HLA class I signal peptide polymorphism determines the level of CD94/NKG2-HLA-E-mediated regulation of effector cell responses.

Human leukocyte antigen (HLA)-E binds epitopes derived from HLA-A, HLA-B, HLA-C and HLA-G signal peptides (SPs) and serves as a ligand for CD94/NKG2A and CD94/NKG2C receptors expressed on natural killer and T cell subsets. We show that among 16 common classical HLA class I SP variants, only 6 can be efficiently processed to generate epitopes that enable CD94/NKG2 engagement, which we term 'functional SPs'. The single functional HLA-B SP, known as HLA-B/-21M, induced high HLA-E expression, but conferred the lowest receptor recognition. Consequently, HLA-B/-21M SP competes with other SPs for providing epitope to HLA-E and reduces overall recognition of target cells by CD94/NKG2A, calling for reassessment of previous disease models involving HLA-B/-21M. Genetic population data indicate a positive correlation between frequencies of functional SPs in humans and corresponding cytomegalovirus mimics, suggesting a means for viral escape from host responses. The systematic, quantitative approach described herein will facilitate development of prediction algorithms for accurately measuring the impact of CD94/NKG2-HLA-E interactions in disease resistance/susceptibility.

hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50.

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

SARS-CoV-2 papain-like protease plays multiple roles in regulating cellular proteins in the endoplasmic reticulum.

Nsp3s are the largest nonstructural proteins of coronaviruses. These transmembrane proteins include papain-like proteases (PLpro) that play essential roles in cleaving viral polyproteins into their mature units. The PLpro of SARS-CoV viruses also have deubiquitinating and deISGylating activities. As Nsp3 is an endoplasmic reticulum (ER)-localized protein, we asked if the deubiquitinating activity of SARS-CoV-2 PLpro affects proteins that are substrates for ER-associated degradation (ERAD). Using full-length Nsp3 as well as a truncated transmembrane form we interrogated, by coexpression, three potential ERAD substrates, all of which play roles in regulating lipid biosynthesis. Transmembrane PLpro increases the level of INSIG-1 and decreases its ubiquitination. However, different effects were seen with SREBP-1 and SREBP-2. Transmembrane PLpro cleaves SREBP-1 at three sites, including two noncanonical sites in the N-terminal half of the protein, resulting in a decrease in precursors of the active transcription factor. Conversely, cleavage of SREBP-2 occurs at a single canonical site that disrupts a C-terminal degron, resulting in increased SREBP-2 levels. When this site is mutated and the degron can no longer be interrupted, SREBP-2 is still stabilized by transmembrane PLpro, which correlates with a decrease in SREBP-2 ubiquitination. All of these observations are dependent on PLpro catalytic activity. Our findings demonstrate that, when anchored to the ER membrane, SARS-CoV-2 Nsp3 PLpro can function as a deubiquitinating enzyme to stabilize ERAD substrates. Additionally, SARS-CoV-2 Nsp3 PLpro can cleave ER-resident proteins, including at sites that could escape analyses based on the established consensus sequence.

Fractional Quantum Hall States of the A Phase in the Second Landau Level.

A proposal of the existence of an Anomalous phase (A phase) [Das et al., Phys. Rev. Lett. 131, 056202 (2023)PRLTAO0031-900710.1103/PhysRevLett.131.056202] at the experimental range of moderate Landau-level-mixing strength has recently been made for the 5/2 state. We here report that the gapped A phase is generic to the sequence of spin-polarized fractional quantum Hall states with filling fractions ν=n/(nm-1) and ν=1-n/(nm-1), (n≥1,m≥3), that exhausts almost all the observed states and also predicts some states in the second Landau level for GaAs systems. Our proposed trial wave functions for all these states have remarkably high overlaps with the corresponding exact ground states and can support non-Abelian quasiparticle excitations with charge e/[2(nm-1)]. By analyzing edge modes, we predict experimentally verifiable thermal Hall conductance 2.5(π^{2}k_{B}^{2}T/3h) for all the states in these sequences.

The role of transcription factors in the acquisition of the four latest proposed hallmarks of cancer and corresponding enabling characteristics.

With the recent description of the molecular and cellular characteristics that enable acquisition of both core and new hallmarks of cancer, the consequences of transcription factor dysregulation in the hallmarks scheme has become increasingly evident. Dysregulation or mutation of transcription factors has long been recognized in the development of cancer where alterations in these key regulatory molecules can result in aberrant gene expression and consequential blockade of normal cellular differentiation. Here, we provide an up-to-date review of involvement of dysregulated transcription factor networks with the most recently reported cancer hallmarks and enabling characteristic properties. We present some illustrative examples of the impact of dysregulated transcription factors, specifically focusing on the characteristics of phenotypic plasticity, non-mutational epigenetic reprogramming, polymorphic microbiomes, and senescence. We also discuss how new insights into transcription factor dysregulation in cancer is contributing to addressing current therapeutic challenges.

Anomalous Reentrant 5/2 Quantum Hall Phase at Moderate Landau-Level-Mixing Strength.

A successful probing of the neutral Majorana mode in recent thermal Hall conductivity measurements opines in favor of the particle-hole symmetric Pfaffian (PH-Pf) topological order, contrasting the theoretical predictions of Pfaffian or anti-Pfaffian phases. Here we report a reentrant anomalous quantized phase that is found to be gapped in the thermodynamic limit, distinct from the conventional Pfaffian, anti-Pfaffian, or PH-Pf phases, at an intermediate strength of Landau level mixing. Our proposed wave function consistent with the PH-Pf shift in spherical geometry rightly captures the topological order of this phase, as its overlap with the exact ground state is very high and it reproduces low-lying entanglement spectra. A unique topological order, irrespective of the flux shifts, found for this phase, possibly corroborates the experimentally found topological order.

Fam60a defines a variant Sin3a-Hdac complex in embryonic stem cells required for self-renewal.

Sin3a is the central scaffold protein of the prototypical Hdac1/2 chromatin repressor complex, crucially required during early embryonic development for the growth of pluripotent cells of the inner cell mass. Here, we compare the composition of the Sin3a-Hdac complex between pluripotent embryonic stem (ES) and differentiated cells by establishing a method that couples two independent endogenous immunoprecipitations with quantitative mass spectrometry. We define the precise composition of the Sin3a complex in multiple cell types and identify the Fam60a subunit as a key defining feature of a variant Sin3a complex present in ES cells, which also contains Ogt and Tet1. Fam60a binds on H3K4me3-positive promoters in ES cells, together with Ogt, Tet1 and Sin3a, and is essential to maintain the complex on chromatin. Finally, we show that depletion of Fam60a phenocopies the loss of Sin3a, leading to reduced proliferation, an extended G1-phase and the deregulation of lineage genes. Taken together, Fam60a is an essential core subunit of a variant Sin3a complex in ES cells that is required to promote rapid proliferation and prevent unscheduled differentiation.

An Insight Into the Driver Mutations and Molecular Mechanisms Underlying Mucinous Adenocarcinoma of the Rectum.

BACKGROUND: Mucinous adenocarcinoma of the rectum accounts for 10% of all rectal cancers and has an impaired response to neoadjuvant chemoradiotherapy and worse overall survival. To date, insufficient genomic research has been performed on this histological subtype. OBJECTIVE: This study aims to define the mismatch repair deficiency rate and the driver mutations underpinning mucinous adenocarcinoma of the rectum and to compare it with rectal adenocarcinoma not otherwise specified. DESIGN: Immunohistochemistry and sequencing were performed on tumor samples from our tumor biobank. SETTINGS: This study was conducted across 2 tertiary referral centers. PATIENTS: Patients with mucinous adenocarcinoma and rectal adenocarcinoma not otherwise specified who underwent rectal resection between 2008 and 2018 were included. MAIN OUTCOME MEASURES: Mismatch repair status was performed by immunohistochemical staining. Mutations in the panel of oncogenes and tumor suppressor genes were determined by sequencing on the MiSeq V3 platform. RESULTS: The study included 33 patients with mucinous adenocarcinoma of the rectum and 100 patients with rectal adenocarcinoma not otherwise specified. Those with mucinous adenocarcinoma had a mismatch repair deficiency rate of 12.1% compared to 2.0% in the adenocarcinoma not otherwise specified cohort (p = 0.04). Mucinous adenocarcinoma and adenocarcinoma not otherwise specified rectal tumors had similar mutation frequencies in most oncogenes and tumor suppressor genes. No difference was found in the KRAS mutation rate (50.0% vs 37.1%, p = 0.29) or BRAF mutation rate (6.7% vs 3.1%, p = 0.34) between the cohorts. No difference was found between the cohorts regarding recurrence-free (p = 0.29) or overall survival (p = 0.14). LIMITATIONS: The major limitations of this study were the use of formalin-fixed, paraffin-embedded tissue over fresh-frozen tissue and the small number of patients included, in particular, in the mucinous rectal cohort. CONCLUSIONS: Most mucinous rectal tumors develop and progress along the chromosomal instability pathway. Further research in the form of transcriptomics, proteomics, and analysis of the effects of the mucin barrier may yield valuable insights into the mechanisms of resistance to chemoradiotherapy in this cohort. See Video Abstract at http://links.lww.com/DCR/B464. UNA PERCEPCIN SOBRE MUTACIONES IMPULSORAS Y MECANISMOS MOLECULARES SUBYACENTES AL ADENOCARCINOMA MUCINOSO DEL RECTO: ANTECEDENTES:El adenocarcinoma mucinoso del recto, representa el 10% de todos los cánceres rectales y tiene una respuesta deficiente a la quimioradioterapia neoadyuvante y una peor supervivencia en general. A la fecha, se han realizado muy pocas investigaciones genómicas sobre este subtipo histológico.OBJETIVO:Definir la tasa de deficiencia en la reparación de desajustes y mutaciones impulsoras, que sustentan el adenocarcinoma mucinoso del recto y compararlo con el adenocarcinoma rectal no especificado de otra manera.DISEÑO:Se realizaron inmunohistoquímica y secuenciación en muestras tumorales de nuestro biobanco de tumores.AJUSTE:El estudio se realizó en dos centros de referencia terciarios.PACIENTES:Se incluyeron pacientes con adenocarcinoma mucinoso y adenocarcinoma no especificado de otra manera, sometidos a resección rectal entre 2008 y 2018.PRINCIPALES MEDIDAS DE RESULTADO:El estado de reparación de desajustes se realizó mediante tinción inmunohistoquímica. Las mutaciones en el panel de oncogenes y genes supresores de tumores, se determinaron mediante secuenciación en la plataforma MiSeq V3.RESULTADOS:El estudio incluyó a 33 pacientes con adenocarcinoma mucinoso del recto y 100 pacientes con adenocarcinoma del recto no especificado de otra manera. Aquellos con adenocarcinoma mucinoso, tenían una tasa de deficiencia de reparación de desajustes del 12,1% en comparación con el 2,0% en la cohorte de adenocarcinoma no especificado de otra manera (p = 0,04). El adenocarcinoma mucinoso y el adenocarcinoma no especificado de otra manera, tuvieron frecuencias de mutación similares en la mayoría de los oncogenes y genes supresores de tumores. No se encontraron diferencias en la tasa de mutación de KRAS (50,0% frente a 37,1%, p = 0,29) o la tasa de mutación de BRAF (6,7% frente a 3,1%, p = 0,34) entre las cohortes. No se encontraron diferencias entre las cohortes con respecto a la supervivencia libre de recurrencia (p = 0,29) o la supervivencia global (p = 0,14).LIMITACIONES:Las mayores limitaciones de este estudio, fueron el uso de tejido embebido en parafina y fijado con formalina, sobre el tejido fresco congelado y el pequeño número de pacientes incluidos, particularmente en la cohorte mucinoso rectal.CONCLUSIONES:La mayoría de los tumores rectales mucinosos se desarrollan y progresan a lo largo de la vía de inestabilidad cromosómica. La investigación adicional en forma transcriptómica, proteómica y análisis de los efectos de la barrera de la mucina, puede proporcionar información valiosa sobre los mecanismos de resistencia a la quimioradioterapia, en esta cohorte. Consulte Video Resumen en http://links.lww.com/DCR/B464.

Decreased Antibiotic Susceptibility Driven by Global Remodeling of the Klebsiella pneumoniae Proteome.

Bacteria can circumvent the effect of antibiotics by transitioning to a poorly understood physiological state that does not involve conventional genetic elements of resistance. Here we examine antibiotic susceptibility with a Class A ß-lactamase+ invasive strain of Klebsiella pneumoniae that was isolated from a lethal outbreak within laboratory colonies of Chlorocebus aethiops sabaeus monkeys. Bacterial responses to the ribosomal synthesis inhibitors streptomycin and doxycycline resulted in distinct proteomic adjustments that facilitated decreased susceptibility to each antibiotic. Drug-specific changes to proteomes included proteins for receptor-mediated membrane transport and sugar utilization, central metabolism, and capsule production, whereas mechanisms common to both antibiotics included elevated scavenging of reactive oxygen species and turnover of misfolded proteins. Resistance to combined antibiotics presented integrated adjustments to protein levels as well as unique drug-specific proteomic features. Our results demonstrate that dampening of Klebsiella pneumoniae susceptibility involves global remodeling of the bacterial proteome to counter the effects of antibiotics and stabilize growth.

FKBPL-based peptide, ALM201, targets angiogenesis and cancer stem cells in ovarian cancer.

BACKGROUND: ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours. METHODS: In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA). RESULTS: ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC. CONCLUSION: FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.

Functional characterization of a chr13q22.1 pancreatic cancer risk locus reveals long-range interaction and allele-specific effects on DIS3 expression.

Genome-wide association studies (GWAS) have identified multiple common susceptibility loci for pancreatic cancer. Here we report fine-mapping and functional analysis of one such locus residing in a 610 kb gene desert on chr13q22.1 (marked by rs9543325). The closest candidate genes, KLF5, KLF12, PIBF1, DIS3 and BORA, range in distance from 265-586 kb. Sequencing three sub-regions containing the top ranked SNPs by imputation P-value revealed a 30 bp insertion/deletion (indel) variant that was significantly associated with pancreatic cancer risk (rs386772267, P = 2.30 × 10-11, OR = 1.22, 95% CI 1.15-1.28) and highly correlated to rs9543325 (r2 = 0.97 in the 1000 Genomes EUR population). This indel was the most significant cis-eQTL variant in a set of 222 histologically normal pancreatic tissue samples (ß = 0.26, P = 0.004), with the insertion (risk-increasing) allele associated with reduced DIS3 expression. DIS3 encodes a catalytic subunit of the nuclear RNA exosome complex that mediates RNA processing and decay, and is mutated in several cancers. Chromosome conformation capture revealed a long range (570 kb) physical interaction between a sub-region of the risk locus, containing rs386772267, and a region ∼6 kb upstream of DIS3 Finally, repressor regulatory activity and allele-specific protein binding by transcription factors of the TCF/LEF family were observed for the risk-increasing allele of rs386772267, indicating that expression regulation at this risk locus may be influenced by the Wnt signaling pathway. In conclusion, we have identified a putative functional indel variant at chr13q22.1 that associates with decreased DIS3 expression in carriers of pancreatic cancer risk-increasing alleles, and could therefore affect nuclear RNA processing and/or decay.

Abatacept reduces disease activity and ultrasound power Doppler in ACPA-negative undifferentiated arthritis: a proof-of-concept clinical and imaging study.

OBJECTIVES: No proven treatment exists for ACPA-negative undifferentiated arthritis (UA). The aim of this study was to evaluate whether abatacept is effective in treating poor prognosis, ACPA-negative UA, including its effect on power Doppler on US (PDUS). METHODS: A proof-of-concept, open-label, prospective study of 20 patients with DMARD-naïve, ACPA-negative UA (⩾2 joint synovitis) and PDUS ⩾ 1 with clinical and 20-joint US (grey scale/PDUS) assessments at baseline, 6, 12, 18 and 24 months. All patients received 12 months of abatacept (monotherapy for minimum first 6 months). The primary end point was a composite of the proportion of patients that at 6 months achieved DAS44 remission, a maximum of one swollen joint for at least 3 consecutive months and no radiographic progression (over 0-12 months). RESULTS: Twenty of the 23 patients screened were enrolled [14 female; mean (sd) age 53.4 (11.2) years, symptom duration 7.5 (0.9) months]. Two (10%) achieved the composite primary end point. A reduction in the mean (sd) DAS44 was observed from a baseline value of 2.66 (0.77) to 2.01 (0.81) at 6 months and to 1.78 (0.95) at 12 months. The DAS44 remission rates were 6/20 (30%; 95% CI: 15, 51%) at 6 months and 8/20 (40%; 95% CI: 22, 62%) at 12 months. A striking decrease in the median (interquartile range; IQR) total PDUS score was noted from 10 (4-23) at baseline to 3 (2-12) and 3 (0-5) at 6 and 12 months, respectively. CONCLUSION: This report is a first in potentially identifying an effective therapy, abatacept monotherapy, for poor-prognosis, ACPA-negative UA, supported by a clear reduction in PDUS. These data justify evaluation in a controlled study.

Differential DNA methylation profiles of coding and non-coding genes define hippocampal sclerosis in human temporal lobe epilepsy.

Temporal lobe epilepsy is associated with large-scale, wide-ranging changes in gene expression in the hippocampus. Epigenetic changes to DNA are attractive mechanisms to explain the sustained hyperexcitability of chronic epilepsy. Here, through methylation analysis of all annotated C-phosphate-G islands and promoter regions in the human genome, we report a pilot study of the methylation profiles of temporal lobe epilepsy with or without hippocampal sclerosis. Furthermore, by comparative analysis of expression and promoter methylation, we identify methylation sensitive non-coding RNA in human temporal lobe epilepsy. A total of 146 protein-coding genes exhibited altered DNA methylation in temporal lobe epilepsy hippocampus (n = 9) when compared to control (n = 5), with 81.5% of the promoters of these genes displaying hypermethylation. Unique methylation profiles were evident in temporal lobe epilepsy with or without hippocampal sclerosis, in addition to a common methylation profile regardless of pathology grade. Gene ontology terms associated with development, neuron remodelling and neuron maturation were over-represented in the methylation profile of Watson Grade 1 samples (mild hippocampal sclerosis). In addition to genes associated with neuronal, neurotransmitter/synaptic transmission and cell death functions, differential hypermethylation of genes associated with transcriptional regulation was evident in temporal lobe epilepsy, but overall few genes previously associated with epilepsy were among the differentially methylated. Finally, a panel of 13, methylation-sensitive microRNA were identified in temporal lobe epilepsy including MIR27A, miR-193a-5p (MIR193A) and miR-876-3p (MIR876), and the differential methylation of long non-coding RNA documented for the first time. The present study therefore reports select, genome-wide DNA methylation changes in human temporal lobe epilepsy that may contribute to the molecular architecture of the epileptic brain.

Repeat cycles of rituximab on clinical relapse in ANCA-associated vasculitis: identifying B cell biomarkers for relapse to guide retreatment decisions.

OBJECTIVE: To assess clinical and B cell biomarkers to predict relapse after rituximab in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) using retreatment on clinical relapse strategy. METHODS: 35 patients with AAV received treatment with 2×1000 mg rituximab, repeated on clinical relapse (up to 5 cycles). Disease activity was assessed by Birmingham Vasculitis Activity Score (BVAS) and peripheral B cell subsets using highly sensitive flow cytometry (HSFC) as previously described; both performed at baseline and every 3 months. RESULTS: Response rates were high: >83%, with median time-to-relapse of 82 weeks for cycle 1 (C1) and >54 weeks for all cycles. Prior to rituximab, AAV was characterised by naïve B-lymphopenia compared to healthy controls. This dysregulation was more marked in patients with raised C-reactive protein (CRP) (p<0.05). In C1, no clinical feature predicted relapse. However, repopulation of naïve B cell at 6 months was associated with a reduced risk of relapse (HR: 0.326, 95% 0.114 to 0.930, p=0.036). Relapse rates at 12 and 18 months were 0% and 14% with naïve repopulation at 6 months, and 31% and 54% without naïve repopulation. CONCLUSIONS: Responses to B cell depletion therapy are long-lasting and relapse post-treatment may be predicted by absence of naïve B cell repopulation at 6 months. Naïve B-lymphopenia may be a biomarker of disease activity in AAV.

Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis.

Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV). The virus expresses unique microRNAs (miRNAs), but the targets and functions of these miRNAs are not completely understood. In order to identify human targets of viral miRNAs, we measured protein expression changes caused by multiple KSHV miRNAs using pulsed stable labeling with amino acids in cell culture (pSILAC) in primary endothelial cells. This led to the identification of multiple human genes that are repressed at the protein level, but not at the miRNA level. Further analysis also identified that KSHV miRNAs can modulate activity or expression of upstream regulatory factors, resulting in suppressed activation of a protein involved in leukocyte recruitment (ICAM1) following lysophosphatidic acid treatment, as well as up-regulation of a pro-angiogenic protein (HIF1α), and up-regulation of a protein involved in stimulating angiogenesis (HMOX1). This study aids in our understanding of miRNA mechanisms of repression and miRNA contributions to viral pathogenesis.

Peptide neurotransmitters activate a cation channel complex of NALCN and UNC-80.

Several neurotransmitters act through G-protein-coupled receptors to evoke a 'slow' excitation of neurons. These include peptides, such as substance P and neurotensin, as well as acetylcholine and noradrenaline. Unlike the fast (approximately millisecond) ionotropic actions of small-molecule neurotransmitters, the slow excitation is not well understood at the molecular level, but can be mainly attributed to suppressing K(+) currents and/or activating a non-selective cation channel. The molecular identity of this cation channel has yet to be determined; similarly, how the channel is activated and its relative contribution to neuronal excitability induced by the neuropeptides are unknown. Here we show that, in the mouse hippocampal and ventral tegmental area neurons, substance P and neurotensin activate a channel complex containing NALCN and a large previously unknown protein UNC-80. The activation by substance P through TACR1 (a G-protein-coupled receptor for substance P) occurs by means of a unique mechanism: it does not require G-protein activation but is dependent on Src family kinases. These findings identify NALCN as the cation channel activated by substance P receptor, and suggest that UNC-80 and Src family kinases, rather than a G protein, are involved in the coupling from receptor to channel.

Abatacept or tocilizumab after rituximab in rheumatoid arthritis? An exploratory study suggests non-response to rituximab is associated with persistently high IL-6 and better clinical response to IL-6 blocking therapy.

OBJECTIVES: To evaluate the efficacy and safety of two different targeted approaches-abatacept or tocilizumab-after rituximab therapy in rheumatoid arthritis, and to explain observed difference in efficacy using blood and synovial studies of interleukin 6 (IL-6) and B cells in patients receiving rituximab therapy. METHODS: Consecutive series of patients who had discontinued rituximab therapy owing to inefficacy or toxicity were treated with abatacept (n=16) or tocilizumab (n=35). Clinical response and reasons for discontinuation were evaluated. Serial blood and synovial samples were obtained from a group of 57 and 25 rituximab-treated patients, respectively, and were analysed for B cells and IL-6 using flow cytometry, immunohistochemistry and quantitative real-time PCR. RESULTS: In the abatacept group, mean (SEM) Disease Activity Score in 28 joints calculated using the erythrocyte sedimentation rate (DAS28-ESR) reduced from 5.69 (0.42) at baseline to 4.94 (0.44) at 6 months (p=0.12). In the tocilizumab group: mean (SEM) DAS28- ESR reduced from 5.75 (0.21) at baseline to 3.28 (0.26) at 6 months (p<0.001). This was paralleled by a significant swollen joint count reduction in the tocilizumab (5.47 (0.70) to 2.70 (0.61), p=0.033), but not abatacept (6.23 (1.3) to 4.15 (1.2), p=0.26), group. In the synovium, despite complete depletion of B cells in 19/22 patients, IL-6 mRNA expression was not significantly reduced after rituximab. Blood B cell numbers remained low 12 months after rituximab. Serum IL-6 was raised at baseline and significantly higher in rituximab clinical non-responders (p=0.035) than responders. A significant reduction in serum IL-6 was seen in rituximab clinical responders (p=0.005) but not in non-responders (p=0.237). CONCLUSION: In patients with rheumatoid arthritis for whom rituximab therapy failed despite adequate B cell depletion, IL-6-directed therapy might be a more logical and effective treatment choice than T cell costimulation blockade. Further controlled studies investigating other possible mechanisms are needed to validate these initial findings.

Integrin beta-like 1 is regulated by DNA methylation and increased in heart failure patients.

AIMS: Dynamic alterations in cardiac DNA methylation have been implicated in the development of heart failure (HF) with evidence of ischaemic heart disease (IHD); however, there is limited research into cell specific, DNA methylation sensitive genes that are affected by dysregulated DNA methylation patterns. In this study, we aimed to identify DNA methylation sensitive genes in the ischaemic heart and elucidate their role in cardiac fibrosis. METHODS: A multi-omics integrative analysis was carried out on RNA sequencing and methylation sequencing on HF with IHD (n = 9) versus non-failing (n = 9) left ventricular tissue, which identified Integrin beta-like 1 (ITGBL1) as a gene of interest. Expression of Itgbl1 was assessed in three animal models of HF; an ischaemia-reperfusion pig model, a myocardial infarction mouse model and an angiotensin-II infused mouse model. Single nuclei RNA sequencing was carried out on heart tissue from angiotensin-II infused mice to establish the expression profile of Itgbl1 across cardiac cell populations. Subsequent in vitro analyses were conducted to elucidate a role for ITGBL1 in human cardiac fibroblasts. DNA pyrosequencing was applied to assess ITGBL1 CpG methylation status in genomic DNA from human cardiac tissue and stimulated cardiac fibroblasts. RESULTS: ITGBL1 was >2-fold up-regulated (FDR adj P = 0.03) and >10-fold hypomethylated (FDR adj P = 0.01) in human HF with IHD left ventricular tissue compared with non-failing controls. Expression of Itgbl1 was up-regulated in three isolated animal models of HF and showed conserved correlation between increased Itgbl1 and diastolic dysfunction. Single nuclei RNA sequencing highlighted that Itgbl1 is primarily expressed in cardiac fibroblasts, while functional studies elucidated a role for ITGBL1 in cardiac fibroblast migration, evident in 50% reduced 24 h fibroblast wound closure occurring subsequent to siRNA-targeted ITGBL1 knockdown. Lastly, evidence provided from DNA pyrosequencing supports the theory that differential expression of ITGBL1 is caused by DNA hypomethylation. CONCLUSIONS: ITGBL1 is a gene that is mainly expressed in fibroblasts, plays an important role in cardiac fibroblast migration, and whose expression is significantly increased in the failing heart. The mechanism by which increased ITGBL1 occurs is through DNA hypomethylation.

A structure-based designed small molecule depletes hRpn13Pru and a select group of KEN box proteins.

Proteasome subunit hRpn13 is partially proteolyzed in certain cancer cell types to generate hRpn13Pru by degradation of its UCHL5/Uch37-binding DEUBAD domain and retention of an intact proteasome- and ubiquitin-binding Pru domain. By using structure-guided virtual screening, we identify an hRpn13 binder (XL44) and solve its structure ligated to hRpn13 Pru by integrated X-ray crystallography and NMR to reveal its targeting mechanism. Surprisingly, hRpn13Pru is depleted in myeloma cells following treatment with XL44. TMT-MS experiments reveal a select group of off-targets, including PCNA clamp-associated factor PCLAF and ribonucleoside-diphosphate reductase subunit M2 (RRM2), that are similarly depleted by XL44 treatment. XL44 induces hRpn13-dependent apoptosis and also restricts cell viability by a PCLAF-dependent mechanism. A KEN box, but not ubiquitination, is required for XL44-induced depletion of PCLAF. Here, we show that XL44 induces ubiquitin-dependent loss of hRpn13Pru and ubiquitin-independent loss of select KEN box containing proteins.