Recombinant Assay Research Articles

Rho GTPase-activating protein 10 (ARHGAP10/GRAF2) is a novel autoantibody target in patients with autoimmune encephalitis

BackgroundIn 2010, we described a novel immunoglobulin G (IgG) autoantibody (termed anti-Ca after the index case) targeting Rho GTPase-activating protein 26 (ARHGAP26, also termed GTPase regulator associated with focal adhesion kinase [GRAF], or oligophrenin-like protein 1 [OPHN1L]) in autoimmune cerebellar ataxia (ACA). Later, ARHGAP26-IgG/anti-Ca was reported in patients with limbic encephalitis/cognitive decline or peripheral neuropathy. In several of the reported cases, the syndrome was associated with cancer. ARHGAP10/GRAF2, which is expressed throughout the central nervous system, shares significant sequence homology with ARHGAP26/GRAF. Mutations in the ARHGAP10 gene have been linked to cognitive and psychiatric symptoms and schizophrenia.ObjectiveTo assess whether ARHGAP26-IgG/anti-Ca co-reacts with ARHGAP10.MethodsSerological testing for ARHGAP10/GRAF2 autoantibodies by recombinant cell-based assays and isotype and IgG subclass analyses.Results26/31 serum samples (84%) from 9/12 (75%) ARHGAP26-IgG/anti-Ca-positive patients and 4/6 ARHGAP26-IgG/anti-Ca-positive CSF samples from four patients were positive also for ARHGAP10-IgG. ARHGAP10-IgG (termed anti-Ca2) remained detectable in the long-term (up to 109 months) and belonged mainly to the complement-activating IgG1 subclass. Median ARHGAP26-IgG/anti-Ca and median ARHGAP10-IgG/anti-Ca2 serum titres were 1:3200 and 1:1000, respectively, with extraordinarily high titres in some samples (ARHGAP26-IgG/anti-Ca: up to 1:1000,000; ARHGAP10-IgG: up to 1:32,000). ARHGAP26/anti-Ca serum titres exceeded those of ARHGAP10-IgG in all samples but one. A subset of patients was positive also for ARHGAP10-IgM and ARHGAP10-IgA. CSF/serum ratios and antibody index calculation suggested intrathecal production of ARHGAP26-IgG/anti-Ca and anti-ARHGAP10. Of 101 control samples, 100 were completely negative for ARHGAP10-IgG; a single control sample bound weakly (1:10) to the ARHGAP10-transfected cells.ConclusionsWe demonstrate that a substantial proportion of patients with ARHGAP26-IgG/anti-Ca-positive autoimmune encephalitis co-react with ARHGAP10. Further studies on the clinical and diagnostic implications of ARHGAP10-IgG/anti-Ca2 seropositivity in patients with autoimmune encephalitis are warranted.

A recombinant monoclonal-based Taenia antigen assay that reflects disease activity in extra-parenchymal neurocysticercosis.

BackgroundAntigen tests for diagnosis and disease monitoring in some types of neurocysticercosis (NCC) are useful but access to testing has been limited by availability of proprietary reagents and/or kits.Methods/Principal findingsThree previously identified IgM-secreting hybridomas whose IgM products demonstrated specificity to Taenia solium underwent variable heavy and light chain sequencing and isotype conversion to mouse IgG. Screening of these recombinantly expressed IgG anti-Ts hybridomas, identified one (TsG10) with the highest affinity to crude Taenia antigen. TsG10 was then used as a capture antibody in a sandwich antigen detection immunoassay in combination with either a high titer polyclonal anti-Ts antibody or with biotinylated TsG10 (termed TsG10*bt). Using serum, plasma, and CSF samples from patients with active NCC and those from NCC-uninfected patients, ROC curve analyses demonstrated that the TsG10-TsG10-*bt assay achieved a 98% sensitivity and 100% specificity in detecting samples known to be antigen positive and outperformed the polyclonal based assay (sensitivity of 93% with 100% specificity). By comparing levels of Ts antigen (Ag) in paired CSF (n = 10) or plasma/serum (n = 19) samples from well-characterized patients with extra-parenchymal NCC early in infection and at the time of definitive cure, all but 2 (1 from CSF and 1 from plasma) became undetectable. There was a high degree of correlation (r = 0.98) between the Ag levels detected by this new assay and levels found by a commercial assay. Pilot studies indicate that this antigen can be detected in the urine of patients with active NCC.Conclusions/SignificanceA newly developed recombinant monoclonal antibody-based Ts Ag detection immunoassay is extremely sensitive in the detection of extra-parenchymal NCC and can be used to monitor the success of treatment in the CSF, serum/plasma and urine. The ability to produce recombinant TsG10 at scale should enable use of this antigen detection immunoassay wherever NCC is endemic.Clinical Trial RegistrationClinicalTrials.gov Identifiers: NCT00001205 - & NCT00001645.

Activation of class 1 integron integrase is promoted in the intestinal environment.

Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.

Generated Randomly and Selected Functionally? The Nature of Enterovirus Recombination.

Genetic recombination in RNA viruses is an important evolutionary mechanism. It contributes to population diversity, host/tissue adaptation, and compromises vaccine efficacy. Both the molecular mechanism and initial products of recombination are relatively poorly understood. We used an established poliovirus-based in vitro recombination assay to investigate the roles of sequence identity and RNA structure, implicated or inferred from an analysis of circulating recombinant viruses, in the process. In addition, we used next-generation sequencing to investigate the early products of recombination after cellular coinfection with different poliovirus serotypes. In independent studies, we find no evidence for a role for RNA identity or structure in determining recombination junctions location. Instead, genome function and fitness are of greater importance in determining the identity of recombinant progeny. These studies provide further insights into this important evolutionary mechanism and emphasize the critical nature of the selection process on a mixed virus population.

Recombinant Limb Assay as in Vivo Organoid Model.

Organ formation initiates once cells become committed to one of the three embryonic germ layers. In the early stages of embryogenesis, different gene transcription networks regulate cell fate after each germ layer is established, thereby directing the formation of complex tissues and functional organs. These events can be modeled in vitro by creating organoids from induced pluripotent, embryonic, or adult stem cells to study organ formation. Under these conditions, the induced cells are guided down the developmental pathways as in embryonic development, resulting in an organ of a smaller size that possesses the essential functions of the organ of interest. Although organoids are widely studied, the formation of skeletal elements in an organoid model has not yet been possible. Therefore, we suggest that the formation of skeletal elements using the recombinant limb (RL) assay system can serve as an in vivo organoid model. RLs are formed from undissociated or dissociated-reaggregated undifferentiated mesodermal cells introduced into an ectodermal cover obtained from an early limb bud. Next, this filled ectoderm is grafted into the back of a donor chick embryo. Under these conditions, the cells can receive the nascent embryonic signals and develop complex skeletal elements. We propose that the formation of skeletal elements induced through the RL system may occur from stem cells or other types of progenitors, thus enabling the study of morphogenetic properties in vivo from these cells for the first time.

Glycoside hydrolase from the GH76 family indicates that marine Salegentibacter sp. Hel_I_6 consumes alpha-mannan from fungi

Microbial glycan degradation is essential to global carbon cycling. The marine bacterium Salegentibacter sp. Hel_I_6 (Bacteroidota) isolated from seawater off Helgoland island (North Sea) contains an α-mannan inducible gene cluster with a GH76 family endo-α-1,6-mannanase (ShGH76). This cluster is related to genetic loci employed by human gut bacteria to digest fungal α-mannan. Metagenomes from the Hel_I_6 isolation site revealed increasing GH76 gene frequencies in free-living bacteria during microalgae blooms, suggesting degradation of α-1,6-mannans from fungi. Recombinant ShGH76 protein activity assays with yeast α-mannan and synthetic oligomannans showed endo-α-1,6-mannanase activity. Resolved structures of apo-ShGH76 (2.0 Å) and of mutants co-crystalized with fungal mannan-mimicking α-1,6-mannotetrose (1.90 Å) and α-1,6-mannotriose (1.47 Å) retained the canonical (α/α)6 fold, despite low identities with sequences of known GH76 structures (GH76s from gut bacteria: <27%). The apo-form active site differed from those known from gut bacteria, and co-crystallizations revealed a kinked oligomannan conformation. Co-crystallizations also revealed precise molecular-scale interactions of ShGH76 with fungal mannan-mimicking oligomannans, indicating adaptation to this particular type of substrate. Our data hence suggest presence of yet unknown fungal α-1,6-mannans in marine ecosystems, in particular during microalgal blooms.

Analytical development insights spanning viral vectors &amp; gene editing

David McCall, Commissioning Editor of Cell &amp; Gene Therapy Insight speaks to Santoshkumar Khatwani, Director Analytical Development, Sangamo Therapeutics, Inc. Dr Santosh Khatwani graduated from the University of Kentucky in 2010 with PhD in Chemistry under the guidance of Prof. Sylvia Daunert. The research work focused on the use of generically engineered recombinant protein and enzymes for bio sensing and biomaterial applications. Furthermore, he obtained postdoctoral training at the University of Minnesota (until 2012) under the supervision of Prof T Andrew Taton and Prof Mark D Distefano with focus on the use of site-specific covalent conjugation for synthesis of protein–DNA conjugates and analytical assays for the generating novel engineered enzymes with improved functional attributes. Dr Khatwani then joined BioVision Inc. in 2012 and served under different capacities until 2017 where he oversaw the manufacture, testing and release of several recombinant protein, enzyme and assays for various metabolically important enzymes. Furthermore, Dr. Khatwani joined the field of cell and gene therapy at multiple companies including Thermofisher Viral Vector Services (formerly Brammer Bio, 2017–2018), Asklepios Biopharmaceuticals (2019) and Sangamo Therapeutics (2018–2019, 2019-present) at various leadership roles where he has led the development of various analytical assays including physicochemical, biophysical and biological assays to support the manufacture and release of viral vectors in cell and gene therapy. Currently Dr. Khatwani is serving as Director of Analytical Development at Sangamo Therapeutics with strong focus on developing analytical solutions and CMC in support of product development at early and late phase of the clinical development.

Lhx2 in germ cells suppresses endothelial cell migration in the developing ovary

LIM-homeobox genes play multiple roles in developmental processes, but their roles in gonad development are not completely understood. Herein, we report that Lhx2, Ils2, Lmx1a, and Lmx1b are expressed in a sexually dimorphic manner in mouse, rat, and human gonads during sex determination. Amongst these, Lhx2 has female biased expression in the developing gonads of species with environmental and genetic modes of sex determination. Single-cell RNAseq analysis revealed that Lhx2 is exclusively expressed in the germ cells of the developing mouse ovaries. To elucidate the roles of Lhx2 in the germ cells, we analyzed the phenotypes of Lhx2 knockout XX gonads. While the gonads developed appropriately in Lhx2 knockout mice and the somatic cells were correctly specified in the developing ovaries, transcriptome analysis revealed enrichment of genes in the angiogenesis pathway. There was an elevated expression of several pro-angiogenic factors in the Lhx2 knockout ovaries. The elevated expression of pro-angiogenic factors was associated with an increase in numbers of endothelial cells in the Lhx2−/− ovaries at E13.5. Gonad recombination assays revealed that the increased numbers of endothelial cells in the XX gonads in absence of Lhx2 was due to ectopic migration of endothelial cells in a cell non-autonomous manner. We also found that, there was increased expression of several endothelial cell-enriched male-biased genes in Lhx2 knockout ovaries. Also, in absence of Lhx2, the migrated endothelial cells formed an angiogenic network similar to that of the wild type testis, although the coelomic blood vessel did not form. Together, our results suggest that Lhx2 in the germ cells is required to suppress vascularization in the developing ovary. These results suggest a need to explore the roles of germ cells in the control of vascularization in developing gonads. Preprint version of the article is available on BioRxiv at https://doi.org/10.1101/2022.03.07.483280.

AtGH3.10 is another jasmonic acid-amido synthetase in Arabidopsis thaliana.

Jasmonoyl-isoleucine (JA-Ile) is a key signaling molecule that activates jasmonate-regulated flower development and the wound stress response. For years, JASMONATE RESISTANT1 (JAR1) has been the sole jasmonoyl-amino acid synthetase known to conjugate jasmonic acid (JA) to isoleucine, and the source of persisting JA-Ile in jar1 knockout mutants has remained elusive until now. Here we demonstrate through recombinant enzyme assays and loss-of-function mutant analyses that AtGH3.10 functions as a JA-amido synthetase. Recombinant AtGH3.10 could conjugate JA to isoleucine, alanine, leucine, methionine, and valine. The JA-Ile accumulation in the gh3.10-2 jar1-11 double mutant was nearly eliminated in the leaves and flower buds while its catabolism derivative 12OH-JA-Ile was undetected in the flower buds and unwounded leaves. Residual levels of JA-Ile, JA-Ala, and JA-Val were nonetheless detected in gh3.10-2 jar1-11, suggesting the activities of similar promiscuous enzymes. Upon wounding, the accumulation of JA-Ile and 12OH-JA-Ile and the expression of JA-responsive genes OXOPHYTODIENOIC ACID REDUCTASE3 and JASMONATE ZIM-DOMAIN1 observed in WT, gh3.10-1, and jar1-11 leaves were effectively abolished in gh3.10-2 jar1-11. Additionally, an increased proportion of undeveloped siliques associated with retarded stamen development was observed in gh3.10-2 jar1-11. These findings conclusively show that AtGH3.10 contributes to JA-amino acid biosynthesis and functions partially redundantly with AtJAR1 in sustaining flower development and the wound stress response in Arabidopsis.

Focal Adhesion Kinase Drives Resistance to Therapy in HPV-Negative Head and Neck Squamous Cell Carcinoma in a p53-Dependent Manner

<h3>Purpose/Objective(s)</h3> Focal adhesion kinase (FAK) expression is associated with radioresistance and worse outcome in human papillomavirus negative [HPV (-)] head and neck squamous cell carcinoma (HNSCC). However, the relative importance of FAK in driving therapeutic resistance is unclear. FAK function can be inhibited by wild type (wt) p53, but TP53 is mutated in over 80% of HPV (-) HNSCC. Based on these observations, we tested the hypothesis that mutant (mut) TP53 permits or potentially drives FAK-mediated therapy resistance. <h3>Materials/Methods</h3> <i>In vivo</i> shRNA screening in 3 human HNSCC xenograft flank models was performed using libraries focused on known "druggable" proteins combined with RT or carboplatin. An <i>in vivo</i> immunodeficient flank model using HN31 (mut TP53) cells expressing FAK shRNA was used to assess <i>in vivo</i> radioresponse. The relationship between p53, FAK, and radioresponse was examined <i>in vitro</i> using HNSCC cells: UMSCC (p53 null) with wt or mut TP53 expressed, HN30 (wt TP53), and HN31 (mut TP53). FAK function was modulated in these cells using siRNA, shRNA, or chemical inhibition (defactinib); response to radiation (RT) or carboplatin was evaluated via immunoblot for DNA damage repair proteins, survival clonogenic assay, MTT assay, DCFDA reactive oxygen species (ROS) assay, and fluorescence-based homologous recombination (HR) and non-homologous end joining (NHEJ) reporter assays. FAK and p53 interactions were evaluated via immunoprecipitation. Two cohorts of HPV (-) HNSCC tumors treated with RT, single institution (n=81) and TCGA (n=324), were stratified by TP53 mutational status and FAK copy number/mRNA expression. <h3>Results</h3> <i>In vivo</i> shRNA screening identified FAK as an excellent target for both radio- and chemosensitization. Inhibition of FAK via shRNA in the HN31 (mut TP53) cell line led to radiosensitization in a xenograft flank model. Defactinib treatment led to increased sensitivity to RT or carboplatin in mut TP53 (HN31) but not wt TP53 (HN30) tumor cell lines. In UMSCC1 cells treated with defactinib, mut TP53 expression resulted in significant radiosensitization, but wt TP53 expression did not. Both wt p53 and mut p53 retained the ability to bind FAK. Basal FAK phosphorylation was higher in cells expressing mut p53 compared to wt p53. FAK inhibition combined with radiation led to higher levels of ɣ-H2AX and ROS and decreased HR and NHEJ repair in mut TP53 expressing cells but had minimal effect in wt TP53 cells. High levels of FAK copy number and gene expression were associated with worse disease-free survival in mut TP53 HPV (-) HNSCC, but not wt TP53 tumors in both patient cohorts. <h3>Conclusion</h3> FAK is a potentially important target for therapeutic sensitization, primarily in mutated TP53 HPV (-) HNSCC.

Metabolic Activation of Perampanel Mediated by CYP1A2

Perampanel (PRP), a noncompetitive α-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptor antagonist with high selectivity, has been used as a new adjuvant for the treatment of fractional seizures with or without primary generalized tonic-clonic seizures and secondary generalized seizures in epilepsy patients over the age of 12. Adverse events such as liver injury have been reported during the clinical application of PRP. The purpose of the study is to explore the in vitro and in vivo metabolic activation of PRP. Two GSH conjugates were detected in rat liver microsomal incubations containing PRP, GSH, and NADPH. The two GSH conjugates were both obtained from the bile of rats and rat primary hepatocytes after exposure to PRP. Similar microsomal incubations complemented with N-acetylcysteine (NAC) in place of GSH offered two NAC conjugates. As expected, the NAC conjugates were detected in the urine of PRP-treated rats. One of the two NAC conjugates was identified as NAC conjugate 12 verified by chemical synthesis. The individual human recombinant P450 enzyme incubation assay demonstrated that CYP1A2 dominated the catalysis for the metabolic activation of PRP. Pretreatment with α-naphthoflavone (NTF) decreased the formation of PRP-derived GSH conjugates in both livers of rats and cultured primary hepatocytes after being treated with PRP. Additionally, NTF was found to decrease the susceptibility of primary hepatocytes to the cytotoxicity of PRP. The findings indicate that PRP was metabolized to the corresponding epoxide, which could participate in PRP-induced cytotoxicity.

Myotropic Activities of Tick Pyrokinin Neuropeptides and Analog in Feeding Tissues of Hard Ticks (Ixodidae).

Neuropeptides regulate many important physiological processes in animals. The G protein-coupled receptors of corresponding small neuropeptide ligands are considered promising targets for controlling arthropod pests. Pyrokinins (PKs) are pleiotropic neuropeptides that, in some insect species, stimulate muscle contraction and modulate pheromone biosynthesis, embryonic diapause, and feeding behavior. However, their function remains unknown in ticks. In this study, we reported the myotropic activity of tick endogenous PKs and a PK agonist analog, PK-PEG8 (MS[PEG8]-YFTPRLa), on feeding tissues of two tick species representing the family Ixodidae lineages, namely, Prostriata (Ixodes scapularis) and Metastriata (Rhipicephalus sanguineus). First, we predicted the sequences of two periviscerokinins (PVK), one with a derived ending RNa and five PKs encoded by the CAPA peptide precursor from R. sanguineus and found the encoded PKs were identical to those of R. microplus identified previously. The pharynx-esophagus of both tick species responded with increased contractions to 10 μM of the endogenous PK as well as to PK-PEG8 but not to the scrambled PK peptide, as expected. A dose-dependent myotropic activity of the PK-PEG8 was found for both tick species, validating the analog activity previously found in the pyrokinin recombinant receptor assay. In agreement with the tissue activity elicited, we quantified the relative transcript abundance of R. sanguineus PK receptor in unfed female ticks and found it was the highest in the feeding tissues extracted from the capitulum and lowest in the reproductive tissue. This is the first report of the activity of pyrokinins in ticks. These findings strongly indicate the potential role of PKs in regulating tick blood feeding and therefore, making the tick PK receptor a potential target for interference.

Development of a recombinant pB602L-based indirect ELISA assay for detecting antibodies against African swine fever virus in pigs

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a devastating disease of domestic and wild pigs. There is no effective vaccine, and the control of the disease relies mainly on surveillance and early detection of infected pigs. Previously, serological assays, such as ELISA, have been developed mainly based on recombinant structural viral proteins of ASFV, including p72, p54, and p30. However, the antibodies against these proteins do not provide efficient protection against ASFV infection in pigs. Therefore, new serological assays that can be applied for clinical diagnosis and evaluating serological immune response in vaccinated pigs are still required. In this study, we expressed and purified a recombinant pB602L protein. The purified pB602L protein was then used as an antigen to develop an indirect ELISA assay. This assay has no cross-reaction with the anti-sera against the 15 most common pig pathogens in China, such as classical swine fever virus, pseudorabies virus, and porcine parvovirus. This assay and a commercial ELISA kit were then used to detect 60 field pig serum samples, including an unknown number of anti-ASFV sera. The coincidence of the two assays was 95%. Furthermore, the pB602L-based ELISA was employed to test the antibody responses to the seven-gene-deleted ASFV strain HLJ/18-7GD in pigs. The results showed that the antibody levels in all vaccinated pigs, starting from the 10th day post-inoculation, have increased continuously during the observation period of 45 days. Our results indicate that this pB602L-based indirect ELISA assay can be employed potentially in the field of ASFV diagnosis.

Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity.

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.

Identification of Natural Product Sulfuretin Derivatives as Inhibitors for the Endoplasmic Reticulum Redox Protein ERO1α.

The flavin adenine dinucleotide containing Endoplasmic Reticulum Oxidoreductase-1 α (ERO1α) catalyzes the formation of de novo disulfide bond formation of secretory and transmembrane proteins and contributes towards proper protein folding. Recently, increased ERO1α expression has been shown to contribute to increased tumor growth and metastasis in multiple cancer types. In this report we sought to define novel chemical space for targeting ERO1α function. Using the previously reported ERO1α inhibitor compound, EN-460, as a benchmark pharmacological tool we were able to identify a sulfuretin derivative, T151742 which was approximately two-fold more potent using a recombinant enzyme assay system (IC50 = 8.27 ± 2.33 μM) compared to EN-460 (IC50= 16.46 ± 3.47 μM). Additionally, T151742 (IC50 = 16.04 μM) was slightly more sensitive than EN-460 (IC50= 19.35μM) using an MTT assay as an endpoint. Utilizing a cellular thermal shift assay (CETSA), we determined that the sulfuretin derivative T151742 demonstrated isozyme specificity for ERO1α as compared to ERO1β and showed no detectable binding to the FAD containing enzyme LSD-1. T151742 retained activity in PC-9 cells in a clonogenicity assay while EN-460 was devoid of activity. Furthermore, the activity of T151742 inhibition of clonogenicity was dependent on ERO1α expression as CRISPR edited PC-9 cells were resistant to treatment with T151742. In summary we identified a new scaffold that shows specificity for ERO1α compared to the closely related paralog ERO1β or the FAD containing enzyme LSD-1 that can be used as a tool compound for inhibition of ERO1α to allow for pharmacological validation of the role of ERO1α in cancer.

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation.

Cell differentiation is the fine-tuned process of cell commitment leading to the formation of different specialized cell types during the establishment of developing tissues and organs. This process is actively maintained in adulthood. Cell differentiation is an ongoing process during the development and homeostasis of organs. Understanding the early steps of cell differentiation is essential to know other complex processes such as morphogenesis. Thus, recombinant chicken limbs are an experimental model that allows the study of cell differentiation and pattern generation under embryonic patterning signals. This experimental model imitates an in vivo environment; it assembles reaggregated cells into an ectodermal cover obtained from an early limb bud. Later, ectoderms are transferred and implanted in a chick embryo receptor to allow its development. This assay was mainly used to evaluate mesodermal limb bud cells; however, it can be applied to other stem or progenitor cells from other organisms.

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Chicken Recombinant Limbs Assay to Understand Morphogenesis, Patterning, and Early Steps in Cell Differentiation

Retrospective Analysis of a New Antigen Assay to Detect Anti-Transglutaminase Antibodies for Diagnosis and Monitoring of Celiac Disease

BACKGROUND The improved diagnosis and monitoring of celiac disease (CD) requires increasingly sensitive and specific serological markers. The development of new antigens ( neo-epitopes) able to detect different sets of anti-transglutaminase antibodies requires studies to demonstrate their advantages. OBJECTIVE The goal of this study was to evaluate a new antigen for the detection of anti-tTG antibodies that combines tissue transglutaminase (tTG) and gliadin peptides. This protein complex occurs under physiological conditions in vivo at the time of deamination. PATIENTS AND METHODS In this retrospective study, serum samples collected from patients from January to December 2018 were analyzed. Results of the determinations for neo-epitope anti-tTG antibodies (manufacturer's cut-off values: negative &lt;12 U/mL, indeterminate 12-18 U/mL; positive &gt;18 U/mL) were recorded along with age, sex, and duodenal biopsy results. Neo-epitope assay results were correlated with clinical and laboratory data and final CD diagnoses, using the manufacturer's cut-off value and a proposed alternate cut-off value. RESULTS A total of 3820 neo-epitope anti-tTg determinations were analyzed. The percentage of values in the indeterminate zone was low (3.6%), with very few values (0.9%) at the exact manufacturer's cut-off limit (18 U/mL). This indicated a good resolution capacity for positives and negatives. The percentage of false positives was lower compared to a previous study (2013 study: 3.6%, vs current study (2018): 0.5%). A new positivity cut-off value was proposed: 20 U/mL, which increased the positive predictive value of the test. CONCLUSIONS The neo-epitope assay is a more precise tool than recombinant human transglutaminase assay for the diagnosis and monitoring of celiac patients. It has a greater ability to resolve positive and negative results (“minimum indeterminate area”). Its greater sensitivity could detect situations (presentation and/or dietary transgression) where conventional techniques show negative or weakly positive serology.

Development and evaluation of NS1 protein based i-ELISA for serodiagnosis of Bluetongue disease

Bluetongue (BT) is an important viral disease of small ruminants and causes major economic losses in India. For serodiagnosis of BT, recombinant antigen-based assays are considered as alternatives to conventional approaches such as serum neutralization test. The present study aimed at the development of an indirect- enzyme-linked immunosorbent assay (i-ELISA) using bluetongue (BTV) recombinant NS1. NS1 gene of BTV encoding a mature NS1 protein was produced as a recombinant protein (rNS1) in Escherichia coli. The immunogenic potential of purified rNS1 was confirmed by detecting specific NS1 antibody responses in sera collected from immunized sheep using ELISA. An i-ELISA based on rNS1 was developed and optimized. In comparison to a commercially available c-ELISA kit, in the detection of BTV specific antibodies, this new assay exhibited a diagnostic sensitivity of 91.2%. This study highlighted the potential utility of rNS1 protein as a safe and novel diagnostic reagent in comparison to live virus antigen, in the development of serodiagnostic assay for surveillance of BTV infection in sheep and goats.

Conformational dynamics promotes disordered regions from function-dispensable to essential in evolved site-specific DNA recombinases

Protein intrinsically disordered regions (IDRs) play pivotal roles in molecular recognition and regulatory processes through structural disorder-to-order transitions. To understand and exploit the distinctive functional implications of IDRs and to unravel the underlying molecular mechanisms, structural disorder-to-function relationships need to be deciphered. The DNA site-specific recombinase system Cre/loxP represents an attractive model to investigate functional molecular mechanisms of IDRs. Cre contains a functionally dispensable disordered N-terminal tail, which becomes indispensable in the evolved Tre/loxLTR recombinase system. The difficulty to experimentally obtain structural information about this tail has so far precluded any mechanistic study on its involvement in DNA recombination. Here, we use in vitro and in silico evolution data, conformational dynamics, AI-based folding simulations, thermodynamic stability calculations, mutagenesis and DNA recombination assays to investigate how evolution and the dynamic behavior of this IDR may determine distinct functional properties. Our studies suggest that partial conformational order in the N-terminal tail of Tre recombinase and its packing to a conserved hydrophobic surface on the protein provide thermodynamic stability. Based on our results, we propose a link between protein stability and function, offering new plausible atom-detailed mechanistic insights into disorder-function relationships. Our work highlights the potential of N-terminal tails to be exploited for regulation of the activity of Cre-like tyrosine-type SSRs, which merits future investigations and could be of relevance in future rational engineering for their use in biotechnology and genomic medicine.