#22), ‘Emerald’ (#20), ‘Cosmopolitan’ (#14), ‘Berkeley’ (#5), and ‘Star’ (#44) (Ethyl acetate (cessions . In thism/z 99.08) and hexanal (m/z 101.096), diminished during storage in a genotype dependent manner with cultivars, such as ‘Jersey’ (#22), ‘Toro’ (#46), ‘Star’ (#44), or ‘Berkeley’ (#5) characterized by a SI lower than -2, and, on the contrary, several nearly stable cultivars, such as ‘Darrow’ (#16), ‘Misty’ (#28), or ‘Bluecrop’ (#7) (m/z 99.08) despite their high values at harvest. On the contrary, other cultivars with low values at harvest, like ‘Biloxy’ (#6), ‘Centra Blue’ (#10), ‘Eliott’ (#19), ‘Rubel’ (#39), or ‘Sky Blue’ (#42), showed positive SI values and the highest concentrations after storage. In addition, reinforcing the high variability among the Vaccinium germplasm, there were also accessions with stable hexenal content during storage, independent of the concentration assessed at harvest, such as ‘Atlantic’ (#2), ‘Bluecrop’ (#7), ‘Brigitta Blue’ (#9), ‘Chandler’ (#12), ‘Darrow’ (#16), or ‘Northblue’ (#30).Aldehyde and terpene content normally decrease during the last ripening phases of blueberry fruit, as previously demonstrated by op’ (#7) . Based om/z 137.133) were the VOC class most negatively affected by fruit storage , ‘Rubel’ (#39), ‘Nui’ (#32), ‘Hortblue Poppins’ (#35), or ‘Atlantic’ (#2) were highly ranked at harvest but showed very low values after storage.Another relevant mass peak for the characterization of blueberry VOC profile was VOC profiling of the blueberry germplasm collection was further analyzed by separately considering the assessments at harvest and after storage, as formerly described for texture. Differently from texture results, the overall VOC profile variability was only partially explained by considering the first two principle components  and to the storage index (SI). Due to the high multidimensionality of VOC profiles, clusters, based on Euclidean’s distances, were not always properly separated over the bidimensional space produced by PCA analysis. In addition, excluding the three Vaccinium virgatum accessions, the profiling based on VOC production did not reveal any significant association with the genetic molecular profile based on six SSRs , terpenes (m/z 137.134), methanol (m/z 33.033) and methyl acetate (m/z 75.043). Clusters “Cl_1” and “Cl_4” were composed by cultivars with high content of C6 aldehydes and terpenes, such as ‘Toro’(#46), ‘Northland’(#31), ‘Puru’ (#37), ‘Cosmopolitan’ (#14) or ‘Jersey’(#22). On the contrary, accessions belonging to “Cl_3” and “CL_2”, for instance ‘Atlantic’ (#2), ‘Biloxi’ (#6), ‘Southern Belle’ (#43), or ‘Legacy’ (#25), had a less intense VOC profile. The three V.virgatum accessions which clustered into “Cl_5”, namely ‘Centra Blue’ (#10), ‘Centurion’ (#11), and ‘Sky Blue’ (#42), were characterized by very low concentration of terpenes and C6 aldehydes, and high content of acetaldehyde (m/z 45.031), ethanol (m/z 47.043) and esters .Five clusters of cultivars were statistically distinguishable at harvest based on VOC composition , C6 alcohols , terpenes (m/z 137.134), and with VOCs that were not so relevant to discern cultivars at harvest, principally ethanol (m/z 47.043), acetaldehyde (m/z 45.031), and several ester compounds . Based on that, Vaccinium accessions of this study were grouped into four clusters. Accessions with the most modified VOC profile were clustered into “Cl_3”. That cluster is composed only of five accessions, namely ‘Berkeley’(#5), ‘Cosmopolitan’(#14), ‘Emerald’(#20), ‘Jersey’(#22), and ‘Star’(#44), characterized by the highest concentrations of ethanol, acetaldehyde, methyl acetate (m/z 75.043) and ethyl acetate (m/z 89.055). This alteration of the VOC profile after postharvest storage may be linked to a raise in fermentation processes due to fruit overripening. Differently, VOC profile of “Cl_2” accessions was for the most part characterized by low concentrations of ethanol and acetaldehyde together with high contents of some ester compounds , especially the cultivars ‘Aurora’(#3), ‘Brigitta Blue’ (#9), ‘North Blue’(#30), ‘O’Neal’ (#33), and ‘Rubel’(#39). Cultivars of the two remaining clusters, “Cl_1” and “Cl_4”, were defined by low concentrations of most compounds, except for C6 aldehydes  and C6 alcohols .After fruit storage, the VOC variability expressed by the first two components of the PCA analysis increased up to 61% . Nonetheless, Vaccinium accessions considered in this study were statistically divided into five clusters based on Euclidean’s distances . Clusters “Cl_3” and “Cl_4” grouped cultivars (i.e. ‘Biloxi’ (#6), ‘Brigitta Blue’ (#9), ‘Emerald’ (#20), ‘Jersey’(#22), or ‘Star’ (#44)) characterized by high SI values of masses related to esters , acetaldehyde (m/z 45.031), and ethanol (m/z 47.043), but low SI values of C6 aldehydes . The latter cluster, “Cl_5”, included only the three V.virgatum cultivars  that differed from the other accessions for the higher stability of C6 aldehydes , C6 alcohols (m/z 83.085 and 85.100), ethanol (m/z 47.043), and acetaldehyde (m/z 45.031).The first two PCA components, assessed over the SI values, explained only 39% of the VOC profile variability  fruit ripening led to the induction in the expression of genes associated with cell wall modifications . Nonetheless, considering each quality trait independently i.e. . The conUntil now only the research of Analytical methodologies considered in this study may result in powerful tools for phenotyping quality traits and, at the same time, in developing genetic markers that help to screen blueberry populations. The phenotyping approach suggested in our research, detailed in Analytical detection of traits concerning fruit taste perception, for most part acidity and sweetness, can still be based on refractometer and titrator. Despite the simplicity and lack of sophistication, results obtained by these techniques are properly linked with consumer perception . NonetheFruit texture, is also one of the main quality traits driving consumer preference . In our Bearing in mind that the aim of VOC assessment is to obtain an objective estimation of the aroma perceived by the consumer during fruit consumption, we consider worthless the application of too aggressive chemical extraction methodologies (i.e. liquid-liquid extraction with hexane or dichloromethane). These methodologies are only necessary for the quantification of compounds at extremely low concentrations that, in case of blueberry fruit, might be under the perception threshold of the consumer. Furthermore, we need high resolution analytical techniques, able to identify and quantify, at once, molecules with different polarity and molecular weight (i.e. methanol and sesquiterpenes) present in a broad concentration range (from ppt to ppm). The extreme complexity of food aroma composition is a challenging issue for any existing analytical technology. The rapid development of mass spectrometry (MS) application in metabolomic studies had a significant impact in the field of VOC analysis. Most of the progresses of MS techniques are focused on instrumental improvements of mass resolution, mass accuracy, sensitivity, and enhanced reproducibility. PTR-MS is particularly suited to develop reliable food VOC fingerprints because it provides handier analytical information (concentration estimation and reduced fragmentation) in comparison with the application of MS-e-noses based on electron impact ionization . PTR-ToFVaccinium flavor improvement. A better understanding of genes and enzymes involved in the VOC production and textural modification is still needed. This could lead to genetic and environmental manipulations to optimize aroma at the time of consumption, following shipping and marketing. To this end, future breeding programs focused of prolonged fruit post-harvest storage need to consider blueberry VOC modification. This can be achieved only with a more informed and precise identification of the best performing cultivars to be used as superior parental lines in combination with a reliable phenotyping methodology and molecular markers.This investigation, together with recent findings on blueberry flavour , suggestAll datasets presented in this study are included in the article/BF designed the research, analyzed and interpreted data, and wrote the manuscript. FM carried out the genetic structure analysis. IK helped with PTR-ToF-MS data analysis. MA assessed the texture analysis and sampled the blueberries. FB guided the PTR-ToF-MS analysis and edited the manuscript. LG coordinated the work design and edited the manuscript.This work was financially supported by the AdP of the PAT (Provincia Autonoma di Trento) and partially by the project AppleBerry (L6/99 of the PAT).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Appropriate use criteria (AUC) have been developed in response to growth in cardiac imaging utilization and concern regarding associated costs. Cardiac computed tomography angiography (CCTA) has emerged as an important modality in the evaluation of coronary artery disease, however its appropriate utilization in actual practice is uncertain. Our objective was to determine the appropriate utilization of CCTA in a large quaternary care institution and to compare appropriate utilization pre and post publication of the 2013 AUC guidelines. We hypothesized that the proportion of appropriate CCTA utilization will be similar to those of other comparable cardiac imaging modalities and that there would be a significant increase in appropriate use post AUC publication.We employed a retrospective cohort study design of 2577 consecutive patients undergoing CCTA between January 1, 2012 and December 30, 2016. An appropriateness category was assigned for each CCTA. Appropriateness classifications were compared pre- and post- AUC publication via the chi-square test.p < 0.001). The increase in appropriate utilization, when extrapolated to the Medicare population of the United States, was associated with potential cost savings of approximately $57 million per year.Overall, 83.5% of CCTAs were deemed to be appropriate based on the AUC. Before the AUC publication, 75.0% of CCTAs were classified as appropriate whereas after the AUC publication, 88.0% were classified as appropriate (We report a high rate of appropriate use of CCTA and a significant increase in the proportion of CCTAs classified as appropriate after the AUC publication. The Appropriate Use Criteria (AUC) were introduced by the American College of Cardiology Foundation in response to growing concern regarding potential overutilization of non-invasive cardiac imaging –3. The aCardiac computed tomography has become an increasingly utilized imaging modality for a variety of indications, with assessment of coronary artery disease by angiography  being the most common , 7. PrioIn this study, we sought to determine the proportion of appropriate CCTA utilization in a quaternary care institution pre- and post-publication of the AUC and to identify a potential shift toward more appropriate use of CCTA. In addition, we aimed to estimate potential cost savings associated with this potential shift. We hypothesized that there would be a significantly higher percentage of CCTAs deemed to be appropriate when ordered after, compared to before, the publication of the AUC and that there will be a significant cost savings associated with increased appropriate use.We conducted a retrospective cohort analysis of consecutive patients undergoing CCTA for the evaluation of coronary artery disease between January 1, 2012 and December 30, 2016 at Sunnybrook Health Sciences Centre, a large quaternary care Canadian medical centre. These patients were referred from 168 referring physicians. Of these referring physicians, 138 (82%) were cardiologists, 21 (13%) were internal medicine specialists and 9 (5%) were cardiovascular surgeons. Because we aimed to include only those patients who underwent coronary computed tomography angiography for the indication of CAD (i.e. CCTAs), patients receiving cardiac CTs for other indications were excluded  to confirm that the test was indeed performed as well as its timing. Demographic information and indication for the CCTAs were obtained from patient charts, prior imaging reports and information on the medical imaging test requisition. Following data collection, appropriateness of each CCTA was classified categorically as “appropriate”, “may be appropriate” or “rarely appropriate” in a manner previously described and validated , 14–16. p value of < 0.05 was used as an indication of statistical significance. All statistical analyses were performed using SAS version 9.3 .Descriptive statistics were utilized to describe the patient population. Characteristics of the patient population were compared using the chi square test for categorical values and the Mann–Whitney test for continuous variables. Appropriateness categories were compared between pre- and post- AUC publication groups via the chi-square test for both the overall cohort as well as for the subgroups of symptomatic and asymptomatic patients. A two tailed We could not identify reliable estimates for the number of CCTAs performed for the entire population of the United States. Further, there exist re-imbursement discrepancies amongst different private insurance plans. Thus, we elected to estimate cost savings for the Medicare population of the United States (those aged 65 years and older). Estimates of unit costs for CCTA and absolute number of CCTAs performed were determined via the 2018 Medicare re-imbursement rate and based on prior research reporting the temporal trends in utilization for CCTA amongst the Medicare population –19. PoteA total of 2,577 consecutive CCTA patient files from January 1, 2012 and December 30, 2016 were reviewed. 889 of these CCTAs were performed prior to the 2013 AUC publication while 1688 were performed after the publication.p ≥ 0.05, see Table The patient demographics are presented in Table p < 0.001, see Fig. In the overall cohort, 83.5% of CCTAs were classified as “appropriate” based on the AUC, 9.0% of cases were deemed “may be appropriate” and 7.5% of cases were deemed “rarely appropriate”. In the pre-AUC publication group, only 75.0% of CCTAs were deemed “appropriate”, compared to 12.4% of CCTAs deemed as “may be appropriate” and 12.6% as “rarely appropriate”. In contrast, in the post-publication group , 88.0% CCTs were deemed to be “appropriate”, while 7.2% were deemed to be “may be appropriate” and 4.9% were deemed to be “rarely appropriate” .There were 2,294 symptomatic patients in our cohort. These were patients who had either chest pain or an anginal equivalent. Of these, 1975 (86.1%) were deemed to be “appropriate”, 224 (9.8%) were deemed “may be appropriate” and 95 (4.1%) were deemed “rarely appropriate”. In the pre-AUC publication group, 530 patients (75.9%) were deemed “appropriate”, compared to 106 (15.2%) who were deemed “may be appropriate” and 62 (8.8%) who were deemed “rarely appropriate”. In contrast, in the post-AUC group, 1445 (90.5%) were deemed “appropriate”, 118 (7.4%) were deemed “may be appropriate” and 33 (2.1%) were deemed “rarely appropriate” (p < 0.001).There were only 283 asymptomatic patients in our cohort. Of these, 177 (62.5%) were deemed “appropriate”, 7 (2.5%) were deemed “may be appropriate” and 99 (35.0%) were deemed “rarely appropriate”. In the pre-AUC group, 137 (71.7%) were deemed “appropriate”, 4 (2.1%) were deemed “may be appropriate” and 50 (26.8%) were deemed “rarely appropriate”. In contrast, in the post AUC group, 40 (43.5%) were deemed “appropriate”, 3 (3.3%) were deemed “may be appropriate” and 49 (53.3%) were deemed “rarely appropriate”  when compared to the older data. In this study, we found a higher proportion of appropriate CCTAs than what was reported in previous studies, although we found similar improvements in appropriateness in response to AUC publication. The relatively high number of appropriate CCTAs when compared to other studies may be in part related to the controlled access of the technology in our jurisdiction [There have been a small number of previous studies examining the effect of AUC publication on appropriateness of CCTAs –13. For sdiction , 22. Oursdiction –25. Furtsdiction , 25.Ordering physicians in the United States will be required to consult the AUC when ordering advanced imaging tests such as CCTAs and cardiac MRI scans starting January 1st, 2021. Our worIt is important to interpret the results of this study in the context of its limitations. First, in this study, CCTAs were scanned at one institution, potentially limiting results from being transferable to other jurisdictions. However, the institution is a quaternary care centre which receives patient referrals from four other community and academic hospitals and a wide array of outpatient clinics. Second, there are inherent limitations to this study due to its retrospective nature. For example, we did not evaluate appropriateness prospectively. The classification for each test was completed using retrospective patient data and occasionally by contacting the referring physician when needed. However, our classification methods were similar to those used in other studies with reported success rates for classifying patients > 95% , 15. TheOur study consisting of approximately 2,500 consecutive CCTAs performed at a Canadian quaternary medical care institution describes an overall high rate of appropriate CCTA utilization coupled with an increase in appropriate utilization following the publication of the 2013 AUC. The AUC publication was also associated with significant decrease in the percentage of tests deemed rarely appropriate which translated to a potential estimated annual cost saving of approximately $57 million when extrapolated to the Medicare population of the United States.


Using the preferred linear model, we found significant increased risks of coronary heart disease (CHD) mortality and CVD mortality as well as combined fatal and non-fatal CHD, CVD, carotid atherosclerosis disease and hypertension in those exposed to drinking water with an As concentration of 10 µg/L compared to the referent (drinking water As concentration of 1 µg/L) population. Notwithstanding limitations included, the observed significant increased risks of CVD endpoints arising from As concentrations in drinking water between 1 µg/L and the 10 µg/L suggests further lowering of this guideline value should be considered.To the best of our knowledge, a dose-response meta-analysis of the relationship between cardiovascular disease (CVD) and arsenic (As) exposure at drinking water As concentrations lower than the WHO provisional guideline value (10 µg/L) has not been published yet. We conducted a systematic review and meta-analyses to estimate the pooled association between the relative risk of each CVD endpoint and low-level As concentration in drinking water both linearly and non-linearly using a random effects dose-response model. In this study, a significant positive association was found between the risks of most CVD outcomes and drinking water As concentration for both linear and non-linear models ( Originating from either geological or anthropogenic activities, arsenic (As) has been widely recognized since the 1950s as one of the most serious human carcinogens . UbiquitThe relationship between CVD and As exposure at As concentrations in drinking water greater than 100 µg/L is well established ,18,20,21Existing meta-analyses and systematic reviews that include the lower end of the As exposure range have not clearly and quantitatively specified any increased risk below 10 µg/L of water As concentration possibly because of a lack of epidemiological data. These reviews notably include a) a qualitative systematic review of the epidemiologic evidences on the association of CVD and low, moderate and high As exposure , b) a meDue to the recent publication of a number of key epidemiological studies  buildingWe searched ISI Web of Science for studies published before December 2019 on the association between CVD risks including overall CVD, coronary heart disease (CHD), stroke, carotid atherosclerosis disease, hypertension and associated clinic markers and As exposure and for the general population. The medical subject headings (MeSH) used were “arteriosclerosis”, “atherosclerosis", “carotid artery diseases”, “cardiovascular diseases”, “cerebrovascular disease”, “cerebrovascular disorders”, “coronary artery disease”, “heart diseases”, “hypertension”, “infarct”, “ischemia”, “ischemic heart disease”, “myocardial infarction”, “peripheral vascular diseases”, “peripheral arterial disease”, “stroke” and were combined with “arsenic”, “arsenic poisoning”, “arsenicals”, “arsenite” and “arsenate”. The terms were combined with the Boolean operators "OR" or "AND". The reference lists and cross-references of eligible studies were also searched as were the bibliographies of recent systematic reviews and meta-analyses reported by Navas-Acien et al. , Phung eStudies not published in English, related to specific scenario like childhood exposure or exposure during pregnancy or occupational exposure, or based on a specific form of As like arsenic trioxide  were excluded. The review of literature was conducted in accordance with the techniques specified by Stroup et al.  for metaBased on the abstract review, studies were firstly excluded if (a) they were case reports or case series; (b) no original data were presented; (c) had no human records; or (d) the outcome measures were not related with mortality or morbidity risk of relevant CVD types and/or clinical indicators considered in this study. The full text of studies which was regarded as potentially eligible was then assessed and further excluded if they were (a) not related with dietary As exposure or not for the general population; (b) no available relative risk  and measures of variability of different types of CVD were mentioned; or c) no categorical As exposure (at least three categories) was used . The metFor each study that appeared to meet the inclusion criteria, we extracted data on the number of deaths or cases, person-years or total number of population, the measured risk values  and their statistical uncertainty ) for each As concentration category. In addition, we recorded the descriptive information including authors’ names, year of study publication, sampling method, sample size, study design, and outcome ascertainment as well as the adjustment for confounders.Similar to the method used by Moon et al. , we combinary As  using Eqn et al. .A random-effects dose-response meta-analysis was conducted to estimate the pooled dose-response relationship between the log-transformed relative risk of each CVD outcome and water As concentrations from the summarized data of multiple studies using the package ‘dosresmeta’ in R statistical software ,65. ThisDose-response relationships for individual studies with at least three concentration categories were plotted using the ‘ggplot’ function in the ‘ggplot2’ package in R and the pooled dose-response relationship for each CVD endpoint was plotted using the ‘matplot’ function in R to visualize the predicted relative risks at higher As level compared with the referent concentration (1 µg/L).2 and adjusted R2) and Akaike’s information criterion (AIC) values via the ‘gof’ function in the ‘dosresmeta’ package in R. Furthermore, dose-response relationships for individual studies were overprinted by the pooled dose-response relationship for each CVD endpoint to visually test the model goodness-of-fit. Heterogeneity was assessed by P-heterogeneity (p < 0.05 being significant), I2-statistics  and Cochran’s Q-statistic [Forest plots were used to show the pooled relative risks (95% CI) for both individual studies and the overall one, comparing risk for chronic exposure to drinking water As of 10 µg/L with 1 µg/L using the ‘forestplot’ function of the ‘metafor’ package in R, with the sizes of the squares of individual study relative risks weighted by the inverse variance of the log-relative risk within each model. Specifically, both the individual and the overall pooled relative risks (95% CIs) in the forest plots were calculated via the dose-response meta-analysis through the ‘dosresmeta’ package in R. Model goodness-of-fit was assessed using deviance tests, coefficient of determination  was plotted against the standard error of log-relative risk, with the funnel centred at the overall model estimate.Finally, sensitivity analyses were conducted. On one hand, to test the reliability and suitability of combining different exposure media, we conducted sensitivity analysis to estimate the relationships between CVD risks and drinking water As both linearly and non-linearly by excluding studies which did not provide water As concentrations directly. On the other hand, with the intention of analysing the effects of low-level As exposure, we also excluded studies with As concentration higher than 100 µg/L and conducted subgroup analysis only for low to moderate As concentrations (taken here to be < 100 µg/L), modelling its linear and non-linear associations with the CVD risks.All our data analysis was performed using R statistical software , version 3.4.3  and R Studio (R Studio Desktop 1.1.423) ,77,78.For our systematic review and meta-analysis, we identified 28 studies which satisfied the inclusion criteria . There wAmong all these studies, there were 13 cohort studies ,80,81,82We have included studies from As-endemic areas, such as Bangladesh ,20,67,70We observed substantial differences in the methods of exposure assessment across studies : while mIn this study, the different CVD outcomes considered were: carotid atherosclerosis ,72,73, CAssociation between CVD risks and As exposure could vary by age, gender, smoking status, alcohol consumption, physical activity, diabetes and obesity status, education level as well as some other socio-economic factors, all of which might be either powerful predictors of CVD risks or important factors in As toxicity ,92,93,94Of the 28 selected studies, 22 studies included dose-response meta-analysis of mortality risk, of which there were seven for CHD, eight for CVD and seven for stroke. The 21 studies on combined fatal and non-fatal CVD risks included three studies on carotid atherosclerosis disease, four studies on CHD, two studies on CVD, eight studies on hypertension and four studies on stroke. Apart from those quantifying the risk of different CVD types, we also considered four studies for CVD clinic markers, including two studies for pulse blood pressure and two studies for QT prolongation .p < 0.05) (p < 0.05) was also obtained for the combined fatal and non-fatal risk of CHD, CVD, carotid atherosclerosis disease and hypertension with the relative risks of these four endpoints at 10 µg/L of water As concentration were 1.405 (95% CI: 1.183–1.667), 1.411 (95% CI: 1.242–1.603), 1.936 (95% CI: 1.403–2.671) and 1.231 (95% CI: 1.043–1.452), respectively, compared with 1 µg/L as the reference  . Compare < 0.05) . Similareference .p < 0.05) for CHD and CVD mortality risks as well as the combined fatal and non-fatal risk of CHD and carotid atherosclerosis disease, significant increases in pooled relative risks at 10 µg/L drinking water As compared with 1 µg/L as a reference were only found for the mortality risk of CHD   .2 statistic, Cochran’s Q-statistic and p-values for heterogeneity as presented in p-values < 0.05 and I2 > 50%).Combined with the estimated Ip-value, R2, adjusted R2 and AIC, as well as the difference between dose-response relationships for individual studies and the pooled dose-response relationship for each CVD endpoint, have been used for the goodness-of-fit assessment , deviance tests indicated no overall lack of fit for other CVD endpoints. In addition, there was only a slight increase in the R2 and the adjusted R2 for non-linear models compared with linear ones for most CVD endpoints, indicating that non-linear models explained more the variation of CVD risks. Linear models are considered a better fit for all the CVD endpoints due to their lower AIC values.Deviance, sessment . Accordip-values for Egger’s Regression Test lower than 0.05  ,33,40,97To reduce model misspecification, we conducted both a constant linear and a smooth non-linear model  via a two-stage ’dosresmeta’ function. Somewhat consistent with previous meta-analysis researches ,24, diffp-value for non-linear trend lower than 0.05); it is, therefore, important to bear in mind that the non-linear analysis of As effects should not be neglected, calling for future individual-level studies with sufficient data on low level As exposure and adverse health effects.Importantly, our study could provide guidelines for future modelling assessment in epidemiological studies. We found the linear model better using AIC as the selection criteria depicting the associations of low-level As concentration and CVD outcomes. Such a conclusion has already been confirmed by several epidemiological and biological studies ,53,61. HUnfortunately, our study also has several limitations, the most important of which might be the unavoidable heterogeneity in our meta-analysis. We assumed that the above-mentioned substantial differences in underlying population characteristics, adjustment for confounding, methods for outcome ascertainment and exposure assessment might be responsible for such heterogeneity. It has been well-documented that assessing As exposure via a biomarker of internal dose is of great importance as it could provide an integrated measure of all sources of exposure, not only from drinking water, but also from diet, soil and air particles ,101. It It is also important to understand that even though several perspective studies, such as the ones conducted by D’Ippoliti et al.  and ErsbThese findings shed light, at least in part, on low-level As exposure induced health risks, suggesting that a consideration of downward revision of the WHO provisional guide value may be indicated to protect better human health and to reduce the economic burden arising from CVD related outcomes, even in countries such as the UK, where relatively few individuals consume drinking water with more than 10 µg/L As , but oveOur meta-analysis indicates an overall positive association between CVD risks and low level (1 µg/L to 10 µg/L) As concentration in drinking water. For the general population, those with chronic exposure to As in drinking water at the WHO provisional guideline value limit (10 µg/L) compared to a reference population with 1 µg/L As are modelled to have significant excess CHD (50% (95% CI: 15%–95%)) and CVD (17% (95% CI: 5%–31%)) mortality risks, as well as significant excess combined fata land non-fatal risks of CHD (41 % (95% CI: 18%–67%)), CVD (41% (95% CI: 24%–60%)), carotid atherosclerosis (94% (95% CI: 40%–167%)) and hypertension (23% (95% CI: 4%–45%)). Given the high mortality and morbidity of CVD risks in the world , such an


Rationale: Cardiac fibrosis is observed in nearly every form of myocardial disease. Long non-coding RNAs (lncRNAs) have been shown to play an important role in cardiac fibrosis, but the detailed molecular mechanism remains unknown.Object: We aimed at characterizing lncRNA 554 expression in murine cardiac fibroblasts (CFs) after myocardial infarction (MI) to identify CF-enriched lncRNA and investigate its function and contribution to cardiac fibrosis and function.Methods and Results: In this study, we identified lncRNA NONMMUT022554 (lncRNA 554) as a regulator of MI-induced cardiac fibrosis. We found that lncRNA 554 was significantly up-regulated in the mouse hearts following MI. Further study showed that lncRNA 554 was predominantly expressed in cardiac fibroblasts, indicating a potential role of lncRNA 554 in cardiac fibrosis. In vitro knockdown of lncRNA 554 by siRNA suppressed fibroblasts migration and expression of extracellular matrix (ECM); while overexpression of lncRNA 554 promoted expression of ECM genes. Consistently, lentivirus mediated in vivo knockdown of lncRNA 554 could inhibit cardiac fibrosis and improve cardiac function in mouse model of MI. More importantly, TGF-β1 inhibitor (TEW-7197) could reverse the pro-fibrotic function of lncRNA 554 in CFs. This suggests that the effects of lncRNA 554 on cardiac fibrosis is TGF-β1 dependent.Conclusion: Collectively, our study illustrated the role of lncRNA 554 in cardiac fibrosis, suggested that lncRNA 554 might be a novel target for cardiac fibrosis. Cardiovascular diseases are the leading cause of death in the world . CardiacNon-coding RNAs, including miRNA, lncRNA, and circRNA, are reported to play a pivotal role in cardiovascular diseases . RecentlA research showed that lncRNA NONMMUT022554, which we named as lncRNA 554 in current study, was highly up-regulated in a mouse model of cardiac fibrosis induced by MI . In the The role of TGF-β1/Smad3 signaling pathway in fibrosis has been clearly identified. Although Smad3 has been considered to be a key transcription factor activated in response to many fibrogenic mediators, knockout of Smad3 can cause autoimmune disease . To dateMale C57BL/6 mice  were purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine. Mice freely accessed food and water. All experimental procedures were reviewed and approved by the Experimental Animal Ethics Committee of Guangzhou Medical University.Neonatal mice cardiac fibroblasts (CFs) and cardiomyocytes (CMs) were isolated as described previously . In brie^5 cells/well concentration and then cultured 24 h. And the fusion degree of cells reached 50% when transfected with siRNA. The scrambled control siRNA (NC) and lncRNA 554 siRNA (si-Lnc554) (20 nM) were transfected into cardiac fibroblasts respectively by using RiboFECT CP Transfection Kit. 48 h after transfection, the cells were used for the following experiments.siRNA probe against lncRNA 554 was designed and produced by RiboBio . And the nucleotide sequences of lncRNA 554 siRNA were 5′-GCAGAUUCUUGCCCUACUUTT-3′, 5′-AAGUAGGGCAAGAAUCUGCTT-3’. Before transfection, cardiac fibroblasts were seeded in six well plate at 1.5 × 10 The Lentivirus overexpression lncRNA 554 was synthesized by Genechem . Isolated CFs were seeded at 40% confluence. And the transduction was performed 72 h after seeding. For Lentivirus transfection, Lentivirus-lncRNA 554 (Len-Lnc 554) or Lentivirus-negative control (Len-NC) was added to the cells with a multiplicity of infection of 100 MOI. After 72 h of transfection, the cells were collected.^5 cells/well concentration and then cultured 12 h. Subsequently, a 1 ml pipette tip was employed to draw 3 parallel lines in each hole. The cells were washed with PBS for three times and then serum free medium was added to each well. The medium should be changed every 2 h for 3 times. The cells were cultured at 37°C with 5% CO2. The images were taken in 0, 12, 24 h after culture.According to the best transfection conditions of siRNA, NC and si-Lnc554 were used to transfect cardiac fibroblasts for 48 h. The cells were trypsinized and seeded in six well plate at 5 × 10 in situ hybridization was performed as described previously (TM lncRNA 554 FISH Probe Mix (Red)) at 37°C in the dark overnight using RiboTM Fluorescent In Situ Hybridization Kit . The cells were washed with Wash Buffer I, II, and III at 42°C in the dark. The cells were stained with DAPI in dark and then washed with 1 x PBS three times. All images were obtained with a fluorescence or confocal microscope .Fluorescence eviously . CFs wer−ΔΔCt and normalized to GAPDH as an internal control. The sequences of primers were synthesized by Sangon Biotech Co. Ltd . The sequences of lncRNA 554 primers were forward: 5′-CATGGATGCAGGCAGTGATT-3’; reverse: 5′- GCCTAGAGTTGGCTTGCTTCTT-3’. The sequences of Col 1 primers were forward: 5′-CAATGGCACGGCTGTGTGCG-3’; reverse: 5′-CACTCGCCCTCCCGTCTTT GG-3’. The sequences of Col 3 primers were forward: 5′-TGGCACAGCAGTC CAACGTA-3’; reverse: 5′-AAGGACAGATCCTGAGTCACAGACA-3’. The sequences of Fn 1 primers were forward: 5′-ATGTGGACCCCTCCTGATAGT-3’; reverse: 5′- GCCCAGTGATTTCAGCAAAGG-3’. The sequences of GAPDH primers were forward: 5′- AGGTCGGTGTGAACGGATTTG-3’; reverse: 5′-GGGGTCGTT GATGGCAACA-3’. The sequences of CTGF primers were forward: 5′-CTCCTA CTACGAGCTGAACCAG-3’; reverse: 5′-CCAGAAAGCTCAAACTTGACAGGC-3’.Total RNA was extracted from neonatal mouse CFs or left ventricular infarct border zone of heart using Trizol reagent . For each sample, 1 μg of the total RNA was converted to cDNA according to the manufacture’s instructions using the cDNA Reverse Transcription Kit . RNA levels of lncRNA 554, collagen I (Col 1), collagen III (Col 3), CTGF and fibronectin 1 (Fn 1) were detected using SYBR Green method  on the ABI Stepone Plus Fast Real-time PCR system. After circle reaction, the threshold cycle (Ct) was determined and the relative quantitative expression of lncRNA 554 and ECM mRNAs was calculated using method 2^9 TU/mL for each mouse) including lentivirus vector and lentivirus-negative control  were injected near the ligation line at four sites. The shRNA sequences of lncRNA 554 were: 5′-GCAGATTCTTGCCCTACTT-3′, 5′-AAGTAGGGC AAGAATCTG-3’. The sequences were inserted into GV248 lentiviral vector (Genechem). At 0, 3, 7, 14, and 28 days after the operation, the mice in the MI group were sacrificed to detect the expression of lncRNA 554, and the remaining mice were fed and monitored daily until the second week for further study.Mice were anesthetized with pentobarbital  and were randomly divided into a sham-operated group, an MI group, a lentivirus group (Len-sh-lnc554) and a lentivirus control (Len-scramble) group. Their chests were opened to expose the hearts. The left descending coronary artery (LAD) was ligated with a 7/0 nylon suture to induce myocardial infarction. Myocardial ischemia was confirmed by markedly S-T segment elevation with electrocardiographic measurements. The sham-operated mice underwent the same process as the MI group but without ligation of LAD. After MI surgery, the mice were injected with lentivirus. The procedure was performed as described previously . A totalMasson's trichrome staining and hematoxylin and eosin (H&E) staining were performed to estimate collagen deposition in the hearts. Two weeks after MI surgery, the hearts were dissected out and fixed with 4% paraformaldehyde, embedded in paraffin, and cut into a 5 µm thick cross section. The sections were stained with Masson's trichrome staining kit  and hematoxylin and eosin kit , and collagen deposition was examined using Ortho Microscope . The area of fibrosis in each group was calculated by ImageJ software.Transthoracic echocardiography was performed at 14 days after MI surgery using a Vevo®2,100 High-Resolution Imaging system  to monitor changes in left ventricular function. The mice were anesthetized with isoflurane, two-dimensional ultrasound and M-mode ultrasound measurements were performed. The short axis at the level of the parasternal papillary muscle of each mouse was measured. And some parameters such as left ventricular fractional shortening (LVFS), left ventricular anterior wall end diastolic thickness (LVAWd), left ventricular anterior wall end systolic thickness (LVAWs), left ventricular internal diastolic diameter (LVIDd), left ventricular internal systolic diameter (LVIDs), and left ventricular ejection fraction (LVEF) were measured.t-test, and one-way AVOVA was used for multiple comparisons. The results were considered statistically significant when p < 0.05.The experimental data were analyzed by SPSS 16.0 and GraphPad Prism5.0 software, and the results were expressed as mean ± standard deviation (x ± s). The comparison between the two groups was analyzed by in situ hybridization to label lncRNA 554. The results showed that lncRNA 554 was expressed in both cytoplasm and nucleus of cardiac fibroblasts  had the highest silencing efficiency , and couAs knockdown of lncRNA 554 led to reduction of collagen, we tested whether upregulation of lncRNA554 could increase the expression of collagen-related genes. First of all, in order to screen the best infection conditions, we set up three groups of lentivirus with different concentrations  to infect fibroblasts. The results showed that the efficiency of lentivirus infection was the highest when MOI = 100 was used . Next, win vitro, we went ahead to explore what would happen following lncRNA 554 silencing in vivo. The hearts were injected with lentivirus containing control or lncRNA 554 shRNA after MI. As shown in Having proved that down-regulation of lncRNA 554 significantly reduced the expression of fibrosis-related genes To identify whether knockdown of lncRNA 554 influences cardiac function of infarcted hearts, echocardiography was performed. MI greatly compromised the function of hearts, reflected by decreases in LVAWd  and LVAWin vitro and we found that silence of lncRNA 554 mitigated cardiac fibrosis and protected cardiac functions of MI mice. More importantly, TGF-β1 inhibitor could evidently inhibit the function of lncRNA 554, indicating that pro-fibrotic function of lncRNA 554 was at least partly owing to TGF-β1 signal pathway.In the present study, we revealed a novel function and molecular mechanism of lncRNA 554 in MI-induced cardiac fibrosis. We first observed upregulation of lncRNA 554 on day 3 post MI, and it reached peak on day 14 and stayed high till day 28 post MI. Furthermore, we found that expression of lncRNA 554 was highly enriched in CFs compared to that in cardiomyocytes. Consistently, down-regulation of lncRNA 554 remarkably reduced CFs migration and ECM genes level LncRNAs have recently been reported to regulate a variety of pathophysiological processes of cardiac diseases, including cardiac fibrosis . A recenAs our FISH experiment found that the subcellular localization of lncRNA 554 was in both cytosol and nucleus, our future studies will focus on determining the potential cytosolic and nuclear factors of TGF-β1 pathway that can be regulated by lncRNA 554. Recently, lncRNAs have been reported to act as miRNA sponges in regulating the biological activities of cardiac fibroblasts . Liang eHowever, there are still some limitations of the present study. Firstly, the detailed mechanism of myocardial fibrosis mediated by lncRNA 554 through TGF-β1 signaling pathway, especially which downstream target genes are directly or indirectly affected, needs to be further validated in future studies. In addition, we need to detect the level of lncRNA 554 in patients with MI in the future, and evaluate its clinical value. Besides, since there are many mechanisms and factors leading to cardiac fibrosis, whether lncRNA 554 promotes cardiac fibrosis through other mechanisms, such as inflammation, still needs to be further verified.In summary, our results revealed lncRNA 554 as a critical pro-fibrotic lncRNA that promoted collagen synthesis, and myocardial fibrosis, and the mechanism may involve the activation of TGF-β1 signal pathway. Knockdown of lncRNA 554 represents a promising strategy for the intervention of cardiac fibrosis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.The animal study was reviewed and approved by Experimental Animal Ethics Committee of Guangzhou Medical University.ZH, SH, JW and DH performed the research, HX and PL analyzed the data, BL, XZ, JW, and DH wrote the paper, BL and DL designed the research study. All authors contributed to manuscript revision, read, and approved the submitted version.This study was supported by National Natural Science Foundation of China (81570258), Natural Science Foundation of Guangdong Province of China (2018A030313060), Medical Science and Technology Research Foundation of Guangdong Province of China , Guangzhou Health Science and Technology Project (20201A011072) and College Students' Science and Technology Innovation Project (2019A002).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


As a comparison, reference and control cells fabricated using a PEO-coated polypropylene membrane and a regular separator, respectively, show a poor capacity of 662 mA∙h g−1 and a short cycle life of 50 cycles.In lithium–sulfur cells, the dissolution and relocation of the liquid-state active material (polysulfides) lead to fast capacity fading and low Coulombic efficiency, resulting in poor long-term electrochemical stability. To solve this problem, we synthesize a composite using a gel polymer electrolyte and a separator as a functional membrane, coated with a layer of poly(ethylene oxide) (PEO) and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI). The PEO/LiTFSI-coated polypropylene membrane slows the diffusion of polysulfides and stabilizes the liquid-state active material within the cathode region of the cell, while allowing smooth lithium-ion transfer. The lithium-sulfur cells with the developed membrane demonstrate a high charge-storage capacity of 1212 mA∙h g One sucatteries . Specifiatteries . Moreoveatteries . In inteatteries .An effective approach to solving the foregoing issues is to modify the cell components by using a porous cathode substrate to host the active material ,9 and a In this study, to improve the electrochemical utilization and reversibility of lithium–sulfur cells, we develop a composite functional separator composed of a gel polymer electrolyte and a polypropylene membrane. This functional separator is fabricated using a layer of a mixture containing poly(ethylene oxide) (PEO) and lithium bis(trifluoromethanesulfonyl)imide (LiTFSI) coated on a polypropylene membrane. PEO is a polymer matrix that is commonly used to prepare gel polymer electrolytes  and is cw = 600,000, Sigma Aldrich, St. Louis, MO, USA) and lithium bis(trifluoromethanesulfonyl)imide  at a molar ratio of O/Li at 20:1 were mixed in acetonitrile  and stirred for 24 h to form a transparent gel polymer electrolyte. The well-mixed gel polymer electrolyte was cast as the functional coating material onto a polypropylene membrane , and the coated membrane was then dried in a vacuum oven at 80 °C for 24 h. The as-prepared PEO/LiTFSI-coated polypropylene membrane was cut into circular disks, 19 mm in diameter. As a reference membrane for investigating lithium-ion diffusion issues, a PEO-coated polypropylene membrane was fabricated using the same procedure, but without LiTFSI. Finally, a polypropylene membrane was prepared for assembling control cells.Poly(ethylene oxide) . The morphology and microstructure of the polypropylene membranes with and without coating layers were observed by field-emission scanning electron microscopy .3  dissolved in mixed 1,2-dimethoxyethane  and 1,3-dioxacyclopentane  solution. The polysulfide catholyte was added to a battery current collector to fabricate a polysulfide cathode at a fixed sulfur loading of 2 mg cm−2 and sulfur content of 51 wt % (considering the weight of the whole cathode). Electrochemical lithium–sulfur cells were assembled in an argon-filled glove box by combining the polysulfide cathode, the PEO/LiTFSI-coated polypropylene membrane, and a lithium counter electrode. For the performance comparison, reference and control cells were assembled using a PEO-coated polypropylene membrane and an uncoated separator, respectively. High-loading analysis was conducted on high-loading polysulfide cathodes with sulfur loadings (contents) of 4 mg cm−2 (67 wt %) and 6 mg cm−2 (76 wt %), respectively. The assembled lithium–sulfur cells were allowed to rest at 25 °C for 30 min before electrochemical cycling and analysis. Electrochemical impedance spectroscopy data were obtained from 1 MHz to 100 mHz using an impedance analyzer  at an AC voltage amplitude of 5 mV and open-circuit voltage. Cyclic voltammograms (CV) were recorded using a potentiostat  in the voltage window of 1.8 to 2.8 V at scan rates of 0.02, 0.03, 0.04, and 0.05 mV s−1. Charge/discharge voltage profiles, cycling performance data, polarization analysis, and high-loading performance data were collected in the same range voltage window using a programmable battery cycler  at a regular cycling rate of C/10 (1C = 1675 mA g−1). The cycling capacity and high-loading performance of cells were evaluated on the basis of the weight of sulfur.The electrochemical characteristics and cell performance of the PEO/LiTFSI-coated polypropylene membrane were analyzed by fabricating electrochemical lithium–sulfur cells. A 1.0 M polysulfide catholyte was obtained by mixing sulfur  and lithium sulfide  into a blank electrolyte solution composed of 1.85 M LiTFSI  and 0.2 M LiNO−2. The PEO-coated polypropylene membrane has a uniform PEO coating  at 2.3–2.1 V and, subsequently, from liquid-state polysulfides to solid-state sulfides  at 2.1–1.8 V [−1 and long cyclability, with a high reversible capacity of 534 mA∙h g−1 and stable Coulombic efficiency of >99% over 200 cycles   . The repectively . The afo−2, 4 mg cm−2, and 6 mg cm−2 . Despite the increase in the amount of active material in the cathode, the discharge and charge curves maintain distinguishable upper and lower discharge plateaus, in addition to two continuous charge plateaus. The polarization is also maintained low  of 2 mg cm−2 (51 wt%), 4 mg cm−2 (67 wt %), and 6 mg cm−2 (76 wt %), respectively. After cycling, these high-loading cathodes maintain a high-capacity retention of 60%–70% and attain a high areal capacity of 2.4–4.0 mA∙h cm−2. Thus, the PEO/LiTFSI composite is a functional coating that facilitates good polysulfide retention and smooth lithium-ion transfer. Moreover, a polysulfide cathode with the developed PEO/LiTFSI-coated polypropylene membrane can host a large amount of sulfur without sacrificing its high electrochemical utilization and efficiency.ined low . These r−2 (51 wt%), 4 mg cm−2 (67 wt %), and 6 (76 wt %) mg cm−2, respectively. Despite increasing sulfur loadings, the cells display similar redox reaction peaks with no obvious voltage delays and rapid current drops, illustrating their good electrochemical reversibility. However, the lithium-ion diffusion coefficients decrease slightly from 1.5 × 10−8–5.0 × 10−8 cm2 s−1 to 1.2 × 10−8–3.8 × 10−8 cm2 s−1 with increasing sulfur loading (To verify the foregoing results, the electrochemical reaction kinetics e,f and r loading  because −2 and high sulfur content of 76 wt % has a high areal specific capacity of 4 mA∙h cm−2. In contrast, control and reference cells fabricated using a PEO-coated polypropylene membrane and a regular separator membrane, respectively, exhibit low ion transfer and fast polysulfide relocation, resulting in low charge-storage capacities and short cycle lives. Our findings highlight the importance of the proposed design in developing advanced functional separators for lithium–sulfur batteries.We develop a functional separator fabricated using a thin-film gel polymer electrolyte and a robust polypropylene membrane. Given its excellent polysulfide-blocking capability, the PEO/LiTFSI-coated polypropylene membrane is configured to face the polysulfide cathode, while the added LiTFSI serves as an ion transfer network. Our tests reveal that this cell has low cell resistance and overlapping discharge/charge curves before and after cycling, indicating its improved reaction kinetics and electrochemical utilization. Moreover, the overlapping CV curves and the high lithium-ion diffusion coefficients indicate that the cell exhibits smooth ion transfer and enhanced electrochemical reversibility and stability. As a result, a high-loading polysulfide cathode fabricated using the developed membrane at a high sulfur loading of 6 mg cm


IUCrJ, 7, 287–293Andring ] on the nitrite reductase and nitrous anhydrase activity of carbonic anhydrase.The paper discusses a recent paper [ CA catalyzes a very simple but vital reaction, i.e. the hydration of carbon dioxide, in mammals, plants and bacteria  to nitric oxide (NO), a signaling multiple-functional gaseous molecule in living organisms. Central to scientific research on CA has been its catalytic site that preferentially binds Zn2+, which is redox-inactive, and Cu2+, which is redox-active , the most abundant endogenous intra-cellular antioxidant with high specificity to Zn2+, Cu2+ and other divalent ions including Hg2+ , as performed by the Cu2+-rich ceruloplasmin, or it could reduce nitrite to NO via intermediate Cu+-formation by GSH or ascorbic acid  is one of the oldest, most efficient and best investigated ubiquitous Zn2+-hCAII) in complex with inorganic nitrite (O=N—O−) at 1.2 Å resolution with two Cu2+ centers, analogous to bacterial nitrite reductases, and suggested that Cu2+-hCAII can function as a nitrite reductase, yet without providing experimental evidence -bound human carbonic anhydrase II  from nitrite and GSH suggesting nitrous anhydrase activity of Zn2+-CAII, which was not inhibitable by the CA-inhibitors acetazolamide or dorzolamide , most likely due to the intermediate formation of S-nitrosocysteine (CysSNO), which can readily and abundantly decompose to NO in the presence of Cu+  to nitric oxide (NO)... (Andring et al., 2018et al., 2009et al., 2018et al., 2018et al., 2018et al., 2018et al. (2020i.e., Hanff et al., 2018i.e. Hanff et al., 2018Andring  al. 2020 stated t al. 2020. In the


The successful integration of eye gaze direction and emotion cues from faces is important not only for co-ordinated interactions, but also for the detection of social signals alerting us to threat posed by a conspecific, or elsewhere in our immediate environment. It is now well-established that people with schizophrenia experience aberrant eye gaze and facial emotion processing. These social-cognitive differences might contribute to the maintenance of socially-themed delusions which are characterised by the hyper-attribution of threatening intentions to others. However, no study has directly examined whether the mechanisms which govern the integration of eye gaze and emotion information diverge in schizophrenia, and more importantly, whether this reflects a fundamental ‘bottom-up’ perceptual deficit or a ‘top-down’ cognitive bias. Fifteen outpatients diagnosed with schizophrenia and 21 healthy age- and IQ-matched controls performed an emotion categorisation task (anger/fear) on morphed facial expressions of anger or fear, displaying either direct or averted gaze. Results in both controls and patients replicated the previous finding that combinations of anger with direct gaze, and fear with averted gaze – which signal a relevant threat to the observer – benefited from more accurate emotion recognition than alternate gaze-emotion combinations. Bayesian model selection revealed that for patients this effect was mediated by a shift in decision bias towards emotions which signal self-relevant threat, rather than a change in sensitivity as observed in controls. These results critically highlight a different cognitive mechanism governing gaze and face-cued emotion integration in schizophrenia, which has a top-down influence on the evaluation of perceptual input. However, recent studies using continuous flash suppression (CFS) tasks – which temporarily supress visual stimuli from conscious awareness, and do not require participants to make stimulus judgments – have shown that patients, like healthy controls, demonstrate intact visual prioritisation of faces over non-face stimuli , but simply instruct participants to report whether the eyes are averted to the left or right, have failed to reliably identify a direct gaze bias in patients. Together, these findings again suggest that aberrant gaze processing in schizophrenia likely reflects an evaluative, self-referential bias rather than deficits in visual perception  which may pose a relevant threat to both the observer and the observed . This sharpening of perceptual sensitivity would correspond to a boost in the bottom-up perceptual processing of Threat+ faces, especially when faces show anger and fear emotions at very low intensities , which in turn would enable observers to more readily and accurately categorise the emotion conveyed by the face . Using B1.1.2Alternatively, the decision bias model assumes that gaze direction cues bias the observer's decision about whether a face is angry or fearful in the direction of the combination signalling a higher threat. Maximal effects of this bias would be observed for neutral face expressions, in which direct gaze will bias observers to categorise the face as angry and neutral faces with averted gaze as fearful. This shift in decision bias – which also impacts on the categorisation of neutral faces with no perceptual emotion cues – would correspond to a top-down contextual influence of gaze cues on the recognition of emotional expression.1.2The current study aimed to adopt 22.1This study was approved by the Human Research Ethics Committee at Macquarie University . Participants received payment for their time and provided written consent before participating.2.2Twenty outpatients diagnosed with Schizophrenia or Schizoaffective Disorder (10M/10F) and 21 healthy controls (14M/7F) participated in this study. To classify for inclusion in the study participants could not have had any current or past neurological disease or injury resulting in a concussion or being unconscious for more than 1 h. All participants were additionally screened for any history of substance abuse (as per DSM-V criteria) and were required to have a minimum eight years of formal education. Finally, all participants had normal or corrected-to-normal vision.M = 53.93, SD = 8.37; Control M = 48.14, SD = 13.47; t(34) = −1.47, p = 0.150, BF10 = 0.746). The National Adult Reading Test was administered as a measure of premorbid intelligence  = 0.02, p = 0.986, BF10 = 0.325).Five patients were excluded from analyses because they failed to accurately categorise emotions on at least 60% of trials, resulting in a final sample of 15 patients . This ensured adequate individual data to reliably characterise the mechanisms underlying the influence of gaze direction on emotion categorisation. Groups did not differ significantly on age  were consistent with previous studies involving non-clinical community samples  adapted from the Radboud Faces Database , emotion , and intensity (7 levels of morphs) as within-subjects factors, and group  as a between-subject factor. The same analysis was performed within each group independently. Student 2.5.1To characterise the mechanisms underlying the enhanced categorisation of Threat+ faces, we performed model-based analyses inspired by signal detection theory see . We comp2.5.1.1In our previous studies conducted with healthy adults and adolescents, as well as adolescents with autism , a perce2.5.1.2Alternatively, the decision bias model assumes that gaze direction biases emotion recognition in favour of the interpretation signalling higher threat .exc), which is the probability that participants' behaviour was governed by the model's assumed mechanism, compared to that of the alternative model. Once we identified the winning model for each group (controls and patients), we ran statistical analyses on the maximum-likelihood parameter estimates extracted from the winning model. We report the statistical analyses from the winning model in each group, and depict on the results figure below, only the parameter estimates extracted from these winning models.For our comparison between these two models, we used a Bayesian model selection method based on the model evidence . This method allows us to quantify the support of one model over the other, by estimating which model best fits the observed data. As both compared models have the same number of parameters, we did not use any method to account for the complexity of the model . Fixed-effects and random-effects statistics are reported. The fixed-effect comparison assumes all the participants relied on the same mechanisms to generate their decisions. The random-effects comparison is more conservative and allows each participant to rely on different mechanisms in order to generate their decisions. For the former, we report the Bayes factor as the ratio of model evidence for the compared model . For the33.1F = 91.74, p < 0.001, η2 = 0.175). A main effect of emotion was also observed as anger was better categorised than fear  = 4.19, p = 0.048, η2 = 0.023). Importantly – and in line with our predictions and previous literature – we found evidence for a significant emotion by gaze interaction  = 11.61, p = 0.002, η2 = 0.008). This effect, however, did not interact with group  = 0.0218 p = 0.893, η2 = 0.000), and was significant in both patients  = 5.34, p = 0.037, η2 = 0.007) and controls  = 6.46, p = 0.019, η2 = 0.009) when analysed separately. This emotion by gaze interaction was characterised by more accurate categorisation of Threat+ than Threat− faces in both controls (t(20) = 2.26, p = 0.03, d = 0.494; see t(14) = 2.66, p = 0.018, d = 0.688; see Emotion categorisation accuracy increased with the level of displayed emotions for both groups . Maximum-likelihood estimates of the perceptual sensitivity parameter extracted from the winning model were significantly larger for Threat+ than Threat− faces  = 1.93, p = 0.034, d = 0.423; In order to characterise the mechanisms underlying better performance for Threat+ than Threat− faces, we compared the perceptual sensitivity model  and decision bias model . Our Bayesian model selection analysis revealed that Model 1  explained the control group data better than Model 2 . Maximum-likelihood estimates of the bias parameter extracted from the winning model differed significantly for direct and averted gaze  = 2.54, p = 0.01, d = 0.657; both a change in the sensitivity and bias parameter. Altogether, these results provide further evidence that the effects in each group were specific to a change in sensitivity for controls, and a change in decision bias for patients.Contrastingly, for the patient group, Model 2 (decision bias shift) explained the data better than Model 1  angry when gaze was directed at the participant, and (2) fearful when gaze was averted. In other words, patients were biased to assume that the face displayed the emotion that would be most threatening to themselves given the gaze cue provided. This bias manifests for neutral faces even though they offer no visual evidence for either emotion. This direct evidence for a decision bias mechanism aligns with indirect supporting evidence from the emotion and gaze processing literature in schizophrenia, which until now have largely remained separate.We found that, like healthy controls, patients were more accurate in categorising Threat+ than Threat− faces. Critically, however, Bayesian modelling of the emotion categorisation data revealed that the underlying mechanism driving this effect differed in patients. Previous modelling of emotion categorisation data has revealed that this phenomenon in neurotypical humans is likely driven by a ‘perceptual sensitivity’ mechanism – whereby gaze direction acts as a contextual cue which increases one's ability to sensitively  categorise emotions . This fiPrevious studies revealed that patients with schizophrenia exhibit deficits in emotion categorisation from faces – particularly for negatively-expressed emotions – even when positive symptoms are mild and effectively controlled using medication . In contStudies of gaze processing have also provided evidence for self-relevance biases influencing the perception of gaze direction in schizophrenia. Specifically, substantial evidence has been put forward suggesting that patients are biased to perceive direct gaze when gaze is averted, or in some cases, where there is no physical eye stimulus at all . This bo5genuine threat signals. A bias towards the perception of threat however, can potentially be adaptive when the threat signal is truly present – but maladaptive when this bias leads to the persistent perception of threat in ambiguous or non-threatening situations. Further work is needed to explore this bias in other social information processing paradigms, whilst examining its association with persecutory delusion symptoms in order to verify whether these biases maintain paranoid beliefs, or are shaped by them.Paranoid and persecutory delusions  are a common belief formation aberration experienced in schizophrenia which can cause significant distress for patients see . The curNathan Caruana:Conceptualization, Formal analysis, Writing - review & editing, Writing - review & editing.Christine Inkley:Investigation, Writing - review & editing.Marwa El Zein:Conceptualization, Formal analysis, Writing - review & editing, Writing - review & editing.This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.