Immunology

Treatment of murine lupus with TIGIT-Ig Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Shuowu Liu, Lizhu Sun, Chuqi Wang, Yingshu Cui, Yuee Ling, Tian Li, Fangxing Lin, Wenyan Fu, Min Ding, Shuyi Zhang, Changhai Lei, Shi Hu Abstract The TIGIT (T cell immunoreceptor with Ig and ITIM domains) protein is a co-inhibitory receptor that has been reported to suppress autoreactive T and B cells to trigger immunological tolerance. We generated a new recombinant protein by connecting the extracellular domain of murine TIGIT to the Fc region of the mouse immunoglobulin IgG2a. The fusion protein was then characterized. The results suggested that among mice with lupus that were treated with the TIGIT-Ig fusion protein, the onset of proteinuria was delayed, serum concentrations of autoantibodies, such as antinuclear antibodies, were reduced without a decrease in the total IgG concentrations, and the survival rate was significantly increased compared to those of the controls. In conclusion, TIGIT-Ig administration showed promising results for both the prevention and treatment of autoimmune diseases in mice. This indicates that treatment with recombinant human TIGIT-Ig shows promise as an effective way to treat human autoimmune diseases.

Diminished cytolytic activity of γδ T cells with reduced DNAM-1 expression in neuroblastoma patients Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Xiaolin Wang, Wenjun Mou, Wei Han, Yue Xi, Xi Chen, Hui Zhang, Hong Qin, Huanmin Wang, Xiaoli Ma, Jingang Gui Abstract Neuroblastoma is one of the children's malignant tumors with poor prognosis, as well as high recurrence and metastasis rates after surgical removal and chemotherapy. γδ T-cell based immunotherapy receives increasing attention thanks to their strong cytolytic activity to tumor cells. Our previous data revealed a significant increase in circulating γδ T-cell frequency in NB patients. In the present study, we found that beside a reduction of IFN-γ in serum of NB patients, DNAM-1 expression decreased in both circulating and PAM-expanded NB γδ T cells. Upon PAM stimulation, NB γδ T cells showed a reduced level of cell proliferation. In addition, the cytolytic activity of NB γδ T cells to NB cell lines was proved to be attenuated in a co-culture system. The fact that DNAM-1 neutralizing antibody abolished the tumor cell killing accentuates the indispensable role of DNAM-1 molecule in γδ T-cell cytolytic function.

MCC950 blocks enhanced interleukin-1β production in patients with NLRP3 low penetrance variants Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): E. Schuh, C.J. Groß, D. Wagner, M. Schlüter, O. Groß, T. Kümpfel Abstract Objective To determine the role of the NLRP3 inflammasome by using the selective NLRP3 inhibitor MCC950 in patients with NLRP3 low penetrance variants and clinical symptoms suggestive for an autoinflammatory syndrome including central nervous system (CNS) involvement. Methods Nineteen symptomatic patients with low penetrance NLRP3 variants (Q703K n = 17, V198M n = 2) recruited between 2011 and 2017 were included in this monocentric study. A functional inflammasome activation assay was performed in patients in comparison to healthy controls (HC), including the determination of interleukin-1beta (IL-1β), interleukin-6 (IL-6) and tumor-necrosis factor alpha (TNF-α) secretion in the presence of the NLRP3 selective small-molecule inhibitor MCC950. Detailed clinical features were assessed and anti-IL-1 treatment response was determined. Results Peripheral blood mononuclear cells (PBMC) from patients with low penetrance NLRP3 variants displayed enhanced IL-1β levels following inflammasome activation compared to HC. Furthermore, IL-1β release was NLRP3-dependent as it was blocked by MCC950. The production of IL-6 and TNF-α was also increased in patients with low penetrance NLRP3 variants. Clinically, they presented with a heterogenous spectrum of neurological manifestations, while cranial nerve inflammation was the most common feature. Overall inflammasome activation did not correlate with disease severity. Eight of ten treated patients responded to anti IL-1 treatment, however a complete response was only documented in four patients. Conclusion PBMC of several patients with NLRP3 low penetrance variants and CNS manifestation showed increased NLRP3-specific IL-1β release upon stimulation and elevated NLRP3-independent IL-6 and TNF-α levels as those were not suppressed by MCC950. Our data suggest that beside the possible causal involvement of the NLRP3 inflammasome additional, yet unidentified genetic or environmental factors may contribute to the multi-organ inflammation in our patients and explain the partial response to IL-1 targeting therapies.

Pathogenic roles of anti-C1q antibodies in recurrent pregnancy loss Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Kazumasa Ohmura, Kenji Oku, Tamao Kitaori, Olga Amengual, Ryo Hisada, Masatoshi Kanda, Yuka Shimizu, Yuichiro Fujieda, Masaru Kato, Toshiyuki Bohgaki, Tetsuya Horita, Shinsuke Yasuda, Mayumi Sugiura-Ogasawara, Tatsuya Atsumi Abstract Recurrent pregnancy loss (RPL) is often considered idiopathic, however excessive complement activation has been observed in pregnancy related manifestations. Anti-C1q antibodies (anti-C1q) are associated with the activation of complement pathway in lupus patients, while it remains unclear in RPL. Firstly, we showed that both the prevalence and titre of anti-C1q were significantly higher in unexplained RPL than in healthy parous individuals. Secondly, we established the murine model of anti-C1q induced pregnancy loss using a monoclonal anti-mouse C1q antibody, JL-1. In mice treated with JL-1, high ratio of pregnancy loss and fetal growth restriction were frequently observed and complement activation occurred. C5a receptor (C5aR) blockade cancelled these pathogenic changes in mice treated with JL-1. In conclusion, our study reveals an association between the prevalence of anti-C1q and RPL. Additionally, our murine model has indicated that anti-C1q can induce reproductive failure, which might be ameliorated by therapy targeting the C5-C5aR axis.

DNA damage accumulation, defective chromatin organization and deficient DNA repair capacity in patients with rheumatoid arthritis Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Vassilis L. Souliotis, Nikolaos I. Vlachogiannis, Maria Pappa, Alexandra Argyriou, Petros P. Sfikakis Abstract We investigated the DNA damage response and repair network in 18 patients with active rheumatoid arthritis and tested the hypothesis that treatment influences this network. A 3-fold increase of endogenous DNA damage (single- and double-strand breaks) was detected in patient-derived peripheral blood mononuclear cells than controls (alkaline comet assay; mean ± SD Olive Tail Moment of 11.8 ± 7.3 versus 4.3 ± 2.2, p < .001). Patients exhibited significantly higher formation of DNA damage (oxidative stress and abasic sites), deficient global genome repair and more condensed chromatin structure than controls. Twelve weeks following treatment, chromatin structure loosened, global genome repair capacity was restored, oxidative stress and abasic sites decreased and levels of endogenous DNA damage reached control values in all 8 patients examined. We conclude that deregulated chromatin organization, deficient DNA repair capacity and augmented formation of DNA damage, which are reversible after treatment, contribute to the accumulation of endogenous DNA damage in rheumatoid arthritis.

Characteristics of regulatory T-cell populations before and after Ty21a typhoid vaccination in children and adults Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Mark E. Rudolph, Monica A. McArthur, Laurence S. Magder, Robin S. Barnes, Wilbur H. Chen, Marcelo B. Sztein Abstract Typhoid fever, caused by the pathogen Salmonella enterica serovar Typhi (S. Typhi), is a serious global health concern. Challenge studies with wild type S. Typhi identified associations between gut-homing regulatory T cells (Treg) and development of typhoid disease. Whether oral live-attenuated Ty21a vaccination induces gut-homing Treg remains unclear. Here, we analyze pediatric and adult Treg pre- and post-Ty21a vaccination in an autologous S. Typhi-antigen presentation model to address this knowledge gap. We show that peripheral memory Treg populations change from childhood to adulthood, but not following Ty21a vaccination. Unsupervised dimensionality reduction with t-distributed stochastic neighbor embedding (tSNE) identifies homing, memory, and functional features which evidence age-associated maturation of multifunctional S. Typhi-responsive Treg, which were not impacted by Ty21a vaccination. These findings improve understanding of pediatric regulatory T cells, while identifying age-related differences in S. Typhi-responsive Treg, which may aid in the development of improved pediatric vaccination strategies against S. Typhi.

Autoimmune/inflammatory syndrome induced by adjuvants (ASIA) demonstrates distinct autoimmune and autoinflammatory disease associations according to the adjuvant subtype: Insights from an analysis of 500 cases Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Abdulla Watad, Nicola Luigi Bragazzi, Dennis McGonagle, Mohammed Adawi, Charlie Bridgewood, Giovanni Damiani, Jaume Alijotas-Reig, Enrique Esteve-Valverde, Mariana Quaresma, Howard Amital, Yehuda Shoenfeld Abstract Background We investigated the pattern of reported immune diseases in the international ASIA syndrome registry. Methods Data from 500 subjects exposed to adjuvants from the ASIA syndrome international registry were analysed. Results The patient mean age was 43 ± 17 years and 89% were female. Within the reported immune diseases, 69% were well-defined immune diseases (autoimmune, autoinflammation, and mixed pattern diseases). Among the well-defined immune diseases following the exposure to adjuvants, polygenic autoimmune diseases were significantly higher than autoinflammatory disorders (92.7% vs 5.8%, respectively, p < 0.001). Polygenic autoimmune diseases such as connective tissue diseases were significantly linked to the exposure to HBV vaccine (OR 3.15 [95%CI 1.08–9.23], p = 0.036). Polygenic autoinflammatory diseases were significantly associated with the exposure to influenza vaccination (OR 10.98 [95%CI 3.81–31.67], p < 0.0001). Conclusions Immune conditions following vaccination are rare, and among these, polygenic autoimmune diseases represent the vast majority of the well-defined immune diseases reported under the umbrella ASIA syndrome. However, vaccines benefit outweighs their autoimmune side effects.

Evaluating laboratory criteria for combined immunodeficiency in adult patients diagnosed with common variable immunodeficiency Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Caroline von Spee-Mayer, Verena Koemm, Claudia Wehr, Sigune Goldacker, Gerhard Kindle, Alla Bulashevska, Michele Proietti, Bodo Grimbacher, Stephan Ehl, Klaus Warnatz Abstract Some patients diagnosed with common variable immunodeficiency (CVID) actually suffer from combined immunodeficiency (CID) and therefore may require a different, CID-adapted treatment. Several CD4 T-cell-based criteria have been proposed in the past to identify patients with CID within the cohort of adult CVID patients. In this monocentric study, we used retrospective immunological and clinical data of 238 CVID patients to compare four different proposals of how to define CID among CVID patients. We demonstrate that none of the current definitions sufficiently separates CID from CVID patients and that the relative reduction of naïve CD4 T cells <10% has the highest sensitivity of all tested markers for patients with clinical complications often associated with CID. Thus, a very low percentage of naïve CD4 T cells in any adult CVID patient should raise suspicion, but is not sufficient to define CID among CVID patients.

Pathogenic NFKB2 variant in the ankyrin repeat domain (R635X) causes a variable antibody deficiency Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Paul Tuijnenburg, Hana Lango Allen, Godelieve J. de Bree, Sinisa Savic, Machiel H. Jansen, Claire Stockdale, Ilenia Simeoni, Ineke J.M. ten Berge, Ester M.M. van Leeuwen, NIHR BioResource, James E. Thaventhiran, Taco W. Kuijpers Abstract Genetic studies are identifying an increasing number of monogenic causes of Common Variable Immunodeficiency (CVID). Pathogenic variants in the C-terminus of NFKB2 have been identified in the subset of CVID patients whose immunodeficiency is associated with ectodermal dysplasia and central adrenal insufficiency. We describe 2 unrelated CVID pedigrees with 4 cases of pathogenic stop gain variants (c.1903C > T) in the ankyrin repeat domain (ARD) of NF-κB2, leading to a premature truncation of the protein at p.Arg635Term (R635X). By immunophenotyping and functional ex vivo B- and T-cell experiments we characterized the variant by reduced class-switched memory B-cell counts and immature plasmablasts, unable to produce IgG and IgA. Features of a poor proliferative T-cell response and reduced expansion of CD4+CXCR5+ T cells was only observed in the two clinically affected index cases without any clear clinical correlate. In conclusion, pathogenic stop variants in the ARD of NFKB2 can cause 'infection-only' CVID with an abnormal B-cell phenotype and a variable clinical penetrance.

Identification of autoantibodies using human proteome microarrays in patients with IPEX syndrome Publication date: June 2019 Source: Clinical Immunology, Volume 203 Author(s): Akihiro Hoshino, Hirokazu Kanegane, Masanori Nishi, Ikuya Tsuge, Kiriko Tokuda, Ichiro Kobayashi, Kohsuke Imai, Tomohiro Morio, Masatoshi Takagi Abstract Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is one of the inborn errors of immunity, characterized by impaired function of the regulatory T cells. Clinical manifestations of IPEX syndrome are characterized by various autoimmune diseases with autoantibodies. The comprehensive analysis for autoantibodies using human proteome microarrays in the four patients with IPEX syndrome was performed. The numbers of the highly expressed autoantibody showing relative log2 ratios greater than 1 were 1876, 513, 234 and 831 (mean: 864), respectively. Some novel autoantibodies which could explain the phenotypes of patients, adrenal dysfunction, muscular hypotonia, afibrinogenemia, enteropathy and pancytopenia were identified. Various kinds of autoantibodies targeting testis-specific antigens were also identified. Human proteome microarray is a powerful tool to understand the pathophysiology of IPEX syndrome. The larger cohort analysis using this method will provide further understanding of the impaired immune tolerance in humans.

Neurogenetics

Association of ATXN2 intermediate-length CAG repeats with amyotrophic lateral sclerosis correlates with the distributions of normal CAG repeat alleles among individual ethnic populations Abstract Intermediate-length CAG repeats in ATXN2 have been widely shown to be a risk factor for sporadic amyotrophic lateral sclerosis (SALS). To evaluate the association of ATXN2 intermediate-length CAG repeat alleles with an increased risk of SALS, we investigated distributions of CAG repeat alleles in 394 patients with SALS and 490 control individuals in the Japanese population. In the intermediate-length repeat units of 29 or more, we identified one SALS patient with 31 repeat units and two control individuals with 30 repeat units. Thus, no significant differences in the carrier frequency of intermediate-length CAG repeat alleles were detected between patients with SALS and control individuals. When we investigated the distribution of "large normal alleles" defined as ATXN2 CAG repeats ranging from 24 up to 33 in the Japanese population compared with those in other populations in previous studies, the frequency of large normal alleles was significantly higher in the European and North American series than in the Japanese series. Moreover, these frequencies in the Turkish, Chinese, Korean, and Brazilian (Latin American) series were also higher than that in the Japanese series. These results raise the possibility that the frequencies of large normal alleles in individual populations underlie the frequencies of ALS risk alleles in the corresponding populations.

Truncating biallelic variant in DNAJA1 , encoding the co-chaperone Hsp40, is associated with intellectual disability and seizures Abstract Intellectual disability poses a huge burden on the health care system, and it is one of the most common referral reasons to the genetic and child neurology clinic. Intellectual disability (ID) is genetically heterogeneous, and it is associated with several other neurological conditions. Exome sequencing is a robust genetic tool and has revolutionized the process of molecular diagnosis and novel gene discovery. Besides its diagnostic clinical value, novel gene discovery is prime in reverse genetics, when human mutations help to understand the function of a gene and may aid in better understanding of the human brain and nervous system. Using WES, we identified a biallelic truncating variant in DNAJA1 gene (c.511C>T p.(Gln171*) in a multiplex Saudi consanguineous family. The main phenotype shared between the siblings was intellectual disability and seizure disorder.

Rs10230207 genotype confers changes in HDAC9 and TWIST1 , but not FERD3L in lymphoblasts from patients with intracranial aneurysm Abstract Intracranial aneurysms (IA) are weakened outpouchings of the arterial wall in the cerebrovasculature. Rupture of an IA often leads to devastating consequences. The early identification of IA patients is crucial for management of their condition. A genetic variant at rs10230207, located nearby the HDAC9, TWIST1, and FERD3L genes, is associated with IA. HDAC9 is a class IIa histone deacetylase that mediates vascular smooth muscle cell dysfunction. TWIST1 is a mechanosensitive transcription factor and its expression is reduced in unstable carotid atherosclerotic plaques. In this study, the expression of the HDAC9, TWIST1, and FERD3L genes was characterized and associated with the presence of the rs10230207 genetic variant. Allelic discrimination and gene expression analysis were performed using lymphoblasts from 85 population controls and 109 IA patients. Subjects that were heterozygous (GT) within rs10230207 were 4.32 times more likely to have an IA than those that were homozygous for the reference allele (GG; 95%CI 1.23 to 14.16). Subjects that were homozygous (TT) were 8.27 times more likely to have an IA than those that were GG (95%CI 2.45 to 27.85). While the presence of the risk allele was not associated with changes in FERD3L gene expression, the risk allele was associated with increased HDAC9 and decrease in TWIST1 mRNA expression. The significant inverse correlation between HDAC9 and TWIST1 gene expression suggests that changes in the expression of both of genes may contribute to the formation of IAs.

Primary familial brain calcification caused by a novel homozygous MYORG mutation in a consanguineous Italian family Abstract Primary familial brain calcification (PFBC) is a rare disorder mostly characterized by calcium deposits in the basal ganglia and a wide spectrum of neurologic and psychiatric symptoms, typically inherited as an autosomal dominant trait. Recently, MYORG was reported as the first autosomal recessive causal gene in PFBC patients of Chinese and Middle Eastern origin. Herein, we describe the first PFBC patient of European descent found to carry a novel homozygous MYORG mutation (p.N511Tfs*243). Interestingly, the patient's father, a heterozygous carrier of the same mutation, showed diffuse bilateral cerebral calcifications with no symptoms other than very mild postural tremor.

Celia's encephalopathy and c.974dupG in BSCL2 gene: a hidden change in a known variant Abstract Celia's encephalopathy (progressive encephalopathy with/without lipodystrophy (PELD)) is a childhood neurodegenerative disorder with a fatal prognosis before the age of 10, due to the variant c.985C>T in the BSCL2gene that causes a cryptic splicing site leading to skipping of exon 7. For years, different authors have reported cases of congenital generalized lipodystrophy due to the variant c.974dupG in BSCL2 associated with neurological manifestations of variable severity, although some of them clearly superimposable to PELD. To identify the molecular mechanisms responsible for these neurological alterations in two patients with c.974dupG. Clinical characterization, biochemistry, and neuroimaging studies of two girls carrying this variant. In silico analysis, PCR amplification, and BSCL2 cDNA sequencing. BSCL2-201 transcript expression, which lacks exon 7, by qPCR in fibroblasts from the index case, from a healthy child as a control and from two patients with PELD, and in leukocytes from the index case and her parents. One with a severe encephalopathy including a picture of intellectual deficiency, severe language impairment, myoclonic epilepsy, and lipodystrophy as described in PELD, dying at 9 years and 9 months of age. The other 2-year-old patient showed incipient signs of neurological involvement. In silico and cDNA sequencing studies showed that variant c.974dupG gives rise to skipping of exon 7. The expression of BSCL2-201 in fibroblasts was significantly higher in the index case than in the healthy child, although less than in the case with homozygous PELD due to c.985C>T variant. The expression of this transcript was approximately half in the healthy carrier parents of this patient. The c.974dupG variant leads to the skipping of exon 7 of the BSCL2 gene and is responsible for a variant of Celia's encephalopathy, with variable phenotypic expression.

Overlap of polymicrogyria, hydrocephalus, and Joubert syndrome in a family with novel truncating mutations in ADGRG1/GPR56 and KIAA0556 Abstract Genetic mutations associated with brain malformations can lead to a spectrum of severity and it is often difficult to determine whether there are additional pathogenic variants contributing to the phenotype. Here, we present a family affected by a severe brain malformation including bilateral polymicrogyria, hydrocephalus, patchy white matter signal changes, and cerebellar and pontine hypoplasia with elongated cerebellar peduncles leading to the molar tooth sign. While the malformation is reminiscent of bilateral frontoparietal polymicrogyria (BFPP), the phenotype is more severe than previously reported and also includes features of Joubert syndrome (JBTS). Via exome sequencing, we identified homozygous truncating mutations in both ADGRG1/GPR56 and KIAA0556, which are known to cause BFPP and mild brain-specific JBTS, respectively. This study shows how two independent mutations can interact leading to complex brain malformations.

Pathogenic variants in AIMP1 cause pontocerebellar hypoplasia Abstract Aminoacyl-tRNA synthetase-interacting multifunctional protein 1 (AIMP1) is a non-catalytic component of the multi-tRNA synthetase complex which catalyzes the ligation of amino acids to the correct tRNAs. Pathogenic variants in several aminoacyl-tRNA synthetases genes have been linked to various neurological disorders, including leukodystrophies and pontocerebellar hypoplasias (PCH). To date, loss-of-function variants in AIMP1have been associated with hypomyelinating leukodystrophy-3 (MIM 260600). Here, we report a novel frameshift AIMP1 homozygous variant (c.160delA,p.Lys54Asnfs) in a child with pontocerebellar hypoplasia and simplified gyral pattern, a phenotype not been previously described with AIMP1 variants, thus expanding the phenotypic spectrum. AIMP1 should be included in diagnostic PCH gene panels.

Facioscapulohumeral muscular dystrophy (FSHD) molecular diagnosis: from traditional technology to the NGS era Abstract Facioscapulohumeral muscular dystrophy (FSHD) is a genetic neuromuscular disorder which mainly affects the muscles of the face, shoulder, and upper arms. FSHD is generally associated with the contraction of D4Z4macrosatellite repeats on 4q35 chromosome or mutations in SMCHD1, which are responsible of the toxic expression of DUX4 in muscle tissue. Despite the recent application of NGS techniques in the clinical practice, the molecular diagnosis of FSHD is still performed with dated techniques such as Southern blotting. The diagnosis of FSHD requires therefore specific skills on both modern and less modern analytical protocols. Considering that clinical and molecular diagnosis of FSHD is challenging, it is not surprising that only few laboratories offer a comprehensive characterization of FSHD, which requires the education of professionals on traditional techniques even in the era of NGS. In conclusion, the study of FSHD provides an excellent example of using classical and modern molecular technologies which are equally necessary for the analysis of DNA repetitive traits associated with specific disorders.

Linkage analysis and whole exome sequencing reveals AHNAK2 as a novel genetic cause for autosomal recessive CMT in a Malaysian family Abstract Charcot-Marie-Tooth (CMT) disease is a form of inherited peripheral neuropathy that affects motor and sensory neurons. To identify the causative gene in a consanguineous family with autosomal recessive CMT (AR-CMT), we employed a combination of linkage analysis and whole exome sequencing. After excluding known AR-CMT genes, genome-wide linkage analysis mapped the disease locus to a 7.48-Mb interval on chromosome 14q32.11–q32.33, flanked by the markers rs2124843 and rs4983409. Whole exome sequencing identified two non-synonymous variants (p.T40P and p.H915Y) in the AHNAK2 gene that segregated with the disease in the family. Pathogenic predictions indicated that p.T40P is the likely causative allele. Analysis of AHNAK2 expression in the AR-CMT patient fibroblasts showed significantly reduced mRNA and protein levels. AHNAK2 binds directly to periaxin which is encoded by the PRX gene, and PRX mutations are associated with another form of AR-CMT (CMT4F). The altered expression of mutant AHNAK2 may disrupt the AHNAK2-PRX interaction in which one of its known functions is to regulate myelination.

PTCD3 mutations cause Leigh-like rather than Leigh syndrome

Genes Genomics

SM-RCNV: a statistical method to detect recurrent copy number variations in sequenced samples Abstract Background Copy number variation (CNV) is an important form of genomic structural variation and is linked to dozens of human diseases. Using next-generation sequencing (NGS) data and developing computational methods to characterize such structural variants is significant for understanding the mechanisms of diseases. Objective The objective of this study is to develop a new statistical method of detection recurrent CNVs across multiple samples from genomic sequences. Methods A statistical method is carried out to detect recurrent CNVs, referred to as SM-RCNV. This method uses a statistic associated with each location by combining the frequency of variation at one location across whole samples and the correlation among consecutive locations. The weights of the frequency and correlation are trained using real datasets with known CNVs. P-value is assessed for each location on the genome by permutation testing. Results Compared with six peer methods, SM-RCNV outperforms the peer methods under receiver operating characteristic curves. SM-RCNV successfully identifies many consistent recurrent CNVs, most of which are known to be of biological significance and associated with diseased genes. The validation rate of SM-RCNV in the CEU call set and YRI call set with Database of Genomic Variants are 258/328 (79%) and (157/309) 51%, respectively. Conclusion SM-RCNV is a well-grounded statistical framework for detecting recurrent CNVs from multiple genomic sequences, providing valuable information to study genomes in human diseases. The source code is freely available at https://sourceforge.net/projects/sm-rcnv/.

Enzymatic construction of shRNA library from oligonucleotide library Abstract Background Short hairpin RNAs (shRNAs) expressed from vectors have been used as an effective means of exploiting the RNA interference (RNAi) pathway in mammalian cells. Genome-scale screening with shRNA libraries has been used to investigate the relationship between genotypes and phenotypes on a large scale. Although several methods have been developed to construct shRNA libraries, their broad application has been limited by the high cost of constructing these libraries. Objective We develop a new method that efficiently constructs a shRNA library at low cost, using treatments with several enzymes and an oligonucleotide library. Methods The library of shRNA expression cassettes, which were cloned into a lentiviral plasmid, was produced through several enzymatic reactions, starting from a library of 20,000 different short oligonucleotides produced by microarray-based oligonucleotide synthesis. Results The NGS sequence analysis of the library shows that 99.8% of them (19,956 from 20,000 sequences) were contained in the library: 63.2% of them represent the correct sequences and the rest showed one or two base pair differences from the expected sequences. Conclusion Considering the ease of our method, shRNA libraries of new genomes and of specific populations of genes can be prepared in a short period of time for genome-scale RNAi library screening.

Comparative transcriptome analyses provide insights into the adaptation mechanisms to acute salt stresses in juvenile Sinonovacula constricta Abstract Background Sinonovacula constricta is an economically important bivalve species in China, Korea and Japan that widely resides in estuarine and coastal areas where salinity fluctuates rapidly. However, little is known about its adaptation mechanisms to acute salt stresses. Objective To reveal the underlying molecular mechanisms involved in acute salt stresses in juvenile S. constricta. Methods Nine cDNA libraries (triplicate each trial) were established from juvenile S. constricta, which were subjected to low salinity (5 psu), optimal salinity (15 psu) and high salinity (25 psu) for 6 h, respectively. Results Illumina sequencing generated 478,587,310 clean reads totally, which were assembled into 427,057 transcripts of 246,672 unigenes. Compared with the control, 1259 and 2163 differentially expressed genes (DEGs) were identified under acute low and high salt stresses, respectively. GO and KEGG enrichment analyses of DEGs revealed that several key metabolic modulations were mainly responsible for the acute salt stresses. According to the significantly highlighted KEGG pathways, some key DEGs were identified and discussed in details. Notably, based on which, some potential osmolytes were further speculated. Conclusion Here, we carried out a unique report of comparative transcriptome analyses in juvenile S. constricta in response to acute salt stresses. The identified DEGs and their significantly enriched GO terms and KEGG pathways were critical for understanding and further investigating the underlying the physical and biochemical performances, and ultimately facilitated S. constricta breeding. Besides, the transcriptome data greatly enriched the genetic information of S. constricta, which were valuable for promoting its molecular biology researches.

Genetic risk score combining six genetic variants associated with the cellular NRF2 expression levels correlates with Type 2 diabetes in the human population Abstract Background Type 2 diabetes (T2D) is known as an inflammatory disease. NRF2 (Nuclear Factor Erythroid 2 Like2) encodes a transcription factor that binds to antioxidant response elements (AREs) and regulates the expression of genes involved in many antioxidant responses. Objective This study aimed to gain insight into individual anti-inflammatory activity to prevent T2D development in humans. Methods We performed a genome-wide association study (GWAS) to identify genetic variants influencing NRF2 expression in LCLs (lymphoblastoid cell lines) generated from 74 different individuals. Association analyses between T2D or its related traits and genetic risk score (GRS) calculated by combining genetic variants detected from GWAS for cellular NRF2 expression were performed using data from 8715 subjects. The T2D prediction model using GRS was evaluated by measuring the area under the curve (AUC) of the receiver operating characteristics (ROC) curve. Results Our GWAS identified six genetic variants (SNP) showing suggestive evidence of associations with cellular NRF2 expression (P < 10− 6). Logistic regression analysis demonstrated that GRS was associated with an increased risk of T2D (P value = 0.003, OR = 1.13). In addition, linear regression analyses showed positive associations between GRS and fasting glucose (P value = 0.028, β = 0.62), 2-h glucose (P value = 0.0004, β = 1.13) and HbA1C (P value = 0.033, β = 0.03). In the T2D prediction model using GRS, the AUC of the ROC curve was 0.69. Conclusion This study highlights genetic variants associated with cellular NRF2 expression and suggests that the GRS of NRF2 expression-associated variants is likely to be a useful indicator of T2D development in the human population.

Markov chain Monte Carlo simulation of a Bayesian mixture model for gene network inference Abstract Background Simultaneous measurement of gene expression level for thousands of genes contains the rich information about many different aspects of biological mechanisms. A major computational challenge is to find methods to extract new biological insights from this wealth of data. Complex biological processes are often regulated under the various conditions or circumstances and associated gene interactions are dynamically changed depending on different biological contexts. Thus, inference of such dynamic relationships between genes with consideration of biological conditions is very challenging. Method In this study, we propose a comprehensive and integrated approach to infer the dynamic relationships between genes and evaluate this approach on three distinct gene networks. Results This study demonstrates the advantage of integrating Markov chain Monte Carlo (MCMC) simulation into a Bayesian mixture model to overcome the high-dimension, low sample size (HDLSS) problem as well as to identify context-specific biological modules. Such biological modules were identified through the summarization of sampled network structures obtained from MCMC simulation. Conclusion This novel approach gives a comprehensive understanding of the dynamically regulated biological modules.

Identification of a novel plant RNA virus species of the genus Amalgavirus in the family Amalgaviridae from chia ( Salvia hispanica ) Abstract Background Chia (Salvia hispanica) is a flowering plant in the family Lamiaceae, which produces seeds that are a rich source of various nutritional compounds. Objective To identify a novel RNA virus potentially associated with chia. Methods Transcriptome data obtained from developing chia seeds were assembled into contigs. Sequence contigs containing an open reading frame (ORF) that showed amino acid identities with a viral RNA-dependent RNA polymerase (RdRp) were identified and analyzed. Results A genomic sequence of a novel plant RNA virus named Salvia hispanica RNA virus 1 (ShRV1) was identified in a chia seed transcriptome dataset. The ShRV1 genome sequence has two ORFs that showed high sequence identities with ORFs of known members of the genus Amalgavirus in the family Amalgaviridae. Amalgaviridae is a family of positive-sense double-stranded non-segmented RNA viruses that infect plants, fungi, and animals. The ShRV1 genome encodes two proteins: a putative replication factory matrix-like protein from ORF1 and an RdRp from the fused ORF of ORF1 and ORF2 by a + 1 programmed ribosomal frameshifting (PRF) mechanism. A conserved + 1 PRF motif sequence UUU_CGU was found at the ORF1/ORF2 boundary. A comparison of 31 amalgavirus ORF1 + 2 fusion proteins revealed that only three positions were repeatedly used as a + 1 PRF site during amalgavirus evolution. Conclusion ShRV1 is a novel virus found to be associated with chia and may be useful for studying the molecular features of amalgaviruses.

Inhibition of TNFα-induced interleukin-6 gene expression by barley ( Hordeum vulgare ) ethanol extract in BV-2 microglia Abstract Background Inflammation in the central nervous system is closely associated with pathological neurodegenerative diseases as well as psychiatric disorders. Prolonged activation of microglia can produce many inflammatory mediators, which may result in pathological neurotoxic side effects. Interleukin (IL)-6 serves as a hallmark of the injured brain. Objective Whole grains are known to contain many bioactive components. However, little information is available about anti-neuroinflammatory effects of grains in the CNS. This study aims to investigate the effect of Hordeum vulgareethanol extract (HVE) on the suppression of IL-6 expression in BV2 microglia. Methods Inhibitory effects of HVE on IL-6 expression were analyzed by immunoblot anaysis, immunofluoresce microscopic analysis, reverse transcription-polymerase chain reaction, and luciferase promoter reporter assay. Results HVE inhibited TNFα-induced phosphorylation of IKKα/β, IκB, and p65/RelA NF-κB. TNFα-induced IL-6 mRNA expression and promoter activity were reduced by HVE. Point mutation of NF-κB-binding site within the IL-6 gene promoter abolished TNFα-induced reporter activity, whereas exogenous expression of p65 NF-κB enhanced IL-6 promoter activity. Conclusion NF-κB-binding site within the IL-6 promoter region is a HVE target element involved in the inhibition of TNFα-induced IL-6 gene transcription. HVE inhibits TNFα-induced IL-6 expression via suppression of NF-κB signaling in BV2 microglial cells.

Molt-dependent transcriptome analysis of claw muscles in Chinese mitten crab Eriocheir sinensis Abstract Background Molting is a critical developmental process for crustaceans, during which the claw muscles undergo periodic atrophy and restoration. But the mechanism underlying this special muscle reshuffle around ecdysis is not yet thoroughly understood. Objective To investigate the molecular mechanism underlying the muscle's reshuffle over the molting cycle of Chinese mitten crab Eriocheir sinensis. Methods The Illumina high-throughput sequencing technique were used to sequence the transcriptome of the whole claw muscles from Chinese mitten crab Eriocheir sinensis in three molting stages (inter-molt C stage, pre-molt D3−4 and post-molt A–B stage); the de novo assembly, annotation and functional evaluation of the contigs were performed with bioinformatics tools. Results Totally 129,149 unigenes, 128,190 CDS, 33,770 SSRs and a large number of SNP sites were obtained, and 3700 and 12,771 differentially expressed genes (DEGs) were identified respectively in A–B and D3−4 stage compared with that in C stage. The identified DEGs were enriched to 746 and 1 408 GO terms respectively in A–B and D3–4 stage compared with C stage (p ≤ 0.05). KEGG pathway analysis showed that the DEGs were significantly enriched in 14 and 11 pathways in A–B vs C comparison and D3−4 vs C comparison (p ≤ 0.05), respectively. These DEGs are involved in several biological processes critical for the animal's growth and development, such as gene expression, protein synthesis, muscle development, new cuticle reconstruction, oxidation–reduction process and glycolytic process. Conclusion The data generated in this study is the first transcriptomic resource from the muscles of Chinese mitten crab, which would facilitate to characterize key molecular processes underlying crab muscle's growth and development during the molting cycles.

Pattern recognition receptors and their interactions with bacterial type III effectors in plants Abstract Innate immune signaling of plants is initiated by pattern recognition receptors (PRRs) at the plasma membrane. Upon pathogen attack, PRRs recognize pathogen-associated molecular patterns (PAMPs) via ectodomain and lead to signaling cascade via cytoplasmic kinase domain. PAMP-triggered immunity (PTI) activates basal defense responses sufficient to confer broad-spectrum disease resistance by inhibiting pathogen entry and growth. On the other hand, one of the major virulence factors in plant-pathogenic bacteria is type III secretion system, which can deliver effector proteins into the host cell and modulate host cellular processes. Most type III effectors are implicated in PTI suppression, and PRRs have been identified as targets of multiple type III effectors. Mutants defective in T3SS lack pathogenicity in many bacterial species, revealing that T3SS-mediated PTI suppression is critical for host colonization and subsequent disease development. This review summarizes molecular basis of bacterial pathogen perception by plant PRRs and also interaction between PRRs and type III effectors during early stages of plant-pathogen interaction.

De novo transcriptomic analysis of gonad of Strongylocentrotus nudus and gene discovery for biosynthesis of polyunsaturated fatty acids Abstract Background Strongylocentrotus nudus is an important cultured sea urchin species in north China, because its gonad is rich in unsaturated fatty acids, particularly long polyunsaturated fatty acids (LC-PUFAs). These PUFAs play pleiotropic and crucial roles in a wide range of biological process. Objective However, the genes contributing to biosynthesis PUFAs have not been elucidated yet, and the molecular mechanism relative to the difference in PUFA composition between male and female gonad as been revealed but the corresponding has not been understood. Methods In this paper, solexa sequencing based transcriptomic approach was used to identify and characterize the key genes relative to PUFA synthesis and further conducted different expressed genes between male and female gonad. Results A total of 130,124 transcripts and 189330 unigenes were de novo assembled from 64.32 Gb data. Next, these unigenes were subjected to functional annotation by mapping to six public databases, and this process revealed a lot of genes involving in lipid metabolism. In addition, three types of fatty acids front-end desaturase and three species of very long fatty acids elongase were identified and the pathway for PUFA biosynthesis was hypothesized. Last, comparative analysis revealed the higher expression level of Δ5 desaturase, Δ6 desaturase, ELOVL-4, -6 and -7 in male gonad compared with female. Conclusion This results could plausible explain the differ in composition of PUFAs between male and female gonad of sea urchin.

Biology & Philosophy

Measurement in biology is methodized by theory Abstract We characterize access to empirical objects in biology from a theoretical perspective. Unlike objects in current physical theories, biological objects are the result of a history and their variations continue to generate a history. This property is the starting point of our concept of measurement. We argue that biological measurement is relative to a natural history which is shared by the different objects subjected to the measurement and is more or less constrained by biologists. We call symmetrization the theoretical and often concrete operation which leads to considering biological objects as equivalent in a measurement. Last, we use our notion of measurement to analyze research strategies. Some strategies aim to bring biology closer to the epistemology of physical theories, by studying objects as similar as possible, while others build on biological diversity.

Teleosemantics, selection and novel contents Abstract Mainstream teleosemantics is the view that mental representation should be understood in terms of biological functions, which, in turn, should be understood in terms of selection processes. One of the traditional criticisms of teleosemantics is the problem of novel contents: how can teleosemantics explain our ability to represent properties that are evolutionarily novel? In response, some have argued that by generalizing the notion of a selection process to include phenomena such as operant conditioning, and the neural selection that underlies it, we can resolve this problem. Here, we do four things: we develop this suggestion in a rigorous way through a simple example, we draw on recent neurobiological research to support its empirical plausibility, we defend the move from a host of objections in the literature, and we sketch how the picture can be extended to help us think about more complex "conceptual" representations and not just perceptual ones.

Evolution of multicellularity: cheating done right Abstract For decades Darwinian processes were framed in the form of the Lewontin conditions: reproduction, variation and differential reproductive success were taken to be sufficient and necessary. Since Buss (The evolution of individuality, Princeton University Press, Princeton, 1987) and the work of Maynard Smith and Szathmary (The major transitions in evolution, Oxford University Press, Oxford, 1995) biologists were eager to explain the major transitions from individuals to groups forming new individuals subject to Darwinian mechanisms themselves. Explanations that seek to explain the emergence of a new level of selection, however, cannot employ properties that would already have to exist on that level for selection to take place. Recently, Hammerschmidt et al. (Nature 515:75–79, 2014) provided a 'bottom-up' experiment corroborating much of the theoretical work Paul Rainey has done since 2003 on how cheats can play an important role in the emergence of new Darwinian individuals on a multicellular level. The aims of this paper are twofold. First, I argue for a conceptual shift in perspective from seeing cheats as (1) a 'problem' that needs to be solved for multi-cellularity to evolve to (2) the very 'key' for the evolution of multicellularity. Secondly, I illustrate the consequences of this shift for both theoretical and experimental work, arguing for a more prominent role of ecology and the multi-level selection framework within the debate then they currently occupy.

The Social Amplification View of facial expression Abstract I offer a novel view of the mechanisms underlying the spontaneous facial expression of emotion. According to my Social Amplification View (SAV), facial expressions result from the interplay of two processes: an emotional process that activates specific facial muscles, though not always to the point of visible contraction, followed by a social cognitive process that amplifies these activations so that they may function more effectively as social signals. I argue that SAV outperforms both the Neurocultural View and the Behavioral Ecology View, as well as previously proposed syntheses of these views, in accounting for various empirical findings.

Race and reference Abstract The biological race debate is at an impasse. Issues surrounding hereditarianism aside, there is little empirical disagreement left between race naturalists and anti-realists about biological race. The disagreement is now primarily semantic. This would seem to uniquely qualify philosophers to contribute to the biological race debate. However, philosophers of race are reluctant to focus on semantics, largely because of their worries about the 'flight to reference'. In this paper, I show how philosophers can contribute to the debate without taking the flight to reference. Drawing on the theory of reference literature and the history of meaning change in science, I develop some criteria for dealing with cases where there is uncertainty about reference. I then apply these criteria to the biological race debate. All of the criteria I develop for eliminating putative kinds are met in the case of 'race' as understood by twentieth century geneticist Theodosius Dobzhansky and his contemporary proponents, suggesting that we should eliminate it from our biological ontology.

Overcoming the underdetermination of specimens Abstract Philosophers of science are well aware that theories are underdetermined by data. But what about the data? Scientific data are selected and processed representations or pieces of nature. What is useless context and what is valuable specimen, as well as how specimens are processed for study, are not obvious or predetermined givens. Instead, they are decisions made by scientists and other research workers, such as technicians, that produce different outcomes for the data. Vertebrate fossils provide a revealing case of this data-processing, because they are embedded in rock that often matches the fossils' color and texture, requiring an expert eye to judge where the fossil/context interface is. Fossil preparators then permanently define this interface by chiseling away the material they identify as rock. As a result, fossil specimens can emerge in multiple possible forms depending on the preparator's judgment, skill, and chosen tools. A prepared fossil then is not yet data but potential data, following Leonelli's (Philos Sci 82:810–821, 2015. https://doi.org/10.1086/684083) relational framework in which data are defined as evidence that scientists have used to support a proposed theory. This paper draws on ethnographic evidence to assess how scientists overcome this underdetermination of specimens, as potential data, in addition to the underdetermination of theories and of data, to successfully construct specimen-based knowledge. Among other strategies, paleontology maintains a division of labor between data-makers and theory-makers. This distinction serves to justify the omission of preparators' nonstandard, individualized techniques from scientific publications. This separation has benefits for both scientists and technicians; however, it restricts knowledge production by preventing scientists from understanding how the pieces of nature they study were processed into researchable specimens.

Contingency's causality and structural diversity Abstract What is the relationship between evolutionary contingency and diversity? The evolutionary contingency thesis emphasizes dependency relations and chance as the hallmarks of evolution. While contingency can be destructive of, for example, the fragile and complex dynamics in an ecosystem, I will mainly focus on the productive or causal aspect of contingency for a particular sort of diversity. There are many sorts of diversities: Gould is most famous for his diversity-to-decimation model, which includes disparate body plans distinguishing different phyla. However, structural diversity construed more broadly spans scales, such as organization in and among cells, structural arrangements and biomechanics on various scales, and even the profile of ancestor-descendent relationships or community structure of interactions within ecosystems. By focusing on stochastic processes in contingent evolution, I argue that contingency causes structural diversity. Specifically, I focus on the plurality of structural types of cells, genetic codes, and phyla diversity as case studies.

Let me tell you 'bout the birds and the bee-mimicking flies and Bambiraptor Abstract Scientists have been arguing for more than 25 years about whether it is a good idea to collect voucher specimens from particularly vulnerable biological populations. Some think that, obviously, scientists should not be harvesting (read: killing) organisms from, for instance, critically endangered species. Others think that, obviously, it is the special job of scientists to collect precisely such information before any chance of retrieving it is forever lost. The character, extent, longevity, and span of the ongoing disagreement indicates that this is likely to be a hard problem to solve. Nonetheless, the aim of this paper is to help field biologists figure out what do to when collecting a voucher specimen risks extinction. Here I present and assess varying practices of specimen collection for both extant (i.e., neontological) and extinct (i.e., paleontological) species in order to compare and contrast cases where extinction risk both is and is not a problem. When it comes to taking vouchers from extant species at some risk of extinction, I determine that those advocating for conservative approaches to collection as well as those advocating for liberal information-gathering practices have good reasons to assess things in the way they each do. This means that there is unlikely to be a decisive, one-size-fits-all response to this problem. Still, progress can be made. We can acknowledge the risks of proceeding in either manner, as well as the uncertainty about how best to proceed (which will be deep in some cases). We can proceed as thoughtfully as possible, and be ready to articulate a rationale for whichever procedure is used in any particular case.

Evidential reasoning in historical sciences: applying Toulmin schemes to the case of Archezoa Abstract This article is a study of the role and use of evidence in the evaluation of claims in the historical sciences. In order to do this, I develop a "snapshot" approach to Toulmin schemas. This framework is applied to the case of Archezoa, an initially supported then eventually rejected hypothesis in evolutionary biology. From this case study, I criticize Cleland's "smoking gun" account of the methodology of the historical sciences. I argue that Toulmin schemas are conceptually precise tools that allow for the building of enriched reconstructions of evidential reasoning. From the application of this framework, I discuss three ways in which the construction and use of facts in the historical sciences are theory-laden. Despite its inherent limits, TS are heuristically useful tools to identify epistemic moves that could be further investigated. It also sheds light on the positive roles of speculation in the historical sciences. Finally, I argue that it provides a context-specific and individuated understanding of hypothesis evaluation in the historical sciences. Overall, I think the application of Toulmin schemas to cases of evidential reasoning in the historical sciences is a promising descriptive and heuristic tool for philosophers of science.

Ancient genetics to ancient genomics: celebrity and credibility in data-driven practice Abstract "Ancient DNA Research" is the practice of extracting, sequencing, and analyzing degraded DNA from dead organisms that are hundreds to thousands of years old. Today, many researchers are interested in adapting state-of-the-art molecular biological techniques and high-throughput sequencing technologies to optimize the recovery of DNA from fossils, then use it for studying evolutionary history. However, the recovery of DNA from fossils has also fueled the idea of resurrecting extinct species, especially as its emergence corresponded with the book and movie Jurassic Park in the 1990s. In this paper, I use historical material, interviews with scientists, and philosophical literature to argue that the search for DNA from fossils can be characterized as a data-driven and celebrity-driven practice. Philosophers have recently argued the need to seriously consider the role of data-driven inquiry in the sciences, and likewise, this history highlights the need to seriously consider the role of celebrity in shaping the kind of research that gets pursued, funded, and ultimately completed. On this point, this history highlights that the traditional philosophical and scientific distinctions between data-driven and hypothesis-driven research are not always useful for understanding the process and practice of science. Consequently, I argue that the celebrity status of a particular research practice can be considered as a "serious epistemic strategy" that researchers, as well as editors and funders, employ when making choices about their research and publication processes. This interplay between celebrity and methodology matters for the epistemology of science.

Mycotoxines

The impact of chlorophyllin on deoxynivalenol transport across jejunum mucosa explants obtained from adult pigs Abstract Regardless of the efforts put into preventing or reducing fungal growth, extensive mycotoxin contamination has been reported in animal feeds. In the case of pigs, one of the mycotoxins of major concern is deoxynivalenol (DON). The use of adsorbents as feed additives represents one of the strategies to control mycotoxins' contamination in feedstuff. Therefore, the aim of the study was to verify the ability of chlorophyllin (CHL) to reduce the absorption rate of DON in swine mucosa explants. Intestine was obtained from routinely slaughtered adult pigs. The mucosa explants were studied by means of Ussing chamber technique. The effect of DON (10 and 30 μg/ml) on mucosa viability and permeability and CHL (100 μg/ml) impact on DON (30 μg/ml) absorption was verified. The results revealed that mucosa explants isolated from adult animals remained unaffected for 90 min in the presence of DON in the lower concentration (10 μg/ml). Mycotoxin in the higher dose (30 μg/ml) increased mucosa permeability (decreased transepithelial electrical resistance value) and enhanced paracellular transport of lucifer yellow and mannitol but did not affect lactate dehydrogenase leakage. The introduction of CHL neither diminished the absorption rate of DON across swine mucosa explants nor prevented the toxic effects of DON on intestine. In conclusion, the results confirm the negative effect of DON on pig jejunum mucosa. However, the toxic effect of DON was observed only when it was used in relatively high doses. A promising adsorbent agent, CHL, failed to reduce the intensity of DON transport across intestine under in vitro conditions.

Frequency and levels of mycotoxins in beer from the Mexican market and exposure estimate for deoxynivalenol mycotoxins Abstract The aim of the present study was to evaluate the occurrence of 23 mycotoxins in beer purchased in Mexico and to assess two exposure scenarios in the Mexican population through beer consumption. Multi-mycotoxin analysis of a total of 61 different beers (132 samples) was carried out using UHPLC-MS/MS equipment. Probability density functions were used to describe mycotoxins contamination. The daily intake of mycotoxins was estimated using a semi-probabilistic approach, applying the Monte Carlo method. Deoxynivalenol (DON) and its metabolites (deoxynivalenol-3-glucoside (DON3G) and 3-acetyl-deoxynivalenol (3ADON)) were the mycotoxins found in higher proportions in contaminated samples. None of the other mycotoxins overpassed the limit of quantification (LOQ) of the method. The combined intake of DON and its analogues ranged from 5.24 to 86.59 ng kg−1 bw day−1, which represent from 1.20 to 19.83% of the DON TDI. The results suggest that depending on the individual consumption of beer and depending on the type of beer, the intake of DON via beer could represent a significant percentage of the tolerable daily intake (TDI).

In vitro characterization of hepatic toxicity of Alternaria toxins Abstract Alternaria mycotoxins are secondary fungal metabolites which can contaminate food and feed. They are produced by Alternaria species with alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) as the main representatives for Alternaria mycotoxins in food. Once passing the intestinal barrier, Alternaria toxins can reach the liver to exert yet uncharacterized molecular effects. Therefore, hepatic in vitro systems were used to examine selected Alternaria mycotoxins for their induction of metabolism-dependent cytotoxicity, phosphorylation of the histone H2AX as a surrogate marker for DNA double-strand breaks, and relevant marker genes for hepatotoxicity. Analysis of cell viability as well as the induction of H2AX phosphorylation in the hepatocarcinoma cell line HepG2 revealed a detoxification of 100 μmol/l AME and AOH by pre-treatment with S9 liver homogenate as shown by a decrease in cytotoxicity and H2AX histone phosphorylation to levels observed in control cells. Concentrations up to 100 μmol/l TeA and TEN did not induce H2AX phosphorylation whether metabolized or not. In the metabolically competent human hepatoma cell line HepaRG, no cytotoxicity of Alternaria toxins occurred even at high concentrations up to 100 μmol/l, which indicates a low cytotoxic potential. Induction of gene expression associated with liver toxicity was analyzed by quantitative real-time PCR using a specific hepatotoxicity PCR array in HepaRG cells: here, an evidence was found that 50 μmol/l of AOH, AME, TeA, and TEN might be associated with hepatotoxic effects, necrosis, and the development of diseases like cholestasis and phospholipidosis.

Occurrence of ochratoxin A in typical salami produced in different regions of Italy Abstract A total of 172 different salamis were purchased from farms and small salami factories located in four Italian regions (Piedmont, Veneto, Calabria, and Sicily) and analyzed for the presence of ochratoxin A (OTA). Analysis was performed by high-performance liquid chromatography coupled to a fluorimetric detector (HPLC-FLD). The detection limit (LOD) for the method used was 0.05 μg/kg, while the quantitation limit (LOQ) was 0.20 μg/kg; the average recovery rate was 89.1%. OTA was detected in 22 salamis, and 3 samples exceeded the Italian guidance value for OTA in pork meat (1 μg/kg). In particular, what emerges from this research is the high percentage of spicy salamis among positive samples (68.2%, 15 out of 22), although spicy salamis are only 27.3% of the total number of samples collected and analyzed. Red chili pepper contaminated by OTA could be responsible for the presence of the mycotoxin in these spicy salamis. It follow that, also the control of some ingredients used in the manufacture of these meat products, like spices, should not be neglected.

Transgenic versus conventional corn: fate of fumonisins during industrial dry milling Abstract The aim of this study was to compare the fate of fumonisins in transgenic and non-transgenic corn during industrial dry milling. For this purpose, whole corn samples and their fractions (germ, pericarp, endosperm, corn meal, and grits) were collected from one of the major Brazilian milling plants, totaling 480 samples. There was no significant difference (p > 0.05) between mean fumonisin (FB1 + FB2) levels in transgenic (1130 μg/kg) and non-transgenic (920 μg/kg) whole corn. However, in non-transgenic germ, endosperm and corn meal fraction fumonisin levels were higher (2940 μg/kg, 250 μg/kg and 190 μg/kg, respectively) than in transgenic fractions (2180 μg/kg, 130 μg/kg and 85.0 μg/kg, respectively). Furthermore, the highest percentages of fumonisins were distributed in the germ, corresponding to about 87 and 76% of the total fumonisins present in the whole corn from non-transgenic and transgenic hybrids, respectively. Concerning the endosperm from non-transgenic and transgenic corn, approximately, 23% and 13% of the total fumonisins were retained after the dry milling. Further processing in corn meal (300 to 420 μm particle size) and grits (590 to 1190 μm) decreased the percentages of remaining fumonisins to 4% and 2% (transgenic) and 10% and 3% (non-transgenic corn), respectively. These results suggested that fumonisin concentration was higher in outer and inner non-transgenic fractions when compared to transgenic ones and that the fate of fumonisins during the industrial dry milling could be affected by the transgenic status. However, it was not possible to conclude that the difference was exclusively due to this variable.

Interaction of the mycotoxin metabolite dihydrocitrinone with serum albumin Abstract Citrinin (CIT) is a nephrotoxic mycotoxin produced by Penicillium, Monascus, and Aspergillus species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes is supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (K~105 L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.

Aflatoxin in maize: a review of the early literature from "moldy-corn toxicosis" to the genetics of aflatoxin accumulation resistance Abstract Aflatoxin is a potent toxin produced by Aspergillus flavus Link:Fr, an opportunistic ear-rot pathogen of maize (Zea mays L. subsp. Mays). Prior to the discovery of aflatoxin, A. flavus was considered a minor pathogen and was not a priority for maize breeders or pathologists. Aflatoxin was discovered in England in 1961 following an epidemic in poultry. By the early 1970s, surveys of agricultural commodities in the USA found that maize produced in the Southeast was especially vulnerable to aflatoxin contamination. Aflatoxin contamination was initially treated as a post-harvest issue, but pre-harvest contamination was proven by 1975. Pre-harvest contamination meant that genetically based host-plant resistance was a possible solution. The potential magnitude of the problem became apparent in 1977 when the southeastern US maize crop suffered epidemic aflatoxin contamination. The first experiment demonstrating the heritability of host-plant resistance to aflatoxin accumulation was published in 1978. These events combined to make breeding for reduced aflatoxin contamination both a high priority and a rational breeding objective. This review surveys the early scientific literature in order to place research on the genetics of aflatoxin accumulation in maize into historical context. It tells the story of how multi-disciplinary research began with veterinary diseases of unknown etiology and resulted in host-plant resistance to a previously minor plant pathogen becoming a central public sector breeding objective.

First report of Fusarium foetens as a mycotoxin producer Abstract Fusarium foetens, a pathogen of Begonia plants, has been recently described as a new fungal species. This Fusarium species causes a destructive vascular wilt disease which leads to the death of the plant. Moreover, Fusarium species are known to produce a huge variety of secondary metabolites such as mycotoxins and phytotoxins. Here, we studied the toxicogenic profile of one F. foetens strain, isolated from maize, employing two methods based on the use of ultra-performance liquid chromatography coupled to mass spectrometry-ion trap-time of flight detection. The mycotoxins beauvericin and fusaric acid were detected in a pure culture of F. foetens. In addition, four fusaric acid analogs (10,11-dihidroxyfusaric acid, hydroxyfusaric acid, dehydrofusaric acid, and a hydroxylated unsaturated fusaric acid analog) were tentatively identified on the basis of their accurate mass and fragmentation patterns. Therefore, these preliminary data indicate that F. foetens isolated from maize is able to produce Fusarium mycotoxins including beauvericin and fusaric acid.

Effects of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol on parameters associated with oxidative stress in HepG2 cells Abstract This work studied the effect of deoxynivalenol (DON) and two of its acetylated analogs (3-ADON, 15-ADON) on first indicators of oxidative stress status, namely production of reactive oxygen species (ROS) and induction of lipid peroxidation (LPO), in HepG2 cells. HepG2 cells were exposed to different concentrations of the three toxins, either alone or in combinations, for 24, 48, and 72 h. Results of cytotoxicity obtained in HepG2 cells were correlated with the detection of ROS and LPO. This effect was inversely correlated with ROS while directly correlated with LPO for the assayed mycotoxins in individual treatment. Combinations of two toxins containing 15-ADON yielded highest values, while for two-toxin combinations with 3-ADON, the effects were minor. A combination of all three mycotoxins alleviated ROS production and the highest levels in LPO were detected, in association to a final breakdown of adaption of ROS early produced by HepG2. In conclusion, parameters of stress evaluation presented in this study (ROS and LPO), revealed increases in HepG2 cells exposed to DON, 3-ADON, and 15-ADON either individually or combined.

Mycotoxins in poultry feed and feed ingredients in Nigeria Abstract Mycotoxins are toxic secondary fungal metabolites that can negatively affect animal productivity when ingested through feed. In order to assess mycotoxin contamination of poultry feed and feed ingredients vis-a-vis source tracking of feed contamination in Nigeria, 102 samples of feed (n = 30) and feed ingredients (n = 72) were collected from in-house mills of poultry farms across 12 states of Nigeria and analyzed for multiple mycotoxins using LC/MS-MS. One hundred and forty microbial metabolites were detected in the feed and feed ingredients. The most frequent mycotoxin in the feed was fumonisin B1, occurring in 97% of the samples at mean concentration of 1014 μg kg−1. AFB1 occurred in 83% of the feed samples at mean concentration of 74 μg kg−1 and in all feed ingredients except fish meal and other cereals (millet and rice). Feed samples analyzed in this study were contaminated with at least four mycotoxins: aflatoxins and fumonisin co-occurring in 80% of the samples. Peanut cake and maize contributed the most to the levels of aflatoxin and fumonisin, respectively, in the feed. Consequently, there is a need to explore other cereal- and protein-based ingredients for compounding feeds in order to reduce the risk associated with high mycotoxin (e.g. aflatoxin) intake in poultry.