Receptor Variants Research Articles

PmTR database: population matched (pm) germline allelic variants of T-cell receptor (TR) loci

The IMGT database profiles the TR germline alleles for all four TR loci (TRA, TRB, TRG and TRD), however, it does not comprise of the information regarding population specificity and allelic frequencies of these germline alleles. The specificity of allelic variants to different human populations can, however, be a rich source of information when studying the genetic basis of population-specific immune responses in disease and in vaccination. Therefore, we meticulously identified true germline alleles enriched with complete TR allele sequences and their frequencies across 26 different human populations, profiled by “1000 Genomes data”. We identified 205 TRAV, 249 TRBV, 16 TRGV and 5 TRDV germline alleles supported by at least four haplotypes. The diversity of germline allelic variants in the TR loci is the highest in Africans, while the majority of the Non-African alleles are specific to the Asian populations, suggesting a diverse profile of TR germline alleles in different human populations. Interestingly, the alleles in the IMGT database are frequent and common across all five super-populations. We believe that this new set of germline TR sequences represents a valuable new resource which we have made available through the new population-matched TR (pmTR) database, accessible via https://pmtrig.lumc.nl/.

Advances in CAR-T cell therapy for malignant solid tumors.

T cells modified by chimeric antigen receptor (CAR) have the advantage of major histocompatibility complex-independent recognition of tumor-associated antigens, so can achieve efficient response to tumor targets. Chimeric antigen receptor (CAR) T cell therapy has shown a good therapeutic effect in hematological malignancies; however, its efficacy is generally not satisfactory for solid tumors. The reasons include the lack of tumor specific antigen target on solid tumors, the uncertainty of homing ability of engineered T cells and the inhibitory immune microenvironment of tumors. In clinical trials, the targets of CAR-T cell therapy for solid tumors are mainly disialoganglioside (GD2), claudin-18 isoform 2 (CLDN18.2), mesenchymal, B7 homolog 3 (B7H3), glypican (GPC) 3 and epidermal growth factor receptor variant Ш (EGFRvШ)Ⅲ. Combination of CAR-T cells with oncolytic viruses, tyrosine kinase inhibitors, and programmed death ligand-1 monoclonal antibodies may increase its efficacy. The CAR-T cell therapy for solid tumors can be optimized through gene editing to enhance the activity of CAR-T cells, adding corresponding regulatory components to make the activation of CAR-T cells safer and more controllable, and enhancing the persistence of CAR-T cells. In this article, we review the latest advances of CAR-T cell therapy in solid tumors to provide new insights for clinical application.

Cistrome and transcriptome analysis identifies unique androgen receptor (AR) and AR-V7 splice variant chromatin binding and transcriptional activities

The constitutively active androgen receptor (AR) splice variant, AR-V7, plays an important role in resistance to androgen deprivation therapy in castration resistant prostate cancer (CRPC). Studies seeking to determine whether AR-V7 is a partial mimic of the AR, or also has unique activities, and whether the AR-V7 cistrome contains unique binding sites have yielded conflicting results. One limitation in many studies has been the low level of AR variant compared to AR. Here, LNCaP and VCaP cell lines in which AR-V7 expression can be induced to match the level of AR, were used to compare the activities of AR and AR-V7. The two AR isoforms shared many targets, but overall had distinct transcriptomes. Optimal induction of novel targets sometimes required more receptor isoform than classical targets such as PSA. The isoforms displayed remarkably different cistromes with numerous differential binding sites. Some of the unique AR-V7 sites were located proximal to the transcription start sites (TSS). A de novo binding motif similar to a half ARE was identified in many AR-V7 preferential sites and, in contrast to conventional half ARE sites that bind AR-V7, FOXA1 was not enriched at these sites. This supports the concept that the AR isoforms have unique actions with the potential to serve as biomarkers or novel therapeutic targets.

Vitamin D Receptor and Binding Protein Gene Variants in Patients with Essential Tremor.

Several studies have shown an association between some variants in the vitamin D receptor (VDR) and the GC vitamin D binding protein (GC) genes with the risk for Parkinson's disease or other neurological disorders. VDR rs2228570 has shown an association with essential tremor (ET) in a previous study. The aim of this study is to look for the association between several common variants in these genes and the risk for ET. We genotyped 272 patients diagnosed with familial ET and 272 age-matched controls using specific TaqMan assays for VDR rs2228570, VDR rs731236, VDR rs7975232, VDR rs739837, VDR rs78783628, GC rs7041, and GC rs4588 single nucleotide variants (SNVs). We found an association between GC rs7041 SNV and ET using recessive, codominant, and allelic models. Despite our results did not find an association between VDR rs2228570 and ET, the pooled data with those by a previous report suggest this association under recessive, codominant, and allelic models. None of the SNVs studied was related to the age at onset of tremor in ET patients. Data from the current study suggest an association between GC rs7041 and VDR rs2228570 SNVs and ET risk.

High-resolution melting analysis after nested PCR for the detection of SARS-CoV-2 spike protein G339D and D796Y variations

High-resolution melting (HRM) analysis was performed to detect G339D and D796Y variations in the SARS-CoV-2 Omicron variant spike protein. We employed two-step PCR consisting of the first RT–PCR and the second nested PCR to prepare the amplicon for HRM analysis. The melting temperatures (Tm) of the amplicon from the cDNA of the Omicron variant receptor binding domain (RBD) were 73.1 °C (G339D variation) and 75.1 °C (D796Y variation), respectively. These Tm values were clearly distinct from those of SARS-CoV-2 isolate Wuhan-Hu-1. HRM analysis after the two-step PCR was conducted on Omicron variant-positive specimens. The HRM curve and Tm value obtained with the Omicron variant-positive specimen were coincident with those of the amplicon from cDNA of the Omicron variant RBD. Our study demonstrates the utility of HRM analysis after two-step PCR for the detection of mutations in SARS-CoV-2 gene.

Ghrelin and Cancer: Examining the Roles of the Ghrelin Axis in Tumor Growth and Progression.

Ghrelin, a hormone produced and secreted from the stomach, is prim arily known as an appetite stimulant. Recently, it has emerged as a potential regulator/biomarker of cancer progression. Inconsistent results on this subject make this body of literature difficult to interpret. Here, we attempt to identify commonalities in the relationships between ghrelin and various cancers, and summarize important considerations for future research. The main players in the ghrelin family axis are unacylated ghrelin (UAG), acylated ghrelin (AG), the enzyme ghrelin O-acyltransferase (GOAT), and the growth hormone secretagogue receptor (GHSR). GOAT is responsible for the acylation of ghrelin, after which ghrelin can bind to the functional ghrelin receptor GHSR-1a to initiate the activation cascade. Splice variants of ghrelin also exist, with the most prominent being In1-ghrelin. In this review, we focus primarily on the potential of In1-ghrelin as a biomarker for cancer progression, the unique characteristics of UAG and AG, the importance of the two known receptor variants GHSR-1a and 1b, as well as the possible mechanisms through which the ghrelin axis acts. Further understanding of the role of the ghrelin axis in tumor cell proliferation could lead to the development of novel therapeutic approaches for various cancers.

Association between beta-2 adrenergic receptor variants and clinical outcomes in children and adolescents with acute asthma.

ABSTRACTObjective To investigate whether different genotypes of p.Arg16Gly, p.Gln27Glu, p.Arg19Cys and p.Thr164Ile variants interfere in response to treatment in children and adolescents with moderate to severe acute asthma.Methods This sample comprised patients aged 2 to 17 years with a history of at least two wheezing episodes and current moderate to severe asthma exacerbation. All patients received multiple doses of albuterol and ipratropium bromide delivered via pressurized metered-dose inhaler with holding chamber and systemic corticosteroids. Hospital admission was defined as the primary outcome. Secondary outcomes were changes in forced expiratory volume in the first second after 1 hour of treatment, and for outpatients, length of stay in the emergency room. Variants were genotyped by sequencing.Results A total of 60 patients were evaluated. Hospital admission rates were significantly higher in carriers of the genotype AA relative to those with genotype AG or GG, within the p.Arg16Gly variant (p=0.03, test χ2, alpha=0.05). Secondary outcomes did not differ between genotypes.Conclusion Hospital admission rates were significantly higher among carriers of the genotype AA within the p.Arg16Gly variant. Trial registration: ClinicalTrials.gov: NCT01323010

M6A-induced repression of SIAH1 facilitates alternative splicing of androgen receptor variant 7 by regulating CPSF1

Androgen receptor splice variant 7 (AR-v7), a constitutively active transcription factor, plays a crucial role in the progression of castration-resistant prostate cancer (CRPC). Here, we found that the cleavage and polyadenylation specificity factor 1 (CPSF1) (the largest subunit of the multi-protein cleavage and polyadenylation specificity complex), regulated by the E3 ubiquitin ligases SIAH1, promoted AR-v7 expression. The data from microarray-based analysis and clinical specimen-based analysis showed that SIAH1 expression was decreased in PCa and was negatively correlated with aggressive phenotypes of PCa. SIAH1 repressed PCa cell growth and invasion under castrate conditions. SIAH1 directly interacted with CPSF1 and promoted ubiquitination and degradation of CPSF1. CPSF1 expression was negatively correlated with SIAH1 expression, but positively with PCa progression. CPSF1 overexpression switched the AR splicing pattern and facilitated the generation of the oncogenic isoform (AR-v7) by binding to the AAUAAA polyadenylation signal contained in AR-cryptic exon CE3. Functionally, SIAH1 acted as a tumor suppressor in PCa pathogenesis by repressing CPSF1-mediated AR-v7 generation. Finally, we demonstrated that m6A methylation was concerned with the repression of SIAH1 in PCa. Our results define SIAH1/CPSF1/AR-v7 axis as a regulatory factor of PCa progression, providing a promising target for treating PCa.

Omicron variant Spike-specific antibody binding and Fc activity are preserved in recipients of mRNA or inactivated COVID-19 vaccines.

The Omicron variant of SARS-CoV-2 has been shown to evade neutralizing antibodies elicited by vaccination or prior infection. Despite the dramatic global spread of the Omicron variant, even among highly vaccinated populations, death rates have not increased concomitantly. These data suggest that immune mechanisms beyond antibody-mediated virus neutralization may protect against severe disease. In addition to neutralizing pathogens, antibodies contribute to control and clearance of infections through Fc-effector mechanisms. Here we probed the ability of vaccine-induced antibodies to drive Fc-effector activity against the Omicron variant using samples from individuals receiving one of three SARS-CoV-2 vaccines. Despite a substantial loss of IgM, IgA, and IgG binding to the Omicron variant Receptor Binding Domain (RBD) in samples from individuals receiving BNT162b2, mRNA-1273, and CoronaVac vaccines, stable binding was maintained against the full-length Omicron Spike protein. Compromised RBD binding IgG was accompanied by a loss of cross RBD-specific antibody Fcγ receptor (FcγR) binding in samples from individuals who received the CoronaVac vaccine, but RBD-specific FcγR2a and FcγR3a binding was preserved in recipients of mRNA vaccines. Conversely, Spike protein-specific antibodies exhibited persistent but reduced binding to FcγRs across all three vaccines, though higher binding was observed in samples from recipients of mRNA vaccines. This was associated with preservation of FcγR2a and FcγR3a binding antibodies and maintenance of Spike protein-specific antibody-dependent natural killer cell activating antibodies. Thus, despite the loss of Omicron neutralization, vaccine-induced Spike protein-specific antibodies continue to drive Fc-effector functions, suggesting a capacity for extra-neutralizing antibodies to contribute to disease control.

Pre-clinical Exploratory Evaluation of Mab806-derived Immunotoxin in Targeting Solid Tumors with High Potency and Selectivity Towards EGFRvIII

The overexpression and mutation of EGFRs are associated with various types of human cancers. Epidermal growth factor receptor variant III (EGFRvIII) is one of the most notable EGFR mutations, and is exclusively present in cancer cells, making it a potential therapeutic target. The present study aims to determine whether recombinant immunotoxin 86 (rIT86) can effectively kill EGFRvIII-expressing cancer cells, while maintaining adequate levels of tolerance.

Application of Surface-Enhanced Raman Spectroscopy to Guide Therapy for Advanced Prostate Cancer Patients

A critical unmet need for advanced prostate cancer (PCa) patients is optimizing systemic treatments to maximize the benefit for individuals. The response of patients with metastatic castration-resistant prostate cancer (mCRPC) to androgen receptor (AR)-directed hormonal treatments (i.e., enzalutamide and abiraterone) is mediated by the expression of a molecular variant of the androgen receptor called androgen receptor variant 7 (AR-V7). Detection and measurement of AR-V7 in mCRPC patients will lead to more informed PCa treatment. Herein, we demonstrate a quantitative nanoparticle-enhanced sandwich antibody assay for the successful ex vivo measurement of AR-V7 protein in serum from mCRPC patients. The nanoparticles are constructed as extrinsic Raman spectroscopy labels (ERLs), and surface-enhanced Raman spectroscopy (SERS) is used for assay readout. Our approach does not require specialized specimen collection materials, circulating tumor cell enrichment, or pretreatment of serum. Calibration of our assay is accomplished by expressing AR-V7 in an appropriate cell line as AR-V7 is not commercially available. We demonstrate a linear calibration curve from cell lysate and correlate lysate protein with mRNA from cultured prostate cancer cells. Finally, we demonstrate a novel pilot-scale application for clinical use by quantitatively measuring AR-V7 in serum of seven advanced PCa patients. Distinct separation of PCa patients by AR-V7 status (positive or negative) was observed. Together, the presence and amount of AR-V7 in serum offer predictive and prognostic value to inform selection between two classes of systemic treatments (i.e., hormones or taxanes). Triaging patients that are AR-V7-positive to other systemic treatments (e.g., taxane-based chemotherapy) can improve progression-free survival and overall survival.

Unravelling the Role of Kinases That Underpin Androgen Signalling in Prostate Cancer.

The androgen receptor (AR) signalling pathway is the key driver in most prostate cancers (PCa), and is underpinned by several kinases both upstream and downstream of the AR. Many popular therapies for PCa that target the AR directly, however, have been circumvented by AR mutation, such as androgen receptor variants. Some upstream kinases promote AR signalling, including those which phosphorylate the AR and others that are AR-regulated, and androgen regulated kinase that can also form feed-forward activation circuits to promotes AR function. All of these kinases represent potentially druggable targets for PCa. There has generally been a divide in reviews reporting on pathways upstream of the AR and those reporting on AR-regulated genes despite the overlap that constitutes the promotion of AR signalling and PCa progression. In this review, we aim to elucidate which kinases—both upstream and AR-regulated—may be therapeutic targets and require future investigation and ongoing trials in developing kinase inhibitors for PCa.

Thrombotic Risk Determined by Protein C Receptor (PROCR) Variants among Middle-Aged and Older Adults: A Population-Based Cohort Study.

The protein C (PC) anticoagulant system has a key role in maintaining hemostatic balance. One missense (Ser219Gly) variant in the PC receptor (PROCR) was associated with venous thromboembolism (VTE) in genome-wide association studies. This study aimed to determine the thrombotic risk of rare and common PROCR variants in a large population-based cohort of middle-aged and older adults. The exonic sequence of PROCR was analyzed for the Ser219Gly variant and other qualifying variants in 28,794 subjects (born 1923-1950, 60% women) without previous VTE, who participated in the Malmö Diet and Cancer study (1991-1996). Incidence of VTE was followed up until 2018. Qualifying variants were defined as loss-of-function or nonbenign (PolyPhen-2) missense variants with minor allele frequencies (MAFs) <0.1%. Re-sequencing identified 36 PROCR variants in the study population (26,210 non-VTE exomes and 2,584 VTE exomes), 11 synonymous, 22 missense, and three loss-of-function variants. Kaplan-Meier analysis of the known Ser219Gly variant (rs867186) showed that homozygosity for this variant increased the risk of disease, whereas heterozygosity showed no effect. Cox multivariate regression analysis revealed an adjusted hazard ratio (HR) of 1.5 (95% confidence interval [CI]: 1.1-2.0). Fifteen rare variants were classified as qualifying and were included in collapsing analysis (burden test and SKAT-O). They did not contribute to risk. However, a Arg113Cys missense variant (rs146420040; MAF = 0.004) showed an increased VTE risk (HR = 1.3; 95% CI: 1.0-1.9). Homozygosity for the Ser219Gly variant and a previously identified functional PROCR variant (Arg113Cys) was associated with VTE. Other variants did not contribute to VTE.

Molecular profiling of melanocortin 4 receptor variants and agouti-related peptide interactions in morbid obese phenotype: a novel paradigm from molecular docking and dynamics simulations

Molecular profiling of melanocortin 4 receptor variants and agouti-related peptide interactions in morbid obese phenotype: a novel paradigm from molecular docking and dynamics simulations

A phase 2 study of onvansertib in combination with abiraterone and prednisone in patients with metastatic castration-resistant prostate cancer (mCRPC).

TPS219 Background: Metastatic CRPC is a leading cause of cancer death worldwide. Although abiraterone (abi) + prednisone in either castration-sensitive or castration-resistant disease increases survival, resistance is universal and generally occurs within 9-16 months of initiating treatment. Polo-like kinase 1 (PLK1) is a serine/threonine protein kinase that regulates mitotic function and is upregulated in prostate cancer following androgen-deprivation therapy (ADT). Onvansertib is an oral and highly-selective PLK1 inhibitor that demonstrated safety and tolerability as a single agent in a Ph1 trial. Preclinical studies showed that PLK1 inhibition enhanced abi anti-tumor effect in cell line models and in patient-derived tumor xenografts via an AR-independent mechanism. Transcriptomic analyses revealed that abi induced mitosis-related gene sets in cells synergistic for abi + onvansertib, and identified an abi-onvansertib synergy gene signature. Methods: The goal of this phase 2 study (NCT03414034) is to observe the effects of onvansertib + abi on disease control, as assessed by prostate-specific antigen (PSA) decline or stabilization after 12 weeks of study treatment, in subjects with mCRPC and early resistance to abi. Patients are enrolled at time of PSA progression while on standard abi. Prior treatment with enzalutamide or apalutamide is not permitted. Three onvansertib dosing schedules are tested in noncomparative arms: arm A (24 mg/m2 on days 1-5 of a 21-day cycle), arm B (18 mg/m2 on days 1-5 of a 14-day cycle), and arm C (12 mg/m2 on days 1-14 of a 21-day cycle). To-date, 24 patients were enrolled in Arm A, and 20 patients each in Arms B and C. With 32 patients in each arm, there will be 90% power to detect a change in disease-control rate from 10% (null) to 30% (alternative). Based on a Simon’s two-stage optimal design, the study will terminate early if &lt; 2 of the first 13 patients in each arm achieve disease control. Exploratory analyses include evaluation of the presence of the androgen receptor variant 7 (AR-V7) in circulating tumor cells and targeted genomic profiling of circulating tumor DNA to assess associations between genomic alterations and response to treatment. Additionally, transcriptomic analysis of archived tumor tissue will be performed to identify gene expression signatures associated with clinical responses. Clinical trial information: NCT03414034.

Small extracellular vesicle-mediated ITGB6 siRNA delivery downregulates the αVβ6 integrin and inhibits adhesion and migration of recipient prostate cancer cells

ABSTRACT The αVβ6 integrin, an epithelial-specific cell surface receptor absent in normal prostate and expressed during prostate cancer (PrCa) progression, is a therapeutic target in many cancers. Here, we report that transcript levels of ITGB6 (encoding the β6 integrin subunit) are significantly increased in metastatic castrate-resistant androgen receptor-negative prostate tumors compared to androgen receptor-positive prostate tumors. In addition, the αVβ6 integrin protein levels are significantly elevated in androgen receptor-negative PrCa patient derived xenografts (PDXs) compared to androgen receptor-positive PDXs. In vitro, the androgen receptor-negative PrCa cells express high levels of the αVβ6 integrin compared to androgen receptor-positive PrCa cells. Additionally, expression of androgen receptor (wild type or variant 7) in androgen receptor-negative PrCa cells downregulates the expression of the β6 but not αV subunit compared to control cells. We demonstrate an efficient strategy to therapeutically target the αVβ6 integrin during PrCa progression by using short interfering RNA (siRNA) loaded into PrCa cell-derived small extracellular vesicles (sEVs). We first demonstrate that fluorescently-labeled siRNAs can be efficiently loaded into PrCa cell-derived sEVs by electroporation. By confocal microscopy, we show efficient internalization of these siRNA-loaded sEVs into PrCa cells. We show that sEV-mediated delivery of ITGB6-targeting siRNAs into PC3 cells specifically downregulates expression of the β6 subunit. Furthermore, treatment with sEVs encapsulating ITGB6 siRNA significantly reduces cell adhesion and migration of PrCa cells on an αVβ6-specific substrate, LAP-TGFβ1. Our results demonstrate an approach for specific targeting of the αVβ6 integrin in PrCa cells using sEVs encapsulating ITGB6-specific siRNAs.

Functional Assessment of Calcium-Sensing Receptor Variants Confirms Familial Hypocalciuric Hypercalcemia.

ContextIn the clinic it is important to differentiate primary hyperparathyroidism (PHPT) from the more benign, inherited disorder, familial hypocalciuric hypercalcemia (FHH). Since the conditions may sometimes overlap biochemically, identification of calcium-sensing receptor (CASR) gene variants causative of FHH (but not PHPT) is the most decisive diagnostic aid. When novel variants are identified, bioinformatics and functional assessment are required to establish pathogenicity.ObjectiveWe identified 3 novel CASR transmembrane domain missense variants, Thr699Asn, Arg701Gly, and Thr808Pro, in 3 probands provisionally diagnosed with FHH and examined the variants using bioinformatics and functional analysis.MethodsBioinformatics assessment utilized wANNOVAR software. For functional characterization, each variant was cloned into a mammalian expression vector; wild-type and variant receptors were transfected into HEK293 cells, and their expression and cellular localization were assessed by Western blotting and confocal immunofluorescence, respectively. Receptor activation in HEK293 cells was determined using an IP-One ELISA assay following stimulation with Ca++ ions.ResultsBioinformatics analysis of the variants was unable to definitively assign pathogenicity. Compared with wild-type receptor, all variants demonstrated impaired expression of mature receptor reaching the cell surface and diminished activation at physiologically relevant Ca++ concentrations.ConclusionThree CASR missense variants identified in probands provisionally diagnosed with FHH result in receptor inactivation and are therefore likely causative of FHH. Inactivation may be due to inadequate processing/trafficking of mature receptor and/or conformational changes induced by the variants affecting receptor signaling. This study demonstrates the value of functional studies in assessing genetic variants identified in hypercalcemic patients.

The GluN2B-Trp373 NMDA Receptor Variant is Associated with Autism-, Epilepsy-Related Phenotypes and Reduces NMDA Receptor Currents in Rats.

Autism spectrum disorder (ASD) is a neurodevelopmental condition with core clinical features of abnormal communication, social interactions, atypical intelligence, and a higher risk of epilepsy. Prior work has suggested that de novo heterozygous mutations in the GRIN2B gene that encodes the GluN2B subunit of N-methyl-D-aspartic acid receptors are likely linked to ASD. However, whether GLuN2B-Trp373 mutation derived from autistic individuals causes ASD-like behavioral aberrations in rats remains to be determined. Here, through in utero electroporation and in vivo studies, we conducted a battery of tests to examine ASD-associated behaviors, cognitive impairments, and susceptibility to pentylenetetrazol-induced seizures. Whole-cell patch recording was utilized to determine whether the GluN2B-Trp373 mutation influences GluN2B-containing NMDA receptor currents in rats. Results show that, behaviorally, GLuN2B-Trp373 mutant rats exhibited core behavioral manifestations of ASD, such as social interaction deficits, increases in stereotyped behaviors and anxiety stereotyped/repetitive, impaired spatial memory, and enhanced risk of pentylenetetrazol-induced seizures, consistent with many of the hallmarks of low-functioning ASD in humans. Functionally, the GluN2B-Trp373 mutation results in reduced GluN2B surface protein expression together with decreased hippocampal NMDA receptor currents. Collectively, our findings highlight that GluN2B-Trp373 mutations can drive the manifestation of ASD-associated symptoms via the suppression of NMDA receptor currents.

Expression and production of pigment epithelium-derived factor (PEDF) and PEDF receptor variants from mammalian and bacterial cells

Human SERPINF1 gene codes for pigment epithelium-derived factor (PEDF), a secreted glycoprotein and member of the SERPIN superfamily. To obtain large amounts of recombinant PEDF proteins, we subcloned the coding sequence of human SERPINF1 mutated versions into the pCEP4 vector and generated stably transfected HEK.Ebna cells. The cells produced and secreted recombinant PEDF proteins into the culturing media. The recombinant PEDF proteins were purified by ion-exchange column chromatography and milligram amounts of highly purified protein were recovered. PEDF has affinity for PEDF-receptor (PEDF-R), a membrane-linked lipase encoded by the PNPLA2 gene. Recombinant PEDF-R truncated versions were obtained from Escherichia coli containing expression vectors with human PNPLA2 cDNAs with 3′end deletions and by induction with isopropyl β-d-1-thiogalactopyranoside. The bacterially derived PEDF-R proteins in insoluble inclusion bodies were solubilized with urea and purified by cation-exchange column chromatography. C-terminally truncated PEDF-R versions containing the ligand binding region retained the ability to bind PEDF. The data demonstrate that mammalian-derived recombinant PEDF and bacterially derived recombinant PEDF-R can be produced and purified in large amounts for further use in structural and biological studies.

New pharmacological perspectives and therapeutic options for opioids: Differences matter.

Opioids remain the major drug class for the treatment of acute, chronic and cancer pain, but have major harmful effects such as dependence and opioid-induced ventilatory impairment. Although no new typical opioids have come onto the market in the past almost 50 years, a plethora of new innovative formulations has been developed to meet the clinical need. This review is intended to shed light on new understanding of the molecular pharmacology of opioids, which has arisen largely due to the genomic revolution, and what new drugs may become available in the coming years. Atypical opioids have and are being developed which not only target the mu opioid receptor but other targets in the pain pathway. Biased mu agonists have been developed but remain 'unbiased' clinically. The contribution of drugs targeting non-mu opioid receptors either alone or as heterodimers shows potential promise but remains understudied. That gene splice variants of the mu opioid receptor produce multiple receptor isoforms in different brain regions, and may change with pain chronicity and phenotype, presents new challenges but also opportunities for precision pain medicine. Finally, that opioids also have pro-inflammatory effects not aligned with mu opioid receptor binding affinity implicates a fresh understanding of their role in chronic pain, whether cancer or non-cancer. Hopefully, a new understanding of opioid analgesic drug action may lead to new drug development and better precision medicine in acute and chronic pain relief with less patient harm.