Beta Domain Research Articles

Microhomology-mediated end-joining Knock-In approaches to delete the allergenic domain of trout parvalbumin beta-1. Preliminary results in F0 animals and feedback

Background Gene editing techniques offer new opportunities to improve important traits in aquaculture. The allergenicity of fish flesh is a major problem in aquaculture. Parvalbumin (Parv) is the most prevalent fish allergen. For instance, in salmonids, a single parvalbumin beta-1 protein (parvb1) has been identified as an allergen in specific patients. Therefore, generating trout carrying two parvb1 alleles deleted from the allergenic peptide-encoding region could prevent allergies in these sensitive individuals. Methods Here, we describe the application of the Crispr/cas9 system in an attempt to delete parvb1 exon 2 encoding the allergenic peptide and, alternatively, to replace exon 2 of parvb1 with exon2 of parvalbumin beta-2 protein (parvb2,) which does not encode the allergenic peptide. Exon skipping and swapping were pursued through microhomology-mediated end-joining (MMEJ) knock-In using specifically designed double-stranded donor DNA. Results Genotyping of approximately 200 F0 fingerlings originating from eggs injected with donor DNA designed for exon 2 skipping led to the identification of only one animal carrying an allele lacking exon 2. Genotyping of approximately 150 fingerlings originating from eggs injected with donor DNA for exon 2 swapping did not result in any trout carrying the expected modified allele. Conclusions These preliminary results indicate the potential difficulties associated with the MMEJ KI experiments performed in farmed fish. Finally, new genomic techniques in aquaculture are further discussed in the context of lively debates taking place in the European parliament regarding a possible revision of the current law that determines the legal status of farm animals modified by genome editing. Gene editing, microhomology-mediated end-joining knock-in, parvalbumin, allergenicity, trout, and genetically modified organisms (GMOs).

Evolution of SARS-CoV-2 Spikes shapes their binding affinities to animal ACE2 orthologs.

Spike-receptor interaction is a critical determinant for the host range of coronaviruses. In this study, we investigated the SARS-CoV-2 WHU01 strain and five WHO-designated SARS-CoV-2 variants of concern (VOCs), including Alpha, Beta, Gamma, Delta, and the early Omicron variant, for their Spike interactions with ACE2 proteins of 18 animal species. First, the receptor-binding domains (RBDs) of Alpha, Beta, Gamma, and Omicron were found to display progressive gain of affinity to mouse ACE2. More interestingly, these RBDs were also found with progressive loss of affinities to multiple ACE2 orthologs. The Omicron RBD showed decreased or complete loss of affinity to eight tested animal ACE2 orthologs, including that of some livestock animals (horse, donkey, and pig), pet animals (dog and cat), and wild animals (pangolin, American pika, and Rhinolophus sinicus bat). These findings shed light on potential host range shift of SARS-CoV-2 VOCs, especially that of the Omicron variant.

Evaluation of serum levels of interferon beta (INF-B) and Nucleotide-binding oligomerization domain 2 (NOD2) gene polymorphism in children with atopic asthma phenotype

Background People of all ages are primarily affected by the chronic inflammatory disease asthma, which is most common in children. A modified International Study of Asthma and Allergies in Childhood (ISAAC) questionnaire found 7.7% of Nile Delta schoolchildren had asthma. This research was to show is their association between the underlying gene polymorphisms of NOD2 and serum level of INF beta and atopic asthma phenotype in Egyptian children and evaluate is their relationship between NOD2 gene polymorphism and the serum level of INF beta. Methods The 127 asthmatic children in this case-control study had typical asthma symptoms, and the 39 age- and sex-matched healthy controls. Subjects were divided in to three groups: Atopic asthma phenotype group: included 60 asthmatic children who had allergic rhinitis and atopic dermatitis as a predominant symptom, Wheezy phenotype group: included 67 asthmatic children presented predominantly with wheezes and controls: 39 healthy controls were chosen because they appeared to be in good health, had no history of symptoms similar to those of asthma, and had no relatives who had allergies or asthma. Results Total serum IgE and eosinophil percentage in control groups showed a positive correlation (r=0.640, P=0.006). In asthmatics, serum INF-β and IgE correlated positively (r=0.217, P=0.031). Increased Serum eosinophilic percentage was associated with decreased serum INF-β and increased serum IgE. Conclusions In comparison to the wheezy phenotype group, the percentage of eosinophils is significantly higher in the atopic asthma phenotype. Serum INF-β and IgE levels went up in tandem with the percentage of eosinophils in the blood. rs2066845 NOD2 genotype The GG genotype increased the risk of asthma in general but not the type of asthma.

Amyloid beta accumulations and enhanced neuronal differentiation in cerebral organoids of Dutch-type cerebral amyloid angiopathy patients.

ADutch-type cerebral amyloid angiopathy (D-CAA) is a hereditary brain disorder caused by a point mutation in the amyloid precursor protein (APP) gene. The mutation is located within the amyloid beta (Aβ) domain of APP and leads to Aβ peptide accumulation in and around the cerebral vasculature. There lack of disease models to study the cellular and molecular pathological mechanisms of D-CAA together with the absence of a disease phenotype in vitro in overexpression cell models, as well as the limited availability of D-CAA animal models indicates the need for a D-CAA patient-derived model. We generated cerebral organoids from four D-CAA patients and four controls, cultured them up to 110 days and performed immunofluorescent and targeted gene expression analyses at two time points (D52 and D110). D-CAA cerebral organoids exhibited Aβ accumulations, showed enhanced neuronal and astrocytic gene expression and TGFβ pathway de-regulation. These results illustrate the potential of cerebral organoids as in vitro disease model of D-CAA that can be used to understand disease mechanisms of D-CAA and can serve as therapeutic intervention platform for various Aβ-related disorders.

Molecular docking study and ADMET prediction of phytoconstituents of Withania Somniferra Flem. in Alzheimer’s disease: an insilico explorative study

AbstractBackground Withania somniferra Flem. (brahmi) is known for its neuroprotective property and protective action against AD. Among different phytochemicals of WS, which phytoconstituents is responsible for this neuroprotection in AD is unknown.MethodProtein Preparation, Ligand Preparation, Molecular Docking on PDB structure of target proteins GSK‐3 beta, CDK‐5, NMDA,SORLA/SORL1,P2×7R, beta catenin, sTLR5 from protein data bank were performed using Schrodinger maestro 12Ligand preparation: Compound was retrieved from pubchem Withaferin‐A and withanolide‐A.Molecular Docking: All in silico calculations and procedures were carried out using Schrödinger software Maestro suite 2019, on an Acer predator laptop with an operating system consisting Linux Ubuntu OS 18.04.02 LTS platform.Pharmacokinetics properties of ligands: admetSAR server (http://lmmd.ecust.edu.cn:8000/) (19), AMDETlabs server (admet.scbdd.com/) and SwissADME server (www.swissadme.ch/) (21) were used for evaluation of pharmacokinetic properties of the ligands (absorption, distribution, metabolism, excretion and toxicity).ResultOut of these 30 ligands, Withaferin‐A (binding energy to the inhibitory domain of gsk3 beta and beta catenin is ‐5.135, ‐7.764 respectively and withanolide‐A binding energy to the inhibitory domain of gsk3 beta and beta catenin is ‐4.371, ‐6.946 and Both the ligands showed good binding profile, favorable pharmacokinetic profile, no violation of drug ability rules. Both the ligands showed permeability through blood brain barrier. Both the ligands shown non Ames toxicity, non carcinogen and weak inhibitor of HERG. Phytochemical of WS (Withaferin‐A and withanolide‐A) showed good binding profile against gsk3beta targets in AD.ConclusionPhytochemical of WS (Withaferin‐A and withanolide‐A) showed good binding profile against Gsk3beta AND Beta catenin targets in AD. We need further studies to evaluate individual efficacy of these phytochemicals in different settings (in‐vitro and in‐vivo).

POU1F1/Pou1f1 c.143-83A > G Variant Disrupts the Branch Site in Pre-mRNA and Leads to Dwarfism.

POU Class 1 Homeobox1 (POU1F1/Pou1f1) is a well-established pituitary-specific transcription factor, and causes, when mutated, combined pituitary hormone deficiency in humans and mice. POU1F1/Pou1f1 has 2 isoforms: the alpha and beta isoforms. Recently, pathogenic variants in the unique coding region of the beta isoform (beta domain) and the intron near the exon-intron boundary for the beta domain were reported, although their functional consequences remain obscure. In this study, we generated mice carrying the Pou1f1 c.143-83A>G substitution that recapitulates the human intronic variant near the exon-intron boundary for the beta domain. Homozygous mice showed postnatal growth failure, with an average body weight that was 35% of wild-type littermates at 12 weeks, which was accompanied by anterior pituitary hypoplasia and deficiency of circulating insulin-like growth factor 1 and thyroxine. The results of RNA-seq analysis of the pituitary gland were consistent with reduction of somatotrophs, and this was confirmed immunohistochemically. Reverse transcription polymerase chain reaction of pituitary Pou1f1 mRNA showed abnormal splicing in homozygous mice, with a decrease in the alpha isoform, an increase in the beta isoform, and the emergence of the exon-skipped transcript. We further characterized artificial variants in or near the beta domain, which were candidate positions of the branch site in pre-mRNA, using cultured cell-basis analysis and found that only c.143-83A>G produced transcripts similar to the mice model. Our report is the first to show that the c.143-83A>G variant leads to splicing disruption and causes morphological and functional abnormalities in the pituitary gland. Furthermore, our mice will contribute understanding the role of POU1F1/Pou1f1 transcripts in pituitary development.

A new strategy for systematically classifying HLA alleles into serological specificities.

HLA serological specificities were defined by the reactivity of HLA molecules with sets of sera and monoclonal antibodies. Many recently identified alleles defined by molecular typing lack their serotype assignment. We surveyed the literature describing the correlation of the reactivity of serologic reagents with AA residues. 20 - 25 AA residues determining epitopes (DEP) that correlated with 82 WHOserologic specificities were identified for HLA class I loci. Thirteen DEP each located in the beta-1 domains that correlated with 24 WHOserologic specificities were identified for HLA-DRB1 and -DQB1 loci. The designation of possible HLA-DPB1, -DQA1, -DPA1, and additional serological specificities that result from epitopes defined by residues located at both -DQA1 and -DQB1 subunits were also examined. HATS software was developed for automated serotype assignments to HLA alleles in one of the three hierarchical matching criteria: (1) all DEP (FULL); (2) selected DEP specific to each serological specificity (SEROTYPE); (3) one AA mismatch with one or more SEROTYPES (INCOMPLETE). Results were validated by evaluating the alleles whose serotypes do not correspond to the first field of the allele name listed in the HLA dictionary. Additional 85 and 21 DEP patterns that do not correspond to any WHO serologic specificities for common HLA class I and DRB1 alleles were identified, respectively. A comprehensive antibody identification panel would allow for accurate unacceptable antigen listing and compatibility predictions in solid organ transplantation. We propose that antibody-screening panels should include all serologic specificities identified in this study.

Catalytic and regulatory subunits of casein kinase 2 in Penaeus vannamei: Cloning, identification, expression profiles and functional analysis

As a highly conserved serine/threonine kinase with catalytic and regulatory subunits distributed ubiquitously in eukaryotic organisms, casein kinase 2 (CK2) is involved in multiple cellular functions, including immune regulation. In this study, two variants of the catalytic subunit (designated PvCK2α-1 and PvCK2α-2) and the regulatory subunit homologs (designated PvCK2β-1 and PvCK2β-2) in Penaeus vannamei were cloned and characterised. PvCK2α-1 and PvCK2α-2 shared the same genomic sequence consisting of six exons and five introns and encoded the same protein of 350 amino acids with an S_TKc domain, although there was a sequence deletion in 3′-UTR in PvCK2α-2 when compared with PvCK2α-1. Because of the sequence deletion in the ORF, PvCK2β-1 and PvCK2β-2 encoded different proteins with a CK_II_beta domain. The gene structures of PvCK2β-1 and PvCK2β-2 were identical and consisted of four exons and three introns. Semi-quantitative RT-PCR analyses revealed that PvCK2α and PvCK2β were constitutively expressed in all P. vannamei tissues tested, with higher levels detected in the immune-related tissues including hemocytes, hepatopancreas, gills and intestine. In these four tissue types, all variants of PvCK2α and PvCK2β were induced upon challenge with white spot syndrome virus (WSSV), Vibrio parahaemolyticus and Staphyloccocus aureus. The inhibition of PvCK2α, PvCK2β-1 and PvCK2βComb (the amount of PvCK2β-1 and PvCK2β-2) significantly reduced the survival rates of P. vannamei after WSSV infection and significantly increased the WSSV viral loads. Knockdown of PvCK2 by RNAi could distinctly decrease the expression of NF-κB related genes. All of these results suggest that PvCK2 plays an important role in the innate immune response to pathogen challenges in P. vannamei, with a positive role in anti-WSSV response which may be mediated through regulating the expression of NF-κB drived antimicrobial peptide genes.

Evaluation of serum levels of interferon beta (INF-Ɓ) and nucleotide-binding oligomerization domain 2 (NOD2) gene polymorphism in relation to asthma phenotypes in children

Evaluation of serum levels of interferon beta (INF-Ɓ) and nucleotide-binding oligomerization domain 2 (NOD2) gene polymorphism in relation to asthma phenotypes in children

Aggregation of high-frequency RBD mutations of SARS-CoV-2 with three VOCs did not cause significant antigenic drift.

Variants of SARS‐CoV‐2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high‐frequency naturally occurring amino acid changes in the receptor‐binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2‐overexpressing 293T cells of different species, LLC‐MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high‐frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high‐frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies.

Antisense Oligonucleotide-Induced Amyloid Precursor Protein Splicing Modulation as a Therapeutic Approach for Dutch-Type Cerebral Amyloid Angiopathy.

Dutch-type cerebral amyloid angiopathy (D-CAA) is a monogenic form of cerebral amyloid angiopathy and is inherited in an autosomal dominant manner. The disease is caused by a point mutation in exon 17 of the amyloid precursor protein (APP) gene that leads to an amino acid substitution at codon 693. The mutation is located within the amyloid beta (Aβ) domain of APP, and leads to accumulation of toxic Aβ peptide in and around the cerebral vasculature. We have designed an antisense oligonucleotide (AON) approach that results in skipping of exon 17, generating a shorter APP isoform that lacks part of the Aβ domain and the D-CAA mutation. We demonstrate efficient AON-induced skipping of exon 17 at RNA level and the occurrence of a shorter APP protein isoform in three different cell types. This resulted in a reduction of Aβ40 in neuronally differentiated, patient-derived induced pluripotent stem cells. AON-treated wild-type mice showed successful exon skipping on RNA and protein levels throughout the brain. These results illustrate APP splice modulation as a promising therapeutic approach for D-CAA.

Melatonin Alleviates Acute Gouty Inflammation In Vivo and In Vitro.

The aim of this study was to identify the effects of melatonin on acute gouty inflammation and to investigate the underlying mechanisms. We found significantly lower serum melatonin levels in gout patients in the acute phase than in those in the remission phase or in normal individuals. The mRNA expression of melatonin receptor 2 (MT2) was also lower in gout patients than in normal individuals. To verify the in-vivo role of melatonin, a gouty arthritis model was established by intraarticular injection of monosodium urate (MSU, 1 mg) crystals into the paws of C57BL/6 mice. Joint inflammation in the mouse model was evaluated by measuring the thickness of the right paw/left paw, and the inflammation index was determined by examining infiltrating neutrophils with haematoxylin and eosin (H&E) staining. Melatonin was found to reduce both paw thickness and the inflammation index in the mouse model, and melatonin also reduced the mRNA levels of interleukin-1 beta (IL-1β), IL-6 and NLR family pyrin domain containing 3 (NLRP3) inflammasome. To mimic gouty inflammation in vitro, mouse peritoneal macrophages were stimulated with lipopolysaccharides (LPS) plus MSU. Melatonin was revealed to reduce IL-1β secretion by stimulated macrophages. The mRNA expression levels of IL-1β and IL-6 were also inhibited by melatonin. Western blot analysis showed that the expression of NLRP3, caspase-1 and pro-IL-1β was also inhibited by melatonin. In conclusion, our study demonstrated that melatonin alleviated gouty inflammation in vivo and in vitro, and the underlying mechanism may involve inhibiting the assembly of the NLRP3 inflammasome.

Modelling the Multifarious Conformations of the Intrinsically Disordered Protein 4E-BP2 with sm-FRET, SAXS & PRE Restraints

Hierarchical phosphorylation of the disordered 4E-BP2 protein stabilizes a binding incompatible 4-stranded beta domain while the C-terminal domain remains disordered. Ensemble descriptions of both phosphorylation states were calculated. The ensembles were restrained using Small-angle X-ray scattering (SAXS) and Paramagnetic Resonance Enhancement (PRE), while the single-molecule Förster Resonance Energy Transfer (smFRET) between residues 32 and 91 was used as validation of the ability of the restrained ensemble to agree with independent experimental evidence.

Increasing AMPK Activity in Human T Cells Enhances Memory Subset Formation without Sacrificing in Vitro Expansion

BACKGROUND: While chimeric antigen receptor (CAR)-T cell therapy has revolutionized the treatment of relapsed/refractory acute lymphoblastic leukemia (ALL), treatment failures continue to occur. In studying therapeutic T cell function, it has become clear that achieving a memory-like phenotype is ideal for CAR-T production. This is likely related to the enhanced oxidative metabolic potential of this subset, which allows for improved persistence and enhanced anti-leukemia activity in vivo. However, current expansion protocols drive T cells towards terminal differentiation, decreasing the number of T cells fit for the in vivo environment. Finding methods to improve the yield of memory-like cells without sacrificing T cell expansion has been challenging. AMP-activated protein kinase (AMPK) is a key metabolic regulator responsible for promoting mitochondrial biogenesis and oxidative metabolism, and is more active in memory T cells at baseline. It is similarly induced by TCR ligation, making it unlikely that it would significantly detract from proliferation. These properties make activation of AMPK a potential candidate pathway for improving the yield of more functional T cells for CAR-T cell therapy. METHODS: AMPK is a heterotrimeric protein complex consisting of alpha, beta, and gamma domains. Functionally, the alpha subunit contains the kinase domain, which is activated by phosphorylation. The gamma subunit controls the phosphorylation, and therefore the activity, of the alpha domain. To increase AMPK signaling in T cells, we cloned the gamma subunit into a lentiviral plasmid containing the elongation factor 1a (EF1a) promoter and a green fluorescent protein (GFP) tag. An empty vector, containing GFP only, served as a negative control. Human T cells were isolated from three separate donors, transduced with our lentiviral construct, and expanded in vitro in the presence of IL-2. AMPK activity was assessed by phosphorylation of Thr172 on the AMPKα subunit as well as phosphorylation of S555 on downstream target Unc-51-like autophagy activating kinase (ULK1) using western blot densitometry, normalized to the total protein amounts. Memory marker expression and mitochondrial density (using Mitotracker Red) were analyzed by flow cytometry. Oxidative metabolism and spare respiratory capacity (SRC) were determined using the Seahorse Metabolic Analyzer. Fold changes for in vitro expansion were calculated by adjusting manual cell counts to reflect GFP positivity and CD4+/CD8+ surface staining. RESULTS: The AMPK gamma subunit was efficiently transduced and expressed by human T cells as measured by GFP expression, qRT-PCR, and western blot analysis. Further, AMPK activity increased in GFP+ cells as indicated by the phosphorylation of AMPKα Thr172 (1.93 +/- 0.05 vs 0.6 +/- 0.09, p<0.001) and ULK1 S555 (1.28 +/- 0.11 vs 0.67 +/- 0.08, p<0.01). Cells transduced with AMPK augmented expression of memory markers CD62L, CD27, and CCR7, with an increased yield of stem cell memory-like T cells marked by co-expression of CD45RA and CD62L (Figure 1). In addition, AMPK-transduced T cells showed a statistically significant increase in mitochondrial density along with notable enhancement of SRC and maximal oxygen consumption rates (Figure 2A,B). Furthermore, the rate of expansion of AMPK-transduced T cells did not differ significantly from Empty-transduced controls, and in fact trended towards increased in both CD4+ and CD8+ cells (Figure 3A). Indeed, the improved rate of expansion in AMPK-transduced CD4+ T cells led to a measurable increase in CD4+ T cell percentages by flow cytometry (Figure 3B). DISCUSSION: Here we present an efficient and direct method to increase AMPK activity in human T cells and demonstrate that increased AMPK activity endows T cells with a variety of characteristics ideal for CAR-T cell therapy. These features include increased memory-marker expression, enhanced SRC and oxidative metabolism, equivalent to augmented in vitro expansion, and improved CD4+ T cell yields. Further studies are ongoing to assess the activity and function of AMPK-transduced CAR-T cells both in vitro and in vivo. Disclosures No relevant conflicts of interest to declare.

Genomics-assisted prediction of salt and alkali tolerances and functional marker development in apple rootstocks

BackgroundSaline, alkaline, and saline-alkaline stress severely affect plant growth and development. The tolerance of plants to these stressors has long been important breeding objectives, especially for woody perennials like apple. The aims of this study were to identify quantitative trait loci (QTLs) and to develop genomics-assisted prediction models for salt, alkali, and salt-alkali tolerance in apple rootstock.ResultsA total of 3258 hybrids derived from the apple rootstock cultivars ‘Baleng Crab’ (Malus robusta Rehd., tolerant) × ‘M9’ (M. pumila Mill., sensitive) were used to identify 17, 13, and two QTLs for injury indices of salt, alkali, and salt–alkali stress via bulked segregant analysis. The genotype effects of single nucleotide polymorphism (SNP) markers designed on candidate genes in each QTL interval were estimated. The genomic predicted value of an individual hybrid was calculated by adding the sum of all marker genotype effects to the mean phenotype value of the population. The prediction accuracy was 0.6569, 0.6695, and 0.5834 for injury indices of salt, alkali, and salt–alkali stress, respectively. SNP182G on MdRGLG3, which changes a leucine to an arginine at the vWFA-domain, conferred tolerance to salt, alkali, and salt-alkali stress. SNP761A on MdKCAB, affecting the Kv_beta domain that cooperated with the linked allelic variation SNP11, contributed to salt, alkali, and salt–alkali tolerance in apple rootstock.ConclusionsThe genomics-assisted prediction models can potentially be used in breeding saline, alkaline, and saline-alkaline tolerant apple rootstocks. The QTLs and the functional markers may provide insight for future studies into the genetic variation of plant abiotic stress tolerance.

Beneficial Effects of Inflammatory Cytokine-Targeting Aptamers in an Animal Model of Chronic Prostatitis.

Non-bacterial prostatitis is an inflammatory disease that is difficult to treat. Oligonucleotide aptamers are well known for their stability and flexibility in conjugating various inflammatory molecules. In this study, we investigated the effects of inflammatory cytokine-targeting aptamers (ICTA), putative neutralizers of TNF-alpha and IL-1 beta activation, on local carrageenan-induced prostate inflammation, allodynia, and hyperalgesia in rats. In vitro evaluation confirmed the binding capability of ICTA. Intraprostatic injection of carrageenan or control vehicle was performed in six-week-old rats, and ICTA (150 µg) or vehicle was administered in the prostate along with carrageenan injection. The von Frey filament test was performed to determine mechanical allodynia, and prostate inflammation was examined seven days after drug administration. Local carrageenan administration resulted in a reduction of the tactile threshold. The levels of mononuclear cell infiltration, pro-inflammatory cytokine interleukin-1 beta (b), caspase-1 (casp-1), and Nucleotide-binding oligomerization domain, Leucine rich Repeat and Pyrin domain containing proteins 1 and 3 (NALP1 and NALP3) in the prostate of rats were increased seven days after carrageenan injection. Treatment with ICTA significantly attenuated the carrageenan-induced hyperalgesia and reduced the elevated levels of proteins including TNF-a and IL-1b in the rats. Apoptosis markers, B-cell lymphoma 2-associated X protein (Bax) and caspase-3, were elevated in ICTA-treated Chronic pelvic pain syndrome (CPPS) rats. These results suggest that ICTA provides protection against local carrageenan-induced enhanced pain sensitivity, and that the neutralization of proinflammatory cytokines may result in inflammatory cell apoptosis.

PVHL-mediated regulation of the anti-angiogenic protein thrombospondin-1 decreases migration of Clear Cell Renal Carcinoma Cell Lines

Thrombospondin-1 (TSP-1) is a multifunctional matrix protein with antitumor activities due in part to its ability to inhibit angiogenesis, which in turn contributes to determine the fate of many tumours. Previous studies have shown that TSP-1 expression supports normal kidney angiostasis, and decreased TSP-1 levels contribute to the angiogenic phenotype of renal cell carcinomas (RCC). The loss of the von Hippel-Lindau tumour suppressor gene (VHL) in these tumours favours stabilization of the Hypoxia Inducible Factors (HIF), which in turn contribute to adapt tumour cells to hostile environments promoting tumour progression. However, HIF-independent regulation of certain genes might also be involved. We have previously shown that TSP-1 is regulated in hypoxia in clear cell RCC (ccRCC) in a HIF-independent manner; however, the effect of VHL protein (pVHL) on TSP-1 expression has not been evaluated. Our results proved that pVHL loss or mutation in its alpha or beta domain significantly decreased TSP-1 levels in ccRCC in a HIF-independent manner. Furthermore, this regulation proved to be important for ccRCC cells behaviour showing that decreased TSP-1 levels rendered ccRCC cells more migratory. This data substantiates a unique regulation pattern for TSP-1 in a pVHL-dependent manner, which may be relevant in the aggressiveness of ccRCC.

High-throughput custom capture sequencing identifies novel mutations in coloboma-associated genes: Mutation in DNA-binding domain of retinoic acid receptor beta affects nuclear localization causing ocular coloboma.

Uveal coloboma is a potentially blinding congenital ocular malformation caused by the failure of optic fissure closure during the fifth week of human gestation. We performed custom capture high‐throughput screening of 38 known coloboma‐associated genes in 66 families. Suspected causative novel variants were identified in TFAP2A and CHD7, as well as two previously reported variants of uncertain significance in RARB and BMP7. The variant in RARB, unlike previously reported disease mutations in the ligand‐binding domain, was a missense change in the highly conserved DNA‐binding domain predicted to affect the protein's DNA‐binding ability. In vitro studies revealed lower steady‐state protein levels, reduced transcriptional activity, and incomplete nuclear localization of the mutant RARB protein compared with wild‐type. Zebrafish studies showed that human RARB messenger RNA partially reduced the ocular phenotype caused by morpholino knockdown of rarga gene, a zebrafish homolog of human RARB. Our study indicates that sequence alterations in known coloboma genes account for a small percentage of coloboma cases and that mutations in the RARB DNA‐binding domain could result in human disease.

Promoter Identification and Transcriptional Regulation of the Goose AMH Gene.

Simple SummaryAnti-Müllerian hormone (AMH) plays a vital role in the development of follicles. We found that the cloning nucleotide sequence of AMH was high homology in geese with other species. Several regulatory elements were identified and transcriptional factors were predicted in the AMH promoter sequence. Through a double-luciferase reporter assay, potential regulatory relationship spanning from −637 to −87 bp were identified. In addition, the mRNA expression of AMH gradually decreased during the development of follicles in geese. In the Chinese hamster ovary (CHO) cell line, the luciferase activity significantly increased by co-expression of AMH and GATA-4. However, when the binding sites of GATA-4 to the promoter of AMH were mutated, the luciferase activity significantly decreased. These results indicated that the transcription of AMH was activated by GATA-4 to inhibit the development of follicles in geese.Anti-Müllerian hormone (AMH) is recognized as a reliable marker of ovarian reserve. However, the regulatory mechanism of goose AMH gene remains poorly understood. In the present study, both the full-length coding sequence (CDS) and promoter sequence of goose AMH have been cloned. Its CDS consisted of 2013 nucleotides encoding 670 amino acids and the amino acid sequence contained two structural domain: AMH-N and transforming growth factor beta (TGF-β) domain. The obtained promoter sequence spanned from the −2386 bp to its transcription start site (ATG). Core promoter regions and regulatory elements were identified as well as transcription factors were predicted in its promoter sequence. The luciferase activity was the highest spanning from the −331 to −1 bp by constructing deletion promoter reporter vectors. In CHO cells, the luciferase activity significantly increased by co-expression of AMH and GATA binding protein 4 (GATA-4), while that significantly decreased by mutating the binding sites of GATA-4 located in the −778 and −1477 bp. Results from quantitative real-time polymerase chain reaction (qPCR) indicated that levels of AMH mRNA in geese granulosa layers decreased gradually with the increasing follicular diameter. Taken together, it could be concluded that the transcriptional activity of AMH was activated by GATA-4 to inhibit the development of small follicles in goose.

Structure, Topology, and Dynamics of Membrane-Inserted Polypeptides and Lipids by Solid-State NMR Spectroscopy: Investigations of the Transmembrane Domains of the DQ Beta-1 Subunit of the MHC II Receptor and of the COP I Protein p24.

MHC class II receptors carry important function in adaptive immunity and their malfunctioning is associated with diabetes type I, chronic inflammatory diseases and other autoimmune diseases. The protein assembles from the DQ alpha-1 and DQ beta-1 subunits where the transmembrane domains of these type I membrane proteins have been shown to be involved in homo- and heterodimer formation. Furthermore, the DQ alpha 1 chain carries a sequence motif that has been first identified in the context of p24, a protein involved in the formation of COPI vesicles of the intracellular transport machinery, to specifically interact with sphingomyelin-C18 (SM-C18). Here we investigated the membrane interactions and dynamics of DQ beta-1 in liquid crystalline POPC phospholipid bilayers by oriented 15N solid-state NMR spectroscopy. The 15N resonances are indicative of a helical tilt angle of the membrane anchor sequence around 20°. Two populations can be distinguished by their differential dynamics probably corresponding the DQ beta-1 mono- and homodimer. Whereas, this equilibrium is hardly affected by the addition of 5 mole% SM-C18 a single population is visible in DMPC lipid bilayers suggesting that the lipid saturation is an important parameter. Furthermore, the DQ alpha-1, DQ beta-1 and p24 transmembrane helical domains were reconstituted into POPC or POPC/SM-C18 lipid bilayers where the fatty acyl chain of either the phosphatidylcholine or of the sphingolipid have been deuterated. Interestingly in the presence of both sphingolipid and polypeptide a strong decrease in the innermost membrane order of the POPC palmitoyl chain is observed, an effect that is strongest for DQ beta-1. In contrast, for the first time the polypeptide interactions were monitored by deuteration of the stearoyl chain of SM-C18. The resulting 2H solid-state NMR spectra show an increase in order for p24 and DQ alpha-1 which both carry the SM recognition motif. Thereby the data are suggestive that SM-C18 together with the transmembrane domains form structures imposing positive curvature strain on the surrounding POPC lipids. This effect is attenuated when SM-C18 is recognized by the protein.