A suppressive oligodeoxynucleotide enhances the efficacy of myelin cocktail/IL-4-tolerizing DNA vaccination and treats autoimmune disease

Targeting pathogenic T cells with Ag-specific tolerizing DNA vaccines encoding autoantigens is a powerful and feasible therapeutic strategy for Th1-mediated autoimmune diseases. However, plasmid DNA contains abundant unmethylated CpG motifs, which induce a strong Th1 immune response. We describe here a novel approach to counteract this undesired side effect of plasmid DNA used for vaccination in Th1-mediated autoimmune diseases. In chronic relapsing experimental autoimmune encephalomyelitis (EAE), combining a myelin cocktail plus IL-4-tolerizing DNA vaccine with a suppressive GpG oligodeoxynucleotide (GpG-ODN) induced a shift of the autoreactive T cell response toward a protective Th2 cytokine pattern. Myelin microarrays demonstrate that tolerizing DNA vaccination plus GpG-ODN further decreased anti-myelin autoantibody epitope spreading and shifted the autoreactive B cell response to a protective IgG1 isotype. Moreover, the addition of GpG-ODN to tolerizing DNA vaccination therapy effectively reduced overall mean disease severity in both the chronic relapsing EAE and chronic progressive EAE mouse models. In conclusion, suppressive GpG-ODN effectively counteracted the undesired CpG-induced inflammatory effect of a tolerizing DNA vaccine in a Th1-mediated autoimmune disease by skewing both the autoaggressive T cell and B cell responses toward a protective Th2 phenotype. These results demonstrate that suppressive GpG-ODN is a simple and highly effective novel therapeutic adjuvant that will boost the efficacy of Ag-specific tolerizing DNA vaccines used for treating Th1-mediated autoimmune diseases.     

Oligonucleotides Containing Acyclic Nucleoside Analogues with Carbamate Internucleoside Linkages

Abstract

Synthesis of 2′-deoxy-2′,3′-secothymidine t and its dimer t?t, where the two 2′-deoxy-2′,3′-secothymidine t units are connected via a carbamate, ?=3′-NH-CO-O-5′, internucleoside linkage has been achieved. These building blocks were protected in the 5′-position, converted into their phosphoramidites, or attached onto CPG, and then used for “chimeric oligonucleotide” synthesis.     

Effect of N,N,N',N'-tetramethylethylenediamine on the migration of proteins in SDS polyacrylamide gels

Changing the concentration of TEMED in SDS polyacrylamide gels was found to affect the migration of proteins. Elevation of TEMED levels caused a generalized decrease in mobility with some proteins being affected more than others. With various brain protein preparations this differential effect could be used to improve the separation of adjacent protein bands. In additon, it was found that a change in the TEMED concentration affected the results of molecular weight determinations. The effect of TEMED was also observed in one non-SDS system.     

A Genetic RNAi Screen for IP3/Ca2+ Coupled GPCRs in Drosophila Identifies the PdfR as a Regulator of Insect Flight

Insect flight is regulated by various sensory inputs and neuromodulatory circuits which function in synchrony to control and fine-tune the final behavioral outcome. The cellular and molecular bases of flight neuromodulatory circuits are not well defined. In Drosophila melanogaster, it is known that neuronal IP₃ receptor mediated Ca²⁺ signaling and store-operated Ca²⁺ entry (SOCE) are required for air-puff stimulated adult flight. However, G-protein coupled receptors (GPCRs) that activate intracellular Ca²⁺ signaling in the context of flight are unknown in Drosophila. We performed a genetic RNAi screen to identify GPCRs that regulate flight by activating the IP₃ receptor. Among the 108 GPCRs screened, we discovered 5 IP₃/Ca²⁺ linked GPCRs that are necessary for maintenance of air-puff stimulated flight. Analysis of their temporal requirement established that while some GPCRs are required only during flight circuit development, others are required both in pupal development as well as during adult flight. Interestingly, our study identified the Pigment Dispersing Factor Receptor (PdfR) as a regulator of flight circuit development and as a modulator of acute flight. From the analysis of PdfR expressing neurons relevant for flight and its well-defined roles in other behavioral paradigms, we propose that PdfR signaling functions systemically to integrate multiple sensory inputs and modulate downstream motor behavior.     

Biosorptive removal of cadmium from contaminated groundwater and industrial effluents

The cadmium removing capacity of a biosorbent Calotropis procera, a perennial wild plant, is reported here. The biomass was found to possess high uptake capacity of Cd(II). Adsorption was pH dependent and the maximum removal was obtained at two different pH i.e. pH 5.0 and 8.0. Maximum biosorption capacity in batch and column mode was found to be 40 and 50.5 mg/g. The adsorption equilibrium (> or =90% removal) was attained within 5 min irrespective of the cadmium ion concentration. Interfering ions viz. Zn(II), As(III), Fe(II), Ni(II) interfered only when their concentration was higher than the equimolar ratio. The Freundlich isotherm best explained the adsorption, yet the monolayer adsorption was also noted at lower concentrations of Cd(II). The FTIR analysis indicates the involvement of hydroxyl (-OH), alkanes (-CH), nitrite (-NO(2)), and carboxyl group (-COO) chelates in metal binding. The complete desorption of the cadmium was achieved by 0.1M H(2)SO(4) and 0.1M HCl. The C. procera based Cd(II) removal technology appears feasible.     

Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion

Fibroblasts often constitute the majority of the stromal cells within a breast carcinoma, yet the functional contributions of these cells to tumorigenesis are poorly understood. Using a coimplantation tumor xenograft model, we demonstrate that carcinoma-associated fibroblasts (CAFs) extracted from human breast carcinomas promote the growth of admixed breast carcinoma cells significantly more than do normal mammary fibroblasts derived from the same patients. The CAFs, which exhibit the traits of myofibroblasts, play a central role in promoting the growth of tumor cells through their ability to secrete stromal cell-derived factor 1 (SDF-1); CAFs promote angiogenesis by recruiting endothelial progenitor cells (EPCs) into carcinomas, an effect mediated in part by SDF-1. CAF-secreted SDF-1 also stimulates tumor growth directly, acting through the cognate receptor, CXCR4, which is expressed by carcinoma cells. Our findings indicate that fibroblasts within invasive breast carcinomas contribute to tumor promotion in large part through the secretion of SDF-1.     

Predicting novel candidate human obesity genes and their site of action by systematic functional screening in Drosophila

The discovery of human obesity-associated genes can reveal new mechanisms to target for weight loss therapy. Genetic studies of obese individuals and the analysis of rare genetic variants can identify novel obesity-associated genes. However, establishing a functional relationship between these candidate genes and adiposity remains a significant challenge. We uncovered a large number of rare homozygous gene variants by exome sequencing of severely obese children, including those from consanguineous families. By assessing the function of these genes in vivo in Drosophila, we identified 4 genes, not previously linked to human obesity, that regulate adiposity (itpr, dachsous, calpA, and sdk). Dachsous is a transmembrane protein upstream of the Hippo signalling pathway. We found that 3 further members of the Hippo pathway, fat, four-jointed, and hippo, also regulate adiposity and that they act in neurons, rather than in adipose tissue (fat body). Screening Hippo pathway genes in larger human cohorts revealed rare variants in TAOK2 associated with human obesity. Knockdown of Drosophila tao increased adiposity in vivo demonstrating the strength of our approach in predicting novel human obesity genes and signalling pathways and their site of action.

This study set out to identify novel gene variants that may contribute to human obesity, by combining human exosome sequencing analyses with systematic functional screening in Drosophila. This identifies a number of novel obesity-associated genes which control adiposity in flies, and uncovers a potential role for the Hippo signaling pathway in obesity.

Obesity is a major risk factor for type 2 diabetes, cardiovascular disease, cancers, and, most recently, COVID-19 [1]. Despite the obvious environmental drivers to weight gain, multiple genetic studies have demonstrated that 40% to 70% of the variation in body weight is attributable to genetic variation [2]. The discovery of genes that contribute to the regulation of human body weight can provide insights into the mechanisms involved in energy homeostasis and identify potential targets for weight loss therapy. Moreover, drug targets supported by human genetic evidence are more likely to transit successfully through the drug discovery pipeline [3].A classical approach to the discovery of pathogenic variants is to investigate consanguineous populations with high degrees of parental relatedness (parents who are first or second cousins) where large portions of the genome are identical by descent as a result of family structure in preceding generations (long regions of homozygosity). Indeed, studies in consanguineous families led to the discovery of the first homozygous loss-of-function mutations in the genes encoding leptin (LEP; [4]) and the leptin receptor (LEPR; [5]) associated with severe obesity. However, at the time, the function of leptin and its receptor had already been established in ob/ob and db/db mice, respectively [6], so the pathogenicity of homozygous mutations that resulted in loss of function in cells was readily established.The situation is more complex when studying homozygous mutations in new candidate genes. Some of these genes may play a direct causal role in the development of obesity, others may increase susceptibility to obesity only in certain contexts, and some genes will play no role at all. Recent large-scale studies in healthy people in outbred populations have revealed that a significant proportion of rare homozygous variants that are predicted to cause a loss of function do not result in a clinically discernible phenotype [7,8]. As such, identifying the subset of genes that may be involved in the regulation of adiposity in large human genetic studies presents a major hurdle.For some diseases, functional screens in cultured cells permit rapid testing of candidate genes, as exemplified by studies of insulin secretion in islet cells for genes associated with type 2 diabetes [9]. However, obesity is a systems-level disorder that cannot be replicated in cells. As such, a functional screen in vivo is needed. Here, we use Drosophila to screen the functional consequences of knocking down expression of candidate human obesity genes and to explore the complex interactions between multiple organ systems that are regulated by environmental and genetic factors.Drosophila has been a useful tool in the functional characterisation of human disease-associated genes [10–12]. Many organ systems and metabolic enzymes are highly conserved in Drosophila, as are the major regulatory mechanisms involved in metabolic homeostasis [13,14]. As in humans, Drosophila accumulate lipids and become obese when raised on a high-fat or high-sugar diet, developing cardiomyopathy and diabetic phenotypes [15,16]. Furthermore, more than 60% of the genes identified in an unbiased genome-wide RNAi screen for increased fat levels in Drosophila have human orthologues [17]. Most studies in Drosophila have performed forward genetic screens resulting in obesity [18] before assessing whether misregulation of the corresponding mammalian orthologue affects adiposity [17]. Another report knocked down Drosophila orthologs of human genes near body mass index (BMI) loci from GWAS studies to identify genes regulating adiposity [19].Here, instead, we chose to take advantage of new data from a cohort of patients carrying rare genetic variants that might cause severe early-onset obesity. We set out to identify, in Drosophila, whether any of these genes are likely to be responsible for the obese phenotype. An additional advantage of working with Drosophila is the potential to identify interacting genes and signalling pathways. We proposed that it would then be possible to search for variants in human orthologues of these genes in larger cohorts of patients, to discover further as yet unidentified genes regulating human obesity.To increase our chances of finding pathogenic variants, we focused on rare homozygous variants identified in probands with severe obesity, many from consanguineous families. After knocking down expression of Drosophila orthologues of candidate human obesity genes, we discovered 4 genes that significantly increased triacylglyceride (TAG) levels. Importantly, none of these genes had been associated previously with human obesity, but the pathways in which they act are known and could be further analysed in Drosophila. Knockdown of further members of one of these signalling pathways, the Hippo pathway, also gave an obesity phenotype, highlighting the success of our approach. We then searched for variants in the novel obesity genes we identified in Drosophila, and their associated signalling pathways, in larger cohorts of unrelated obese people and healthy controls. This uncovered yet another gene, which, when knocked down in Drosophila, increased adiposity. We demonstrate that the cross-fertilisation of human and Drosophila genetics is a powerful system to provide novel insights into the genetic and cellular processes regulating adiposity and may ultimately contribute to strategies for the prevention and treatment of obesity.     

Rational design of therapeutic mAbs against aggregation through protein engineering and incorporation of glycosylation motifs applied to bevacizumab

The aggregation of biotherapeutics is a major hindrance to the development of successful drug candidates; however, the propensity to aggregate is often identified too late in the development phase to permit modification to the protein's sequence. Incorporating rational design for the stability of proteins in early discovery has numerous benefits. We engineered out aggregation-prone regions on the Fab domain of a therapeutic monoclonal antibody, bevacizumab, to rationally design a biobetter drug candidate. With the purpose of stabilizing bevacizumab with respect to aggregation, 2 strategies were undertaken: single point mutations of aggregation-prone residues and engineering a glycosylation site near aggregation-prone residues to mask these residues with a carbohydrate moiety. Both of these approaches lead to comparable decreases in aggregation, with an up to 4-fold reduction in monomer loss. These single mutations and the new glycosylation pattern of the Fab domain do not modify binding to the target. Biobetters with increased stability against aggregation can therefore be generated in a rational manner, by either removing or masking the aggregation-prone region or crowding out protein-protein interactions.