Double-negative-2 B cells are the major synovial plasma cell precursor in rheumatoid arthritis.

B cells are key pathogenic drivers of chronic inflammation in rheumatoid arthritis (RA). There is limited understanding of the relationship between synovial B cell subsets and pathogenic antibody secreting cells (ASCs). This knowledge is crucial for the development of more targeted B-cell depleting therapies. While CD11c+ double-negative 2 (DN2) B cells have been suggested as an ASC precursor in lupus, to date there is no proven link between the two subsets in RA. We have used both single-cell gene expression and BCR sequencing to study synovial B cells from patients with established RA, in addition to flow cytometry of circulating B cells. To better understand the differentiation patterns within the diseased tissue, a combination of RNA-based trajectory inference and clonal lineage analysis of BCR relationships were used. Both forms of analysis indicated that DN2 B cells serve as a major precursors to synovial ASCs. This study advances our understanding of B cells in RA and reveals the origin of pathogenic ASCs in the RA synovium. Given the significant role of DN2 B cells as a progenitor to pathogenic B cells in RA, it is important to conduct additional research to investigate the origins of DN2 B cells in RA and explore their potential as therapeutic targets in place of the less specific pan-B cells depletion therapies currently in use.

Design and Synthesis of Covalent Inhibitors of FabA.

There is an urgent need for the development of new therapeutics with novel modes of action to target Gram-negative bacterial infections, due to resistance to current drugs. Previously, FabA, an enzyme in the bacterial type II fatty acid biosynthesis pathway, was identified as a potential drug target in Pseudomonas aeruginosa, a Gram-negative bacteria of significant clinical concern. A chemical starting point was also identified. There is a cysteine, Cys15, in the active site of FabA, adjacent to where this compound binds. This paper describes the preparation of analogues containing an electrophilic warhead with the aim of covalent inhibition of the target. A wide variety of analogues were successfully prepared. Unfortunately, these analogues did not increase inhibition, which may be due to a loop within the enzyme partially occluding access to the cysteine.

Chloroplast/thylakoid-rich material: A possible alternative to the chemically synthesised flow enhancer polyglycerol polyricinoleate in oil-based systems.

Chloroplasts are abundant organelles in a diverse range of plant materials; they are predominantly composed of multicomponent thylakoid membranes which are lipid and protein rich. Intact or unravelled thylakoid membranes should, in principle, have interfacial activity, but little has been published on their activity in oil-in-water systems, and nothing on their performance on an oil continuous system. In this work different physical methods were used to produce a range of chloroplast/thylakoid suspensions with varying degrees of membrane integrity. Transmission electron microscopy revealed that pressure homogenisation led to the greatest extent of membrane and organelle disruption compared to less energy intensive preparation methods The ability of the derived materials to modulate the flow behaviour of a chocolate model system (65% (w/w) sugar/ sunflower oil (natural amphiphiles removed) suspension) was investigated by acquiring rheological parameters. All chloroplast/thylakoid preparations reduced yield stress, apparent viscosity, tangent flow point and cross over point in a concentration-dependent fashion, although not as significantly as polyglycerol polyricinoleate applied at a commercially relevant concentration in the same chocolate model system. Confocal laser scanning microscopy confirmed presence of the alternative flow enhancer material at the sugar surfaces. This research reveals that low-energy processing methods that do not extensively disrupt thylakoid membranes are applicable to generating materials with marked capacity to affect the flow behaviour of a chocolate model system. In conclusion, chloroplast/thylakoid materials hold strong potential as natural alternatives to synthetic rheology modifiers for lipid-based systems such as PGPR.

Functionalized Soybean Oil- and Vanillin-Based Dual Cure Photopolymerizable System for Light-Based 3D Structuring.

A novel dual cure photopolymerizable system was developed by combining two plant-derived acrylic monomers, acrylated epoxidized soybean oil and vanillin dimethacrylate, as well as the thiol monomer pentaerythritol tetrakis (3-mercaptopropionate). Carefully selected resin composition allowed the researchers to overcome earlier stability/premature polymerization problems and to obtain stable (up to six months at 4 °C) and selectively-polymerizable resin. The resin demonstrated rapid photocuring without an induction period and reached a rigidity of 317.66 MPa, which was more than 20 times higher than that of the other vanillin-based polymers. Improved mechanical properties and thermal stability of the resulting cross-linked photopolymer were obtained compared to similar homo- and copolymers: Young's modulus reached 4753 MPa, the compression modulus reached 1634 MPa, and the temperature of 10% weight loss was 373 °C. The developed photocurable system was successfully applied in stereolithography and characterized with femtosecond pulsed two-beam initiation threshold measurement for the first time. The polymerization threshold of the investigated polymer was determined to be controlled by the sample temperature, making the footprint of the workstations cheaper, faster, and more reliable.

A comparison of DNA repair pathways to achieve a site-specific gene modification of the Bruton's tyrosine kinase gene.

Gene editing utilizing homology-directed repair has advanced significantly for many monogenic diseases of the hematopoietic system in recent years but has also been hindered by decreases between in vitro and in vivo gene integration rates. Homology-directed repair occurs primarily in the S/G2 phases of the cell cycle, whereas long-term engrafting hematopoietic stem cells are typically quiescent. Alternative methods for a targeted integration have been proposed including homology-independent targeted integration and precise integration into target chromosome, which utilize non-homologous end joining and microhomology-mediated end joining, respectively. Non-homologous end joining occurs throughout the cell cycle, while microhomology-mediated end joining occurs predominantly in the S phase. We compared these pathways for the integration of a corrective DNA cassette at the Bruton's tyrosine kinase gene for the treatment of X-linked agammaglobulinemia. Homology-directed repair generated the most integration in K562 cells; however, synchronizing cells into G1 resulted in the highest integration rates with homology-independent targeted integration. Only homology-directed repair produced seamless junctions, making it optimal for targets where insertions and deletions are impermissible. Bulk CD34+ cells were best edited by homology-directed repair and precise integration into the target chromosome, while sorted hematopoietic stem cells contained similar integration rates using all corrective donors.

Real-time quantitation of thyroidal radioiodine uptake in thyroid disease with monitoring by a collar detection device.

Radioactive iodine (RAI) is safe and effective in most patients with hyperthyroidism but not all individuals are cured by the first dose, and most develop post-RAI hypothyroidism. Postoperative RAI therapy for remnant ablation is successful in 80-90% of thyroid cancer patients and sometimes induces remission of nonresectable cervical and/or distant metastatic disease but the effective tumor dose is usually not precisely known and must be moderated to avoid short- and long-term adverse effects on other tissues. The Collar Therapy Indicator (COTI) is a radiation detection device embedded in a cloth collar secured around the patient's neck and connected to a recording and data transmission box. In previously published experience, the data can be collected at multiple time points, reflecting local cervical RAI exposure and correlating well with conventional methods. We evaluated the real-time uptake of RAI in patients with hyperthyroid Graves' disease and thyroid cancer. We performed a pilot feasibility prospective study. Data were analyzed using R© (version 4.0.3, The R Foundation for Statistical Computing, 2020), and Python (version 3.6, Matplotlib version 3.0.3). The COTI was able to provide a quantitative temporal pattern of uptake within the thyroid in persons with Graves' disease and lateralized the remnant tissue in persons with thyroid cancer. The study has demonstrated that the portable collar radiation detection device outside of a healthcare facility is accurate and feasible for use after administration of RAI for diagnostic studies and therapy to provide a complete collection of fractional target radioactivity data compared to that traditionally acquired with clinic-based measurements at one or two time-points.Clinical Trials Registration NCT03517579, DOR 5/7/2018.

Biochemical characterization of protease activity of Nsp3 from SARS-CoV-2 and its inhibition by nanobodies.

Of the 16 non-structural proteins (Nsps) encoded by SARS CoV-2, Nsp3 is the largest and plays important roles in the viral life cycle. Being a large, multidomain, transmembrane protein, Nsp3 has been the most challenging Nsp to characterize. Encoded within Nsp3 is the papain-like protease domain (PLpro) that cleaves not only the viral polypeptide but also K48-linked polyubiquitin and the ubiquitin-like modifier, ISG15, from host cell proteins. We here compare the interactors of PLpro and Nsp3 and find a largely overlapping interactome. Intriguingly, we find that near full length Nsp3 is a more active protease compared to the minimal catalytic domain of PLpro. Using a MALDI-TOF based assay, we screen 1971 approved clinical compounds and identify five compounds that inhibit PLpro with IC50s in the low micromolar range but showed cross reactivity with other human deubiquitinases and had no significant antiviral activity in cellular SARS-CoV-2 infection assays. We therefore looked for alternative methods to block PLpro activity and engineered competitive nanobodies that bind to PLpro at the substrate binding site with nanomolar affinity thus inhibiting the enzyme. Our work highlights the importance of studying Nsp3 and provides tools and valuable insights to investigate Nsp3 biology during the viral infection cycle.

Optimizing Integration and Expression of Transgenic Bruton's Tyrosine Kinase for CRISPR-Cas9-Mediated Gene Editing of X-Linked Agammaglobulinemia.

X-linked agammaglobulinemia (XLA) is a monogenic primary immune deficiency characterized by very low levels of immunoglobulins and greatly increased risks for recurrent and severe infections. Patients with XLA have a loss-of-function mutation in the Bruton's tyrosine kinase (BTK) gene and fail to produce mature B lymphocytes. Gene editing in the hematopoietic stem cells of XLA patients to correct or replace the defective gene should restore B cell development and the humoral immune response. We used the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform to precisely target integration of a corrective, codon-optimized BTK complementary DNA (cDNA) cassette into its endogenous locus. This process is driven by homologous recombination and should place the transgenic BTK under transcriptional control of its endogenous regulatory elements. Each integrated copy of this cDNA in BTK-deficient K562 cells produced only 11% as much BTK protein as the wild-type gene. The donor cDNA was modified to include the terminal intron of the BTK gene. Successful integration of the intron-containing BTK donor led to a nearly twofold increase in BTK expression per cell over the base donor. However, this donor variant was too large to package into an adeno-associated viral vector for delivery into primary cells. Donors containing truncated variants of the terminal intron also produced elevated expression, although to a lesser degree than the full intron. Addition of the Woodchuck hepatitis virus posttranscriptional regulatory element led to a large boost in BTK transgene expression. Combining these modifications led to a BTK donor template that generated nearly physiological levels of BTK expression in cell lines. These reagents were then optimized to maximize integration rates into human hematopoietic stem and progenitor cells, which have reached potentially therapeutic levels in vitro. The novel donor modifications support effective gene therapy for XLA and will likely assist in the development of other gene editing-based therapies for genetic disorders.

Parkinson disease among patients treated for benign prostatic hyperplasia with α1 adrenergic receptor antagonists.

BACKGROUNDRecently the α1 adrenergic receptor antagonist terazosin was shown to activate PGK1, a possible target for the mitochondrial deficits in Parkinson disease related to its function as the initial enzyme in ATP synthesis during glycolysis. An epidemiological study of terazosin users showed a lower incidence of Parkinson disease when compared with users of tamsulosin, an α1 adrenergic receptor antagonist of a different class that does not activate PGK1. However, prior research on tamsulosin has suggested that it may in fact potentiate neurodegeneration, raising the question of whether it is an appropriate control group.METHODSTo address this question, we undertook an epidemiological study on Parkinson disease occurrence rate in 113,450 individuals from the United States with 5 or more years of follow-up. Patients were classified as tamsulosin users (n = 45,380), terazosin/alfuzosin/doxazosin users (n = 22,690), or controls matched for age, sex, and Charlson comorbidity index score (n = 45,380).RESULTSIncidence of Parkinson disease in tamsulosin users was 1.53%, which was significantly higher than that in both terazosin/alfuzosin/doxazosin users (1.10%, P < 0.0001) and matched controls (1.01%, P < 0.0001). Terazosin/alfuzosin/doxazosin users did not differ in Parkinson disease risk from matched controls (P = 0.29).CONCLUSIONThese results suggest that zosins may not confer a protective effect against Parkinson disease, but rather that tamsulosin may in some way potentiate Parkinson disease progression.FUNDINGThis work was supported by Cerevel Therapeutics.

Targeting N-myristoylation for therapy of B-cell lymphomas.

Myristoylation, the N-terminal modification of proteins with the fatty acid myristate, is critical for membrane targeting and cell signaling. Because cancer cells often have increased N-myristoyltransferase (NMT) expression, NMTs were proposed as anti-cancer targets. To systematically investigate this, we performed robotic cancer cell line screens and discovered a marked sensitivity of hematological cancer cell lines, including B-cell lymphomas, to the potent pan-NMT inhibitor PCLX-001. PCLX-001 treatment impacts the global myristoylation of lymphoma cell proteins and inhibits early B-cell receptor (BCR) signaling events critical for survival. In addition to abrogating myristoylation of Src family kinases, PCLX-001 also promotes their degradation and, unexpectedly, that of numerous non-myristoylated BCR effectors including c-Myc, NFκB and P-ERK, leading to cancer cell death in vitro and in xenograft models. Because some treated lymphoma patients experience relapse and die, targeting B-cell lymphomas with a NMT inhibitor potentially provides an additional much needed treatment option for lymphoma.

Thyroid uptake test with portable device (COTI) after 131I tracer administration: proof of concept.

COTI (collar therapy indicator) has been recently introduced for the detection of gamma rays with emphasis on thyroid investigations. The aim of this study was to test the feasibility of a prototype version of COTI including activity detectors with low sensitivity in performing thyroid uptake measurements for a large group of patients. Consequently, thyroid uptake tests were carried out for a total of 89 patients (22 males and 67 females; age: 44 ± 13 years) with thyroid cancer (n = 74), hyperthyroidism (n = 16) at 2 and 24 h after administration of 0.44-2 MBq of 131I. Eight individuals among the thyroid cancer patients were monitored up to 96 h after administration. The COTI device was equipped with two CsI (Tl) detectors, known as LoHi type, sensitive to activity ranges from 0.02 to 30 MBq of 131I. The uptake values from COTI were compared with those measured with a standard probe. It was found that the mean uptake of thyroid activity in thyroid cancer patients was 2.1 ± 1.3% at 2 h when measured with the standard probe, while it was 2.2 ± 1.2% when measured with COTI. In addition, the average uptake at 24 h after administration was 2.5 ± 3.2% and 3.2 ± 3.8% measured with COTI and the standard probe, respectively. A strong correlation was found at 24 h between the results obtained with COTI and the standard probe, while a weaker correlation was seen at 2 h. Overall, there was no significant difference between the results obtained with the standard probe and those obtained with COTI at both 2 and 24 h (Pvalue ≥ 0.05). Besides, 85% of the uptake values measured with COTI were less than those measured with the standard probe at the 24 h after administration. The average uptake value was 0.9 ± 0.8% after 96 h by COTI, and 1.4 ± 1.3% by the standard probe. Pertaining to the hyperthyroidism patients, COTI showed mean uptake values of 20 ± 16% and 23 ± 18% at 2 and 24 h, respectively. In contrast, the standard probe suggested higher mean uptake values of 26 ± 18% and 30 ± 22%, respectively. It is concluded that the prototype of COTI used in the present study has been proved to be a feasible and promising tool in thyroid investigations. It is noted, however, that the next COTI generation should include detectors equipped with collimator and energy discrimination.

In Human Autoimmunity, a Substantial Component of the B Cell Repertoire Consists of Polyclonal, Barely Mutated IgG+ve B Cells.

B cells are critical for promoting autoimmunity and the success of B cell depletion therapy in rheumatoid arthritis (RA) confirms their importance in driving chronic inflammation. Whilst disease specific autoantibodies are useful diagnostically, our understanding of the pathogenic B cell repertoire remains unclear. Defining it would lead to novel insights and curative treatments. To address this, we have undertaken the largest study to date of over 150 RA patients, utilizing next generation sequencing (NGS) to analyze up to 200,000 BCR sequences per patient. The full-length antigen-binding variable region of the heavy chain (IgGHV) of the IgG B cell receptor (BCR) were sequenced. Surprisingly, RA patients do not express particular clonal expansions of B cells at diagnosis. Rather they express a polyclonal IgG repertoire with a significant increase in BCRs that have barely mutated away from the germline sequence. This pattern remains even after commencing disease modifying therapy. These hypomutated BCRs are expressed by TNF-alpha secreting IgG+veCD27-ve B cells, that are expanded in RA peripheral blood and enriched in the rheumatoid synovium. A similar B cell repertoire is expressed by patients with Sjögren's syndrome. A rate limiting step in the initiation of autoimmunity is the activation of B cells and this data reveals that a sizeable component of the human autoimmune B cell repertoire consists of polyclonal, hypomutated IgG+ve B cells, that may play a critical role in driving chronic inflammation.

Assessment of rapeseed oil body (oleosome) lipolytic activity as an effective predictor of emulsion purity and stability.

The lipolytic activity in oil body creams as affected by recovery and washing protocols was investigated. The effect of thermal treatment on the hydrolytic activity and physical stability of fresh and aged (up to 30 days) oil body emulsions was studied. The use of alkaline pH solutions (9.5) to soak and grind rapeseeds were more effective reducing the contamination of oil body material from seed proteins/enzymes, compared with neutral pHs. Soaking and grinding seeds with a NaHCO3 solution (0.1 M, pH 9.5) yielded oil bodies with a similar composition to those prepared in urea (9 M); however, the physical stability over storage was compromised due to the presence of hydrolytic enzymes. Heating a dispersion of oil bodies for 6 mins at 95 °C did not alter the physical properties of oil bodies and significantly reduced lipolytic activity (>90% enzyme inactivation), resulting in a stable emulsion.

The effect of monovalent (Na+, K+) and divalent (Ca2+, Mg2+) cations on rapeseed oleosome (oil body) extraction and stability at pH 7.

Oleosomes are storage vehicles of TAGs in plant seeds. They are protected with a phospholipid-protein monolayer and extracted with alkaline aqueous media; however, pH adjustment intensifies the extraction process. Therefore, the aim of this work was to investigate the extraction mechanism of rapeseed oleosomes at pH 7 and at the presence of monovalent and divalent cations (Na+, K+, Mg2+, and Ca+2). The oleosome yield at pH 9.5 was 64 wt%, while the yield at pH 7 with H2O was just 43 wt.%. The presence of cations at pH 7, significantly enhanced the yield, with K+ giving the highest yield (64 wt.%). The cations affected the oleosome interface and their interactions. The presence of monovalent cations resulted in aggregation and minor coalescence, while divalent cations resulted in extensive coalescence. These results help to understand the interactions of oleosomes in their native matrix and design simple extraction processes at neutral conditions.

Burkholderia pseudomallei d-alanine-d-alanine ligase; detailed characterisation and assessment of a potential antibiotic drug target.

Burkholderia pseudomallei is a serious, difficult to treat Gram-negative pathogen and an increase in the occurrence of drug-resistant strains has been detected. We have directed efforts to identify and to evaluate potential drug targets relevant to treatment of infection by B. pseudomallei. We have selected and characterised the essential enzyme d-alanine-d-alanine ligase (BpDdl), required for the ATP-assisted biosynthesis of a peptidoglycan precursor. A recombinant supply of protein supported high-resolution crystallographic and biophysical studies with ligands (AMP and AMP+d-Ala-d-Ala), and comparisons with orthologues enzymes suggest a ligand-induced conformational change occurring that might be relevant to the catalytic cycle. The detailed biochemical characterisation of the enzyme, development and optimisation of ligand binding assays supported the search for novel inhibitors by screening of selected compound libraries. In a similar manner to that observed previously in other studies, we note a paucity of hits that are worth follow-up and then in combination with a computational analysis of the active site, we conclude that this ligase represents a difficult target for drug discovery. Nevertheless, our reagents, protocols and data can underpin future efforts exploiting more diverse chemical libraries and structure-based approaches.

Identification of inhibitors of an unconventional Trypanosoma brucei kinetochore kinase.

The discovery of 20 unconventional kinetochore proteins in Trypanosoma brucei has opened a new and interesting area of evolutionary research to study a biological process previously thought to be highly conserved in all eukaryotes. In addition, the discovery of novel proteins involved in a critical cellular process provides an opportunity to exploit differences between kinetoplastid and human kinetochore proteins to develop therapeutics for diseases caused by kinetoplastid parasites. Consequently, we identified two of the unconventional kinetochore proteins as key targets (the highly related kinases KKT10 and KKT19). Recombinant T. brucei KKT19 (TbKKT19) protein was produced, a peptide substrate phosphorylated by TbKKT19 identified (KKLRRTLSVA), Michaelis constants for KKLRRTLSVA and ATP were determined (179 µM and 102 µM respectively) and a robust high-throughput compatible biochemical assay developed. This biochemical assay was validated pharmacologically with inhibition by staurosporine and hypothemycin (IC50 values of 288 nM and 65 nM respectively). Surprisingly, a subsequent high-throughput screen of a kinase-relevant compound library (6,624 compounds) yielded few hits (8 hits; final hit rate 0.12%). The low hit rate observed was unusual for a kinase target, particularly when screened against a compound library enriched with kinase hinge binding scaffolds. In an attempt to understand the low hit rate a TbKKT19 homology model, based on human cdc2-like kinase 1 (CLK1), was generated. Analysis of the TbKKT19 sequence and structure revealed no obvious features that could explain the low hit rates. Further work will therefore be necessary to explore this unique kinetochore kinase as well as to assess whether the few hits identified can be developed into tool molecules or new drugs.

Identification of GSK3186899/DDD853651 as a Preclinical Development Candidate for the Treatment of Visceral Leishmaniasis.

The leishmaniases are diseases that affect millions of people across the world, in particular visceral leishmaniasis (VL) which is fatal unless treated. Current standard of care for VL suffers from multiple issues and there is a limited pipeline of new candidate drugs. As such, there is a clear unmet medical need to identify new treatments. This paper describes the optimization of a phenotypic hit against Leishmania donovani, the major causative organism of VL. The key challenges were to balance solubility and metabolic stability while maintaining potency. Herein, strategies to address these shortcomings and enhance efficacy are discussed, culminating in the discovery of preclinical development candidate GSK3186899/DDD853651 (1) for VL.

Discovery and Lead Optimization of Atropisomer D1 Agonists with Reduced Desensitization.

The discovery of D1 subtype-selective agonists with drug-like properties has been an enduring challenge for the greater part of 40 years. All known D1-selective agonists are catecholamines that bring about receptor desensitization and undergo rapid metabolism, thus limiting their utility as a therapeutic for chronic illness such as schizophrenia and Parkinson's disease. Our high-throughput screening efforts on D1 yielded a single non-catecholamine hit PF-4211 (6) that was developed into a series of potent D1 receptor agonist leads with high oral bioavailability and CNS penetration. An important structural feature of this series is the locked biaryl ring system resulting in atropisomerism. Disclosed herein is a summary of our hit-to-lead efforts on this series of D1 activators culminating in the discovery of atropisomer 31 (PF-06256142), a potent and selective orthosteric agonist of the D1 receptor that has reduced receptor desensitization relative to dopamine and other catechol-containing agonists.

Engineering Lineage Potency and Plasticity of Stem Cells using Epigenetic Molecules.

Stem cells are considered as a multipotent regenerative source for diseased and dysfunctional tissues. Despite the promise of stem cells, the inherent capacity of stem cells to convert to tissue-specific lineages can present a major challenge to the use of stem cells for regenerative medicine. We hypothesized that epigenetic regulating molecules can modulate the stem cell's developmental program, and thus potentially overcome the limited lineage differentiation that human stem cells exhibit based on the source and processing of stem cells. In this study, we screened a library of 84 small molecule pharmacological agents indicated in nucleosomal modification and identified a sub-set of specific molecules that influenced osteogenesis in human mesenchymal stem cells (hMSCs) while maintaining cell viability in-vitro. Pre-treatment with five candidate hits, Gemcitabine, Decitabine, I-CBP112, Chidamide, and SIRT1/2 inhibitor IV, maximally enhanced osteogenesis in-vitro. In contrast, five distinct molecules, 4-Iodo-SAHA, Scriptaid, AGK2, CI-amidine and Delphidine Chloride maximally inhibited osteogenesis. We then tested the role of these molecules on hMSCs derived from aged human donors and report that small epigenetic molecules, namely Gemcitabine and Chidamide, can significantly promote osteogenic differentiation by 5.9- and 2.3-fold, respectively. Taken together, this study demonstrates new applications of identified small molecule drugs for sensitively regulating the lineage plasticity fates of bone-marrow derived mesenchymal stem cells through modulating the epigenetic profile of the cells.