Retinoid Binding Research Articles

Conditional Knockouts of Interphotoreceptor Retinoid Binding Protein Suggest Two Independent Mechanisms for Retinal Degeneration and Myopia.

Interphotoreceptor retinoid-binding protein's (IRBP) role in eye growth and its involvement in cell homeostasis remain poorly understood. One hypothesis proposes early conditional deletion of the IRBP gene could lead to a myopic response with retinal degeneration, whereas late conditional deletion (after eye size is determined) could cause retinal degeneration without myopia. Here, we sought to understand if prior myopia was required for subsequent retinal degeneration in the absence of IRBP. This study investigates if any cell type or developmental stage is more important in myopia or retinal degeneration. IBRPfl/fl mice were bred with 5 Cre-driver lines: HRGP-Cre, Chx10-Cre, Rho-iCre75, HRGP-Cre Rho-iCre75, and Rx-Cre. Mice were analyzed for IRBP gene expression through digital droplet PCR (ddPCR). Young adult (P30) mice were tested for retinal degeneration and morphology using spectral-domain optical coherence tomography (SD-OCT) and hematoxylin and eosin (H&E) staining. Function was analyzed using electroretinograms (ERGs). Eye sizes and axial lengths were compared through external eye measurements and whole eye biometry. Across all outcome measures, when bred to IRBPfl/fl, HRGP-Cre and Chx10-Cre lines showed no differences from IRBPfl/fl alone. With the Rho-iCre75 line, small but significant reductions were seen in retinal thickness with SD-OCT imaging and postmortem H&E staining without increased axial length. Both the HRGP-Cre+Rho-iCre75 and the Rx-Cre lines showed significant decreases in retinal thickness and outer nuclear layer cell counts. Using external eye measurements and SD-OCT imaging, both lines showed an increase in eye size. Finally, function in both lines was roughly halved across scotopic, photopic, and flicker ERGs. Our studies support hypotheses that for both eye size determination and retinal homeostasis, there are two critical timing windows when IRBP must be expressed in rods or cones to prevent myopia (P7-P12) and degeneration (P21 and later). The rod-specific IRBP knockout (Rho-iCre75) showed significant retinal functional losses without myopia, indicating that the two phenotypes are independent. IRBP is needed for early development of photoreceptors and eye size, whereas Rho-iCre75 IRBPfl/fl knockout results in retinal degeneration without myopia.

Molecular assessment of the taxonomic status of pine vole species Microtus subterraneus and Microtus majori in southeastern Europe and Turkey

Intra– and inter–specific phylogenetic relationships and the taxonomic status of pine vole populations have been controversial for years, and cryptic species are thought to exist, especially concering the species' distribution area. To clarify the taxonomic status of Turkish populations we analysed mitochondrial (cytochrome–b and cytochrome oxidase–I) and nuclear (interphotoreceptor retinoid binding protein) gene markers, adding GenBank data from Europe and Caucasia. Considering the data obtained based on mean genetic distance, genetic diversity and fixation index values, Bayesian trees and Median–joining networks, we found that M. subterraneus and M. majori are valid species which have diverged since 1.28 Mya. Findings also suggested that although Anatolian, Thracian (Turkish Thrace and Greece) and European populations of M. subterraneus and Anatolian and Caucasian populations of M. majori have been in the process of divergency since 0.528–0.337 Mya correspond to the Pleistocene glacial periods, these intrapopulations do not appear to be different species.Besides, considering the high intraspecific variation in M. subterraneus, it remains likely that new species could be identified in future studies.

The role of microbiome in uveitis in experimental mice model

AbstractUnlike the individual's genetic makeup, gut microbiota can be influenced, and thus used in the disease therapy or even prevention. We have shown that absence or reduction of microbiota modifies the inflammatory response in the murine model of experimental autoimmune uveoretinitis (EAU). Our further purpose of the project was to test influence of probiotics on intensity of intraocular inflammation in EAU. Uveitis was induced in C57BL/6J mice by interphotoreceptor retinoid binding protein in complete Freund's adjuvant and with pertussis toxin. The grade of uveitis was assessed in vivo by using otoscope connected to camera and post mortem on haematoxylin and eosin stained tissues. Bacteria Escherichia coli were administered orally three times a week and saline solution was administered to control group. Different regimens of administration were tested. Interestingly, mice administered with probiotics reached statistically significant lower grade of uveitis in comparison to the control group in clinical and histological evaluation. This may have valuable implications for the control of sight‐threatening uveitis in patients.Supported by research project AZV MZ CR 17‐31248A.Key words: microbiome, probiotics, Escherichia coli, experimental autoimmune uveitis, EAU.

Comparative genetic research on Microtus mystacinus (de Filippi, 1865) distributed in Asia and Europe inferred from mitochondrial (CYTB and COXI) and nuclear (IRBP) gene regions

Abstract The East European vole Microtus mystacinus is the most widespread vole species in Anatolia. It is also frequently seen in watery habitats in a large area of Eurasia. In this paper, an attempt was made to ascertain the level of genetic differentiation between Anatolian (Asian part of Turkey) and Turkish Thracian (European part of Turkey) populations together with additional data from other parts of Asia and Europe by analysing two mitochondrial (cytochrome-b and cytochrome oxidase subunit I) and one nuclear (interphotoreceptor retinoid binding protein) gene regions. Acquired Bayesian Inference trees mostly separated the Asian and European populations of M. mystacinus and the fixation index values implied a significant differentiation between these populations for mitochondrial DNA. On the other hand, the median-joining networks did not show diverging populations, significantly, and the mean genetic distance values among populations were found to be low for both mitochondrial and nuclear DNA. Evolutionary divergence times of Asian and European populations were also calculated and dated back to approximately 0.316–0.111 million years ago, coinciding with the ice ages of the Pleistocene epoch. According to the obtained results, M. mystacinus populations have not diverged enough to form different species; however, there is a separation between Asian and European populations which might result in speciation.

Melatonin, an endogenous hormone, modulates Th17 cells via the reactive-oxygen species/TXNIP/HIF-1α axis to alleviate autoimmune uveitis

BackgroundMelatonin, an indoleamine produced by the pineal gland, plays a pivotal role in maintaining circadian rhythm homeostasis. Recently, the strong antioxidant and anti-inflammatory properties of melatonin have attracted attention of researchers. We evaluated the therapeutic efficacy of melatonin in experimental autoimmune uveitis (EAU), which is a representative animal model of human autoimmune uveitis.MethodsEAU was induced in mice via immunization with the peptide interphotoreceptor retinoid binding protein 1–20 (IRBP1–20). Melatonin was then administered via intraperitoneal injection to induce protection against EAU. With EAU induction for 14 days, clinical and histopathological scores were graded to evaluate the disease progression. T lymphocytes accumulation and the expression of inflammatory cytokines in the retinas were assessed via flow cytometry and RT-PCR, respectively. T helper 1 (Th1), T helper 17 (Th17), and regulatory T (Treg) cells were detected via flow cytometry for both in vivo and in vitro experiments. Reactive-oxygen species (ROS) from CD4 + T cells was tested via flow cytometry. The expression of thioredoxin-interacting protein (TXNIP) and hypoxia-inducible factor 1 alpha (HIF-1α) proteins were quantified via western blot.ResultsMelatonin treatment resulted in notable attenuation of ocular inflammation in EAU mice, evidenced by decreasing optic disc edema, few signs of retinal vasculitis, and minimal retinal and choroidal infiltrates. Mechanistic studies revealed that melatonin restricted the proliferation of peripheral Th1 and Th17 cells by suppressing their transcription factors and potentiated Treg cells. In vitro studies corroborated that melatonin restrained the polarization of retina-specific T cells towards Th17 and Th1 cells in addition to enhancing the proportion of Treg cells. Pretreatment of retina-specific T cells with melatonin failed to induce EAU in naïve recipients. Furthermore, the ROS/ TXNIP/ HIF-1α pathway was shown to mediate the therapeutic effect of melatonin in EAU.ConclusionsMelatonin regulates autoimmune T cells by restraining effector T cells and facilitating Treg generation, indicating that melatonin could be a hopeful treatment alternative for autoimmune uveitis.

Parental uveitis causes elevated hair loss in offspring of C57BL/6J mice

Our previous study demonstrated that parental uveitis in a susceptible population can cause hair loss and increase the susceptibility to experimental autoimmune uveitis (EAU) in offspring. However, it is unclear whether parental uveitis affects the development of offspring in an EAU-moderate-susceptible population. Herein, moderate-susceptible C57BL/6J mice were immunized with inter-photoreceptor retinoid binding protein (IRBP) 651–670 to develop EAU and were kept together for mating. Gross examination and histopathological changes of the offspring gestated with parental uveitis were observed to evaluate the impact of parental uveitis on the development of the offspring. Differentially expressed genes (DEGs) were screened by RNA sequencing in the affected skin and eyeball of the offspring on postnatal day 27. Adult offspring were injected 75 μg IRBP651-670 to evaluate their susceptibility to EAU. Gross examination in the offspring revealed hair loss on postnatal days 11–31. Histopathological observation showed increased melanin granules and hair follicles of skin in the affected offspring with hair loss. Gene Ontology (GO) analysis in the skin revealed differential expression of genes involved in the mitotic cell cycle, response to endogenous stimulus, hair follicle development, and hair cycle. The DEGs in the skin were predominately associated with the cell cycle and peroxisome proliferator-activated receptor (PPAR) signaling pathway. The GO enrichment analysis in the eyeball showed differential expression of genes involved in the nervous system development, camera-type eye photoreceptor cell differentiation, neuron projection morphogenesis, axon development, and calcium-induced calcium release activity; enriched pathways included the circadian entrainment and glutamatergic synapses. No increased susceptibility to EAU in offspring gestated from parental remitting EAU was observed at a low-dose 75 μg IRBP induction. These results suggested that parental uveitis in a moderate-susceptible population could affect the skin development and DEG profiles of skin and eyeball related to the response to endogenous stimulus, the PPAR signaling pathway, and glutamatergic synapse, which provides the molecular evidence to explain the influence of parental uveitis on offspring development.

Hydroxychloroquine Alleviates EAU by Inhibiting Uveitogenic T Cells and Ameliorating Retinal Vascular Endothelial Cells Dysfunction.

PurposeInflammation triggers the activation of CD4+T cells and the breakdown of blood–retinal barrier, thus contributing to the pathology of experimental autoimmune uveitis (EAU). We explored the anti-inflammatory effect of hydroxychloroquine (HCQ) on EAU and the potential mechanisms active in T cells and retinal vascular endothelial cells (RVECs).MethodsC57BL/6J mice were immunized with interphotoreceptor retinoid binding protein 1-20 (IRBP1–20) to induce EAU and then treated with the vehicle or HCQ (100 mg/kg/day). On day 7, 14, 21, 30 and 60 after immunization, clinical scores were evaluated. On day 14, histopathological scores were assessed, and retinas, spleens, and lymph nodes were collected for quantitative polymerase chain reaction or flow cytometry analysis. RVEC dysfunction was induced by tumor necrosis factor α (TNF-α) stimulation. The expression of cytokines, chemokines, adhesion molecules, and lectin-like oxidized LDL receptor-1 (LOX-1)/nuclear factor κB (NF-κB) was measured in RVECs with or without HCQ.ResultsHCQ treatment protected mice from uveitis, evidenced by reduced expression of inflammatory factors, chemokines, and adhesion molecules in the retina. In systemic immune response, HCQ inhibited the activation of naïve CD4+T cells and frequencies of T effector cells, and promoted T regulatory cells. HCQ decreased IRBP1-20–specific T cell responses and proliferation of CD4+T cells in vitro. Further studies established that TNF-α induced RVECs to express inflammatory cytokines, chemokines, and adhesion molecules, whereas HCQ alleviated the alterations via the LOX-1/NF-κB pathways.ConclusionsHCQ alleviates EAU by regulating the Teff/Treg balance and ameliorating RVECs dysfunction via the LOX-1/NF-κB axis. HCQ may be a promising therapeutic candidate for uveitis.

Experimental Autoimmune Uveitis: An Intraocular Inflammatory Mouse Model.

Experimental Autoimmune Uveitis (EAU) is driven by immune cells responding to self-antigens. Many features of this non-infectious, intraocular inflammatory disease model recapitulate the clinical phenotype of posterior uveitis affecting humans. EAU has been used reliably to study the efficacy of novel inflammatory therapeutics, their mode of action and to further investigate the mechanisms that underpin disease progression of intraocular disorders. Here, we provide a detailed protocol on EAU induction in the C57BL/6J mouse - the most widely used model organism with susceptibility to this disease. Clinical assessment of disease severity and progression will be demonstrated using fundoscopy, histological examination and fluorescein angiography. The induction procedure involves subcutaneousinjection of an emulsion containing a peptide (IRBP1-20) from the ocular protein interphotoreceptor retinoid binding protein (also known as retinol binding protein 3), Complete Freund's Adjuvant (CFA) and supplemented with killed Mycobacterium tuberculosis. Injection of this viscous emulsion on the back of the neck is followed by a single intraperitoneal injection of Bordetella pertussis toxin. At the onset of symptoms (day 12-14) and under general anesthesia, fundoscopic images are taken to assess disease progression through clinical examination. These data can be directly compared with those at later timepoints and peak disease (day 20-22) with differences analyzed. At the same time, this protocol allows the investigator to assess potential differences in vessel permeability and damage using fluorescein angiography. EAU can be induced in other mouse strains - both wildtype or genetically modified - and combined with novel therapies offering flexibility for studying drug efficacy and/or disease mechanisms.

The rhodopsin-retinochrome system for retinal re-isomerization predates the origin of cephalopod eyes

BackgroundThe process of photoreception in most animals depends on the light induced isomerization of the chromophore retinal, bound to rhodopsin. To re-use retinal, the all-trans-retinal form needs to be re-isomerized to 11-cis-retinal, which can be achieved in different ways. In vertebrates, this mostly includes a stepwise enzymatic process called the visual cycle. The best studied re-isomerization system in protostomes is the rhodopsin-retinochrome system of cephalopods, which consists of rhodopsin, the photoisomerase retinochrome and the protein RALBP functioning as shuttle for retinal. In this study we investigate the expression of the rhodopsin-retinochrome system and functional components of the vertebrate visual cycle in a polyplacophoran mollusk, Leptochiton asellus, and examine the phylogenetic distribution of the individual components in other protostome animals.ResultsTree-based orthology assignments revealed that orthologs of the cephalopod retinochrome and RALBP are present in mollusks outside of cephalopods. By mining our dataset for vertebrate visual cycle components, we also found orthologs of the retinoid binding protein RLBP1, in polyplacophoran mollusks, cephalopods and a phoronid. In situ hybridization and antibody staining revealed that L. asellus retinochrome is co-expressed in the larval chiton photoreceptor cells (PRCs) with the visual rhodopsin, RALBP and RLBP1. In addition, multiple retinal dehydrogenases are expressed in the PRCs, which might also contribute to the rhodopsin-retinochrome system.ConclusionsWe conclude that the rhodopsin-retinochrome system is a common feature of mollusk PRCs and predates the origin of cephalopod eyes. Our results show that this system has to be extended by adding further components, which surprisingly, are shared with vertebrates.

Taxonomic status of Dahl’s Jird, Meriones dahli, as inferred from cytochrome b and IRBP gene sequences (Mammalia: Rodentia)

Dahl’s Jird, Meriones dahli, is distributed in Armenia and eastern Turkey and was previously considered as a subspecies of M. meridianus based on morphological characters. We examined the taxonomic status of Dahl’s Jird using two gene regions, the mitochondrial cytochrome b (Cytb) gene and the Inter Retinoid Binding Protein (IRBP), a nuclear gene. Phylogenetic trees and median-joining networks confirmed the close relationship between M. dahli and M. meridianus. The evolutionary divergence time between these populations was estimated 750 Kya, indicating that these populations diverged in the Middle Pleistocene. The genetic distance between M. dahli and M. meridianus was 4% for Cytb and 0.6% for IRBP. In conclusion, we characterized the genetic distance using molecular methods. We propose the relegation of M. dahli back to subspecies status as M. m. dahli.

STAT3-Specific Single Domain Nanobody Inhibits Expansion of Pathogenic Th17 Responses and Suppresses Uveitis in Mice.

STAT3 activates transcription of genes that regulate cell growth, differentiation, and survival of mammalian cells. Genetic deletion of Stat3 in T cells has been shown to abrogate Th17 differentiation, suggesting that STAT3 is a potential therapeutic target for Th17-mediated diseases. However, a major impediment to therapeutic targeting of intracellular proteins such as STAT3 is the lack of efficient methods for delivering STAT3 inhibitors into cells. In this study, we developed a novel antibody (SBT-100) comprised of the variable (V) region of a STAT3-specific heavy chain molecule and demonstrate that this 15 kDa STAT3-specific nanobody enters human and mouse cells, and induced suppression of STAT3 activation and lymphocyte proliferation in a concentration-dependent manner. To investigate whether SBT-100 would be effective in suppressing inflammation in vivo, we induced experimental autoimmune uveitis (EAU) in C57BL/6J mice by active immunization with peptide from the ocular autoantigen, interphotoreceptor retinoid binding protein (IRBP651-670). Analysis of the retina by fundoscopy, histological examination, or optical coherence tomography showed that treatment of the mice with SBT-100 suppressed uveitis by inhibiting expansion of pathogenic Th17 cells that mediate EAU. Electroretinographic (ERG) recordings of dark and light adapted a- and b-waves showed that SBT-100 treatment rescued mice from developing significant visual impairment observed in untreated EAU mice. Adoptive transfer of activated IRBP-specific T cells from untreated EAU mice induced EAU, while EAU was significantly attenuated in mice that received IRBP-specific T cells from SBT-100 treated mice. Taken together, these results demonstrate efficacy of SBT-100 in mice and suggests its therapeutic potential for human autoimmune diseases.

Molecular characterization and phylogeography of Mediterranean picarels (Spicara flexuosa, S. maena and S. smaris) along the coasts of Turkey and the Eastern Mediterranean

In this study, molecular taxonomy associated with Spicara flexuosa, Spicara maena, and Spicara smaris was examined with a geographical focus along the coasts of the Turkey and the Mediterranean. In addition, the effects of the Turkish Straits System on the evolutionary history and phylogeography of Spicara flexuosa were investigated. The mitochondrial cytochrome c oxidase I, cytochrome b, and the nuclear Interphotoreceptor retinoid–binding protein genes were used for these purposes. Our results indicated that the distinction of the three taxa under Spicara is possible with the two different mitochondrial DNA markers. Nuclear DNA analyses indicated that reproductive isolation of the S. maena and S. flexuosa was complete. Demographic analyses support the sudden population growth of S. flexuosa after a potential bottleneck. The result of the dating analyses suggested that the three Spicara species were differentiated during the early or middle Pliocene. An absence of genetic structure between Spicara flexuosa subpopulations from Turkey indicated the connectivity, within sampling locations, suggesting that the Turkish Straits System is a corridor for gene flow for this species. As picarel populations have experienced rapid decline in their distribution areas in recent years, separate population studies are necessary to help make informed conservation and management decisions for S. maena and S. flexuosa.

Antiepileptogenic Effect of Retinoic Acid.

Retinoic acid, a metabolite of vitamin A, acts through either genomic or nongenomic actions. The genomic action of retinoids exerts effects on gene transcription through interaction with retinoid receptors such as retinoic acid receptors (RARα, β, and γ) and retinoid X receptors (RXRα, β, and γ) that are primarily concentrated in the amygdala, pre-frontal cortex, and hippocampal areas in the brain. In response to retinoid binding, RAR/RXR heterodimers undergo major conformational changes and orchestrate the transcription of specific gene networks. Previous experimental studies have reported that retinoic acid exerts an antiepileptogenic effect through diverse mechanisms, including the modulation of gap junctions, neurotransmitters, long-term potentiation, calcium channels and some genes. To our knowledge, there are no previous or current clinical trials evaluating the use of retinoic acid for seizure control.

Reversible Michael Addition of an Engineered Cysteine Residue to a Light Activated Ligand, a New Design Strategy for a Synthetic Photoswitchable Fluorescent Protein

Reversible photoswitchable fluorescent probes can transit between two different optical states when triggered by light. Because of the ability to switch, they become useful in live cell super-resolution microscopy (e.g. RESOLFT, psSIM, STORM, PALM), information storage, and optical control of protein activity. Currently, only a handful of fluorescent proteins and synthetic dyes exhibit reversible photoswitchability. Hence, there is a growing interest for new systems with enhanced functionality. We are currently focused on utilizing a synthetic ligand capable of reversible Michael addition of an active cysteine residue, within the binding pocket of human cellular retinoid binding protein (hCRBPII).

Greater Bandicoot Rats (Bandicota indica) are Not Native to Sundaland Based on Deoxyribonucleic Acid (DNA) Analyses

Bandicoot rats (genus Bandicota) are distributed widely across the Indomalay biogeographic realm of tropical East Asia. One widely distributed species, the greater bandicoot rat (Bandicota indica), has a disjunct distribution including both north and south of the biogeographic break at the Isthmus of Kra. We compared genetic variation of greater bandicoot rats from north and south of the Isthmus of Kra using mitochondrial cytochrome b (cyt b, 1140 bp) and nuclear interphotoreceptor retinoid binding protein (IRBP, 801 bp) sequences. We found that the greater bandicoot rat (B. indica) is not native to Sundaland, the region south of the Isthmus of Kra. The species was introduced to the region recently as the genetic divergence with other regions is very low and phylogenies of both genes showed Malaysian greater bandicoot rat very closely related to conspecifics from Lao PDR. Haplotype data revealed all individuals from Malaysia are homogenous, which implied that the species was introduced recently. The greater bandicoot rats in Malaysia are so far only reported in the rice producing regions of Kedah and Perlis, but they may be increasing in number and distribution. A more detailed survey on the distribution and population demographics of Malaysian greater bandicoot rats are needed to support a management plan for this invasive species.

The protective function of invariant natural killer T cells in the relapse of experimental autoimmune uveoretinitis

Experimental autoimmune uveoretinitis (EAU) in mice provides a useful platform to study the pathogenesis and experimental therapeutics of human uveitis. One often used EAU model employs C57BL/6 (B6) mice sensitized with a peptide residue having 1 to 20 amino acids of human interphotoreceptor retinoid binding protein (hIRBP1-20). The model using the B6 background has permitted a liberal use of genetically engineered strains and has provided insights for understanding uveoretinitis. However, this is usually acute/monophasic and does not represent human uveoretinitis that is characterized as a chronic/recurrent disease. Several chronic/recurrent EAU models have been developed; of these, we employed administration of staphylococcal enterotoxin B (SEB) for relapse in the present study, and found that recurrence was induced at day 24 after primary immunization, which is thought to be the convalescent phase. We reported the activation of invariant natural killer T (iNKT)-cells upon primary immunization of the EAU model mice with the ligand RCAI-56, which was found to mitigate the disease in our previous study. Here, we first attempted to ameliorate EAU in the relapse model using a preventive regimen by activating iNKT cells at the same time relapse induction (day 24) or in a regimen after 3 days of relapse induction (day 27). The preventive as well as post-inductive regimens were successful in reducing histopathological scores by inhibiting the Ag-specific Th17-biased response. Collectively, activation of iNKT cells may be useful to mitigate the relapse response of EAU induced with SEB.

A new fungus-mediated RNAi method established and used to study the fatty acid and retinol binding protein function of the plant-parasitic nematode Aphelenchoides besseyi

ABSTRACT RNA interference (RNAi) is a powerful tool for gene functional analysis of plant-parasitic nematodes (PPNs). RNAi involving soaking in a dsRNA solution and in planta methods is commonly applied in the study of gene function in PPNs. However, certain problems restrict the application of these methods. Therefore, more convenient and effective RNAi methods need to be established for different PPNs according to their biological characteristics. In this study, the fatty acid and retinoid binding protein genes (Ab-far-1, Ab-far-4, and combinatorial Ab-far-1 and Ab-far-4) of the rice white tip nematode (RWTN), Aphelenchoides besseyi, were used as target genes to construct a fungal RNAi vector, and the Ab-far-n dsRNA transgenic Botrytis cinerea (ARTBn) were generated using Agrobacterium-mediated transformation technology. After RWTN feeding on ARTBn, the expression of Ab-far-1 and Ab-far-4 in the nematodes was efficiently silenced, and the reproduction and pathogenicity of the nematodes were clearly inhibited. The Ab-far-1 and Ab-far-4 co-RNAi effects were better than the effects when each gene was individually targeted with RNAi. Additionally, the RNAi induced when RWTNs fed on ARTB1 were persistent and heritable. Thus, a new method of fungus-mediated RNAi was established for fungivorous PPNs and was verified as effective and applicable to the study of nematode gene function. This technique will remove the technological bottlenecks and provide a new method to studying the multiple genes with polygene co-RNAi in fungivorous PPNs. This study also provides a theoretical basis and new thought for further study of the gene function in PPNs. Abbreviations: FAR(Fatty acid and retinol-binding proteins); RWTN (The rice white tip nematode, Aphelenchoides besseyi); Ab-far-n (Fatty acid and retinol binding protein gene of A. besseyi); ARTB1 (Ab-far-1 hpRNA transgenic Botrytis cinerea); ARTB4 (Ab-far-4 hpRNA transgenic Botrytis cinerea); ARTB1/4 (combinatorial Ab-far-1 and Ab-far-4 hpRNA transgenic B. cinerea); EVTB (Empty vector transgenic B. cinerea); GRTB (eGFP hpRNA transgenic B. cinerea); WTB (Wild-type B. cinerea).

Synthesis and biological evaluation of an antibacterial azaborine retinoid isostere

Our continued synthetic interest in this class of retinoids, CD437 and its analogs, against methicillin-resistant Staphylococcus aureus (MRSA) has brought us to explore further isosteric substitutions within the scaffold. Although our previous findings have shown promising activity against gram-positive pathogens, their therapeutic viability remained an issue. Specifically, through preliminary analysis, our best performing compound, analog 2, displayed low solubility within serum as well as high affinity for retinoid binding proteins with a concentration dependent relationship. To circumvent this issue, we proposed a class of analogs containing an azaborine substitution in place of the naphthalene moiety. Azaborines have a nitrogen-boron bond substituting a carbon–carbon double bond that alters the electronics of the parent scaffold. This motif has been explored successfully in cancer research but to the best of our knowledge has yet to be applied to antibiotics. Herein, we describe the synthesis of the desired analogs, antimicrobial activity, and surprising physiochemical properties.

Alpha-taxonomy in the cricetid rodent Neomicroxus, a first assessment

Neomicroxus , a recently named genus, comprises small-bodied cricetid rodents patchily distributed in high-Andean ranges from Ecuador to Venezuela. Currently, two species of Neomicroxus are recognized, N. bogotensis , endemic to the Cordillera Oriental in Colombia and Cordillera de Merida and Paramo de Tama in Venezuela, and N. latebricola that occurs northern Andes of Ecuador. The genus is among the most poorly understood Neotropical rodents and to date no formal assessment about its alpha taxonomy was conducted. Based on DNA evidence of the first portion of the mitochondrial cytochrome b gene (cytb) and the first exon of the interphotoreceptor retinoid binding protein (IRBP), as well as craniodental measurements, we explored the divergence degree, genetic structure and phyletic relationships of the two species currently allocated under Neomicroxus . Our analyses support the monophyly of the genus as well as its uncertain tribal affiliation. Neomicroxus was retrieved as structured in two main branches, in agreement with the traditional recognition of two species. The populations referred to N. bogotensis exhibit deep divergence values (> 6 %) pointing to the existence of undescribed species under its concept. In contrast, populations of N. latebricola show a shallow genetic structure although implying recognizable geographical breaks. A moderate degree of genetic and morphological differentiation supports a new subspecies for the western populations of N. latebricola . Our contribution is the first attempt to better understanding the alpha taxonomy of Neomicroxus , highlighting the importance of the geographic complexity as a barrier to the genetic flow in N. bogotensis and the significance of the subspecies concept to formalize the geographic variation recovered in N. latebricola

HcFAR, a functional inhibitor of goat TGF-β1 identified from excretory and secretory products of Haemonchus contortus

Haemonchus contortus has developed complexed and multifaceted mechanisms of immune evasion to enable the survival in the host. Generating excretion and secretion products (ESPs) to subvert or suppress the functions of host cytokines is a newly immune regulatory pattern found during recent years. Transforming growth factor-β (TGF-β) has critical immune regulatory functions in nematode infections. In this study, co-immunoprecipitation (co-IP) assay was used to identify the goat TGF-β1 binding proteins from HcESPs. The interaction between TGF-β1 and nematode fatty acid retinoid binding domain containing protein of H. contortus (HcFAR) was analyzed by glutathione S-transferase (GST)-pull down assay. The suppressive effect of rHcFAR on TGF-β1-induced immunoglobulin A (IgA) secretion was observed by co-incubation of rHcFAR and TGF-β1 with goat peripheral blood mononuclear cells (PBMCs). The IgA concentrations were determined using enzyme linked immunosorbent assay (ELISA) kit. Meanwhile, the suppressive effect of rHcFAR on TGF-β1-induced T helper (Th) 9 differentiation was investigated by co-incubation of rHcFAR, TGF-β1 and interleukin (IL)-4 with goats PBMCs. In parallel, IL-4 was replaced by IL-6 to determine the effects on the Th17 differentiation. The transcriptions of IL-9 and IL-17 in PBMCs were then evaluated by real-time PCR. Finally, we found that HcFAR from HcESPs could bind to goat TGF-β1 in vitro. The ELISA results of IgA showed that 40 μg/mL rHcFAR could suppress the IgA secretion of PBMCs induced by TGF-β1. Additionally, rHcFAR (at 10 μg/mL and 20 μg/mL) could inhibit the mRNA transcription of IL-9 induced by TGF-β1 and IL-4. Meanwhile, rHcFAR could also downregulate the transcription of IL-17 induced by TGF-β1 and IL-6 in a dose-dependent manner. These results indicated that HcFAR was a functional inhibitor of goat TGF-β1 and this information may help contribute to understanding of the relationship between the ESPs and host cytokines.