Reciprocal regulatory balance within the CLEC16A-RNF41 mitophagy complex depends on an intrinsically disordered protein region.

CLEC16A is an E3 ubiquitin ligase that regulates mitochondrial quality control through mitophagy and is associated with over 20 human diseases. CLEC16A forms a complex with another E3 ligase, RNF41, and a ubiquitin-specific peptidase, USP8; however, regions that regulate CLEC16A activity or the assembly of the tripartite mitophagy regulatory complex are unknown. Here, we report that CLEC16A contains an internal intrinsically disordered protein region (IDPR) that is crucial for CLEC16A function and turnover. IDPRs lack a fixed secondary structure and possess emerging yet still equivocal roles in protein stability, interactions, and enzymatic activity. We find that the internal IDPR of CLEC16A is crucial for its degradation. CLEC16A turnover was promoted by RNF41, which binds and acts upon the internal IDPR to destabilize CLEC16A. Loss of this internal IDPR also destabilized the ubiquitin-dependent tripartite CLEC16A-RNF41-USP8 complex. Finally, the presence of an internal IDPR within CLEC16A was confirmed using NMR and CD spectroscopy. Together, our studies reveal that an IDPR is essential to control the reciprocal regulatory balance between CLEC16A and RNF41, which could be targeted to improve mitochondrial health in disease.

The Heart Renaissance.

Ancient societies believed the heart was the most important organ in the body. Ancient religions held that only through the heart could one connect with God. During Europe's Middle Ages there was little to no advances regarding the heart's workings. As the Middle Ages gave way to the Renaissance, scientists and physicians began questioning long-standing theories on the heart. The first accurate descriptions of the heart and its function were written, and the first anatomically correct representations of the heart were drawn.

Oral Ritlecitinib and Brepocitinib for Moderate-to-Severe Ulcerative Colitis: Results From a Randomized, Phase 2b Study.

BACKGROUND & AIMS: The efficacy and safety of ritlecitinib (oral JAK3/TEC family kinase inhibitor) and brepocitinib (oral TYK2/JAK1 inhibitor) as induction therapy were assessed in patients with active, moderate-to-severe ulcerative colitis. METHODS: This phase 2b, parallel-arm, double-blind umbrella study randomized patients with moderate-to-severe ulcerative colitis to receive 8-week induction therapy with ritlecitinib (20, 70, 200 mg), brepocitinib (10, 30, 60 mg), or placebo once daily. The primary endpoint was total Mayo Score (TMS) at week 8. RESULTS: Of 319 randomized patients, 317 received ritlecitinib (n = 150), brepocitinib (n = 142), or placebo (n = 25). The placebo-adjusted mean TMSs (90% confidence interval) at week 8 were -2.0 (-3.2 to -0.9), -3.9 (-5.0 to -2.7), and -4.6 (-5.8 to -3.5) for ritlecitinib 20, 70, and 200 mg, respectively (P = .003, P < .001, P < .001), and -1.8 (-2.9 to -0.7), -2.3 (-3.4 to -1.1), and -3.2 (-4.3 to -2.1) for brepocitinib 10, 30, and 60 mg, respectively (P = .009, P = .001, P < .001). Estimates (90% confidence interval) for placebo-adjusted proportions of patients with modified clinical remission at week 8 were 13.7% (0.5%-24.2%), 32.7% (20.2%-45.3%), and 36.0% (23.6%-48.6%) for ritlecitinib 20, 70, and 200 mg, respectively, and 14.6% (1.9%-25.7%), 25.5% (11.0%-38.1%), and 25.5% (11.0%-38.1%) for brepocitinib 10, 30, and 60 mg, respectively. Adverse events were mostly mild, and there were no serious cases of herpes zoster infection. Infections were observed with brepocitinib (16.9% [12.5%-23.7%]), ritlecitinib (8.7% [5.2%-13.4%]), and placebo (4.0% [0.2%-17.6%]). One death due to myocardial infarction (ritlecitinib) and 1 thromboembolic event (brepocitinib) occurred; both were considered unrelated to study drug. CONCLUSIONS: Ritlecitinib and brepocitinib induction therapies were more effective than placebo for the treatment of moderate-to-severe active ulcerative colitis, with an acceptable short-term safety profile. CLINICALTRIALS: gov number: NCT02958865.

A Novel C-C Chemoattractant Cytokine (Chemokine) Receptor 6 (CCR6) Antagonist (PF-07054894) Distinguishes between Homologous Chemokine Receptors, Increases Basal Circulating CCR6+ T Cells, and Ameliorates Interleukin-23-Induced Skin Inflammation.

Blocking chemokine receptor C-C chemoattractant cytokine (chemokine) receptor (CCR) 6-dependent T cell migration has therapeutic promise in inflammatory diseases. PF-07054894 is a novel CCR6 antagonist that blocked only CCR6, CCR7, and C-X-C chemoattractant cytokine (chemokine) receptor (CXCR) 2 in a ß-arrestin assay panel of 168 G protein-coupled receptors. Inhibition of CCR6-mediated human T cell chemotaxis by (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) was insurmountable by CCR6 ligand, C-C motif ligand (CCL) 20. In contrast, blockade of CCR7-dependent chemotaxis in human T cells and CXCR2-dependent chemotaxis in human neutrophils by PF-07054894 were surmountable by CCL19 and C-X-C motif ligand 1, respectively. [3H]-PF-07054894 showed a slower dissociation rate for CCR6 than for CCR7 and CXCR2 suggesting that differences in chemotaxis patterns of inhibition could be attributable to offset kinetics. Consistent with this notion, an analog of PF-07054894 with fast dissociation rate showed surmountable inhibition of CCL20/CCR6 chemotaxis. Furthermore, pre-equilibration of T cells with PF-07054894 increased its inhibitory potency in CCL20/CCR6 chemotaxis by 10-fold. The functional selectivity of PF-07054894 for inhibition of CCR6 relative to CCR7 and CXCR2 is estimated to be at least 50- and 150-fold, respectively. When administered orally to naïve cynomolgus monkeys, PF-07054894 increased the frequency of CCR6+ peripheral blood T cells, suggesting that blockade of CCR6 inhibited homeostatic migration of T cells from blood to tissues. PF-07054894 inhibited interleukin-23-induced mouse skin ear swelling to a similar extent as genetic ablation of CCR6. PF-07054894 caused an increase in cell surface CCR6 in mouse and monkey B cells, which was recapitulated in mouse splenocytes in vitro. In conclusion, PF-07054894 is a potent and functionally selective CCR6 antagonist that blocks CCR6-mediated chemotaxis in vitro and in vivo. SIGNIFICANCE STATEMENT: The chemokine receptor, C-C chemoattractant cytokine (chemokine) receptor 6 (CCR6) plays a key role in the migration of pathogenic lymphocytes and dendritic cells into sites of inflammation. (R)-4-((2-(((1,4-Dimethyl-1H-pyrazol-3-yl)(1-methylcyclopentyl)methyl)amino)-3,4-dioxocyclobut-1-en-1-yl)amino)-3-hydroxy-N,N-dimethylpicolinamide (PF-07054894) is a novel CCR6 small molecule antagonist that illustrates the importance of binding kinetics in achieving pharmacological potency and selectivity. Orally administered PF-07054894 blocks homeostatic and pathogenic functions of CCR6, suggesting that it is a promising therapeutic agent for the treatment of a variety of autoimmune and inflammatory diseases.

Efficacy and safety of topical brepocitinib cream for mild-to-moderate chronic plaque psoriasis: a phase IIb randomized double-blind vehicle-controlled parallel-group study.

BACKGROUND: Plaque psoriasis (PsO) is an inflammatory skin disease driven, in part, by the activation of Janus kinase (JAK) signalling pathways. OBJECTIVES: To assess the efficacy and safety of multiple doses of topical brepocitinib, a tyrosine kinase 2/JAK1 inhibitor, in participants with mild-to-moderate PsO. METHODS: This phase IIb multicentre randomized double-blind study was conducted in two stages. In stage 1, participants received one of eight treatments for 12 weeks: brepocitinib 0.1% once daily, 0.3% once or twice daily, 1.0% once or twice daily, 3.0% once daily, or vehicle once or twice daily. In stage 2, participants received brepocitinib 3.0% twice daily or vehicle twice daily. The primary endpoint was the change from baseline in Psoriasis Area and Severity Index (PASI) score at week 12, analysed using analysis of covariance. The key secondary endpoint was the proportion of participants who achieved a Physician Global Assessment response [score of clear (0) or almost clear (1) and an improvement of ≥ 2 points from baseline] at week 12. Additional secondary endpoints included the difference vs. vehicle in change from baseline in PASI, using mixed-model repeated measures, and the change from baseline in Peak Pruritus Numerical Rating Scale at week 12. Safety was monitored. RESULTS: Overall, 344 participants were randomized. Topical brepocitinib did not result in statistically significant changes compared with respective vehicle controls in the primary or key secondary efficacy endpoints for any dose group. At week 12, least squares mean change from baseline in PASI score ranged from -1.4 to -2.4 for the brepocitinib once-daily groups vs. -1.6 for vehicle once daily, and from -2.5 to -3.0 for the brepocitinib twice-daily groups vs. -2.2 for vehicle twice daily. From week 8, change from baseline in PASI score separated from vehicle in all brepocitinib twice daily groups. Brepocitinib was well tolerated, with adverse events (AEs) occurring at similar rates across groups. One participant in the brepocitinib 1.0% once-daily group developed a treatment-related AE of herpes zoster in the neck area. CONCLUSIONS: Topical brepocitinib was well tolerated but did not result in statistically significant changes compared with vehicle when administered at the doses evaluated to treat signs and symptoms of mild-to-moderate PsO.

Efficacy and safety of oral ritlecitinib for the treatment of active nonsegmental vitiligo: A randomized phase 2b clinical trial.

BACKGROUND: Vitiligo is a chronic autoimmune disorder characterized by depigmented patches of the skin. OBJECTIVE: To evaluate the efficacy and safety of ritlecitinib, an oral JAK3 (Janus kinase)/TEC (tyrosine kinase expressed in hepatocelluar carcinoma) inhibitor, in patients with active nonsegmental vitiligo in a phase 2b trial (NCT03715829). METHODS: Patients were randomized to once-daily oral ritlecitinib ± 4-week loading dose (200/50 mg, 100/50 mg, 30 mg, or 10 mg) or placebo for 24 weeks (dose-ranging period). Patients subsequently received ritlecitinib 200/50 mg daily in a 24-week extension period. The primary efficacy endpoint was percent change from baseline in Facial-Vitiligo Area Scoring Index at week 24. RESULTS: A total of 364 patients were treated in the dose-ranging period. Significant differences from placebo in percent change from baseline in Facial-Vitiligo Area Scoring Index were observed for the ritlecitinib 50 mg groups with (-21.2 vs 2.1; P < .001) or without (-18.5 vs 2.1; P < .001) a loading dose and ritlecitinib 30 mg group (-14.6 vs 2.1; P = .01). Accelerated improvement was observed after treatment with ritlecitinib 200/50 mg in the extension period (n = 187). No dose-dependent trends in treatment-emergent or serious adverse events were observed across the 48-week treatment. LIMITATIONS: Patients with stable vitiligo only were excluded. CONCLUSIONS: Oral ritlecitinib was effective and well tolerated over 48 weeks in patients with active nonsegmental vitiligo.

Ritlecitinib and brepocitinib demonstrate significant improvement in scalp alopecia areata biomarkers.

BACKGROUND: Janus kinase (JAK) inhibitors have shown encouraging results in the treatment of alopecia areata (AA), an autoimmune form of hair loss, in small, uncontrolled studies and case reports. OBJECTIVE: We conducted a biopsy substudy during the randomized, double-blind, placebo-controlled first 24 weeks of a phase 2a clinical trial that evaluated the efficacy and safety of ritlecitinib, an inhibitor of JAK3 and the tyrosine kinase expressed in hepatocellular carcinoma (TEC) kinase family, and brepocitinib, an inhibitor of tyrosine kinase 2 (TYK2)/JAK1 in the treatment of AA. METHODS: Change in biomarkers in lesional scalp biopsy samples between baseline and weeks 12 and 24 was an exploratory end point, and 46 patients participated from the ritlecitinib (n = 18), brepocitinib (n = 16), and placebo (n = 12) groups. Correlations of biomarkers with hair regrowth, measured using the Severity of Alopecia Tool (SALT) score, were also evaluated. CLINICAL TRIAL REGISTRATION: NCT02974868. RESULTS: At week 24, both ritlecitinib and brepocitinib demonstrated improvement exceeding 100% in the lesional scalp transcriptome toward a nonlesional profile. At week 12, the improvements in scalp tissue were greater with brepocitinib than ritlecitinib; however, at week 24, the improvements were greater with ritlecitinib. CONCLUSIONS: For both ritlecitinib and brepocitinib, improvement in the SALT scores was positively associated with expression of TH1 markers and negatively associated with expression of hair keratins. Larger, long-term clinical trials are warranted.

Efficacy and safety of topical brepocitinib for the treatment of mild-to-moderate atopic dermatitis: a phase IIb, randomized, double-blind, vehicle-controlled, dose-ranging and parallel-group study.

BACKGROUND: Atopic dermatitis (AD) is a prevalent inflammatory, pruritic skin disease. The Janus kinase (JAK) pathway is a treatment target. OBJECTIVES: To assess the efficacy, safety and pharmacokinetics of topical cream brepocitinib, a small-molecule tyrosine kinase 2 (TYK2)/JAK1 inhibitor, in participants with mild-to-moderate AD. METHODS: In this phase IIb, double-blind, dose-ranging study, participants were randomized to receive one of eight treatments for 6 weeks: brepocitinib 0·1% once daily (QD), 0·3% QD or twice daily (BID), 1·0% QD or BID, 3·0% QD, or vehicle QD or BID. The primary endpoint was the percentage change from baseline in the Eczema Area and Severity Index (EASI) total score at week 6. Adverse events (AEs) were monitored. RESULTS: Overall, 292 participants were enrolled and randomized. The brepocitinib 1% QD and 1% BID groups achieved statistically significantly greater (with multiplicity-adjusted P < 0·05 due to Hochberg's step-up method) percentage reductions from baseline in EASI total score at week 6 [least squares mean (90% confidence interval, CI): QD: -70·1 (-82·1 to -58·0); BID: -75·0 (-83·8 to -66·2)] compared with respective vehicle [QD: -44·4 (-57·3 to -31·6); BID: -47·6 (-57·5 to -37·7)]. There was not a dose-dependent trend in AE frequency, and there were no serious AEs or deaths. CONCLUSIONS: Topical brepocitinib is effective and well tolerated in participants with mild-to-moderate AD. What is already known about this topic? Janus kinase (JAK) inhibitors are in development for treatment of atopic dermatitis (AD). The tyrosine kinase 2 and JAK 1 inhibition by brepocitinib may bring a new profile for topical JAK inhibitors for treatment of mild-to-moderate AD. What does this study add? Topical brepocitinib can provide rapid, effective symptom reduction, and could offer a novel alternative to current topical treatments for mild-to-moderate AD.

Oral tyrosine kinase 2 inhibitor PF-06826647 demonstrates efficacy and an acceptable safety profile in participants with moderate-to-severe plaque psoriasis in a phase 2b, randomized, double-blind, placebo-controlled study.

BACKGROUND: Psoriasis treatments lack durable efficacy and have inconvenient administration, highlighting the need for new therapies. OBJECTIVE: To evaluate the efficacy and safety of tyrosine kinase 2 inhibitor, PF-06826647, in moderate-to-severe plaque psoriasis. METHODS: This phase 2b, double-blind study randomized participants to oral, once-daily PF-06826647 (1:1:2:2:2) 50:100:200:400 mg:placebo (16 weeks), then 200 or 400 mg (24 weeks) (NCT03895372). The primary end point was a proportion of participants achieving psoriasis area severity index (PASI) 90 at week 16. Secondary end points (PASI50/75/90/100; Physician's Global Assessment) and safety were assessed to week 40. RESULTS: Overall, 178 participants were treated. A significantly greater proportion of participants (risk difference % [90% CI]) achieved PASI90 in the 200-mg (33.0 [18.0, 47.1], P = .0004) and 400-mg (46.5 [30.6, 60.6], P < .0001; week 16) groups versus placebo. Significant increases from placebo were observed for all secondary end points (200 and 400 mg; weeks 6-16; P < .05); increases were evident to week 40 (categorical data). PF-06826647 was well tolerated and most treatment-emergent adverse events were mild/moderate. Eighteen participants discontinued due to treatment-emergent adverse events (14 arising from laboratory abnormalities). LIMITATIONS: Limitations included the large proportion of White males and non-placebo-controlled extension. CONCLUSION: PF-06826647 200 and 400 mg once daily showed significant efficacy versus placebo at week 16 and was well tolerated over 40 weeks.

Urine Tenofovir Levels Measured Using a Novel Immunoassay Predict Human Immunodeficiency Virus Protection.

New tools are needed to support pre-exposure prophylaxis (PrEP) adherence for human immunodeficiency virus (HIV) prevention, including those that enable real-time feedback. In a large, completed PrEP trial, adequate urine tenofovir levels measured using a novel immunoassay predicted HIV protection and showed good sensitivity and specificity for detectable plasma tenofovir.

Anti-TL1A Antibody PF-06480605 Safety and Efficacy for Ulcerative Colitis: A Phase 2a Single-Arm Study.

BACKGROUND & AIMS: An immune component of inflammatory bowel disease is up-regulated tumor necrosis factor-like ligand 1A (TL1A). Anti-TL1A antibodies such as PF-06480605, a fully human immunoglobulin G1 monoclonal antibody, may have therapeutic potential. METHODS: This Phase 2a, multicenter, single-arm, open-label study (TUSCANY) evaluated safety, tolerability, efficacy, pharmacokinetics, and immunogenicity in PF-06480605-treated participants with moderate to severe ulcerative colitis (UC). Participants received 500 mg intravenous PF-06480605 every 2 weeks, 7 doses total, with a 3-month follow-up period. Primary safety and efficacy endpoints were the incidence of adverse events (AEs) and week 14 endoscopic improvement (EI) (Mayo endoscopic subscore = 0 or 1), respectively. Secondary endpoints included total soluble TL1A (free/drug-bound) (sTL1A), incidence of anti-drug and neutralizing antibodies, PF-06480605 concentrations, and changes in fecal calprotectin and high-sensitivity C-reactive protein. Histology was assessed at week 14. RESULTS: The study enrolled 50 participants; 42 completed. Of 109 treatment-emergent AEs, 18 were treatment-related. The most common AEs were UC disease exacerbation and arthralgia (6 participants each). Four serious AEs, no deaths, and no malignancies were reported. Week 14 EI was observed in a statistically significant proportion of participants (38.2% [uniformly minimum-variance unbiased estimator, per protocol population]). Minimal histologic disease was observed after treatment (Robarts Histopathology Index ≤5: 33.3%; Geboes Index ≤3.2: 47.6%). sTL1A increase over time from baseline indicated sustained target engagement. Forty-one participants (82%) tested positive for anti-drug antibodies and 5 (10%) for neutralizing antibodies. CONCLUSIONS: PF-06480605 demonstrated an acceptable safety profile and statistically significant EI in participants with moderate to severe UC, warranting further study in a larger participant cohort. Tissue histopathology analyses support this conclusion. TRIAL REGISTRATION NUMBER: https://clinicaltrials.gov/NCT02840721.

A phase 2a randomized, placebo-controlled study to evaluate the efficacy and safety of the oral Janus kinase inhibitors ritlecitinib and brepocitinib in alopecia areata: 24-week results.

BACKGROUND: Alopecia areata (AA) is an autoimmune form of hair loss with limited treatments. OBJECTIVE: To evaluate the efficacy and safety of the Janus kinase inhibitors ritlecitinib and brepocitinib in patients who have AA with ≥ 50% scalp hair loss. METHODS: Patients were randomized to once-daily ritlecitinib, brepocitinib, or placebo. The primary efficacy endpoint was a 24-week change from baseline in the Severity of Alopecia Tool (SALT) score. The key secondary efficacy endpoint was the proportion of patients achieving 30% improvement in SALT score (SALT30). RESULTS: The ritlecitinib, brepocitinib, and placebo groups included 48, 47, and 47 patients, respectively. At week 24, least-squares mean difference from placebo in SALT score change from baseline was 31.1 (95% confidence interval [CI], 18.8-43.5) for ritlecitinib and 49.2 (95% CI, 36.6-61.7) for brepocitinib (P < .0001 for both comparisons with placebo). SALT30 was achieved by 50% (90% CI, 38%-62%) of patients receiving ritlecitinib, 64% (90% CI, 51%-75%) receiving brepocitinib, and 2% (90% CI, 0%-9%) receiving placebo. Two patients experienced a serious adverse event (rhabdomyolysis) in the brepocitinib group only. LIMITATIONS: Only a single-dosage regimen of each study drug was included. CONCLUSION: Treatment with ritlecitinib or brepocitinib for 24 weeks was efficacious and generally well tolerated.

Targeted Delivery of Cell Softening Micelles to Schlemm's Canal Endothelial Cells for Treatment of Glaucoma.

Increased stiffness of the Schlemm's canal (SC) endothelium in the aqueous humor outflow pathways has been associated with elevated intraocular pressure (IOP) in glaucoma. Novel treatments that relax this endothelium, such as actin depolymerizers and rho kinase inhibitors, are in development. Unfortunately, these treatments have undesirable off-target effects and a lower than desired potency. To address these issues, a targeted PEG-b-PPS micelle loaded with actin depolymerizer latrunculin A (tLatA-MC) is developed. Targeting of SC cells is achieved by modifying the micelle surface with a high affinity peptide that binds the VEGFR3/FLT4 receptor, a lymphatic lineage marker found to be highly expressed by SC cells relative to other ocular cells. During in vitro optimization, increasing the peptide surface density increased micellar uptake in SC cells while unexpectedly decreasing uptake by human umbilical vein endothelial cells (HUVEC). The functional efficacy of tLatA-MC, as measured by decreased SC cell stiffness compared to non-targeted micelles (ntLatA-MC) or targeted blank micelles (tBL-MC), is verified using atomic force microscopy. tLatA-MC reduced IOP in an in vivo mouse model by 30-50%. The results validate the use of a cell-softening nanotherapy to selectively modulate stiffness of SC cells for therapeutic reduction of IOP and treatment of glaucoma.

On the Need to Develop Guidelines for Characterizing and Reporting Intrinsic Disorder in Proteins.

Since the early 2000s, numerous computational tools have been created and used to predict intrinsic disorder in proteins. At present, the output from these algorithms is difficult to interpret in the absence of standards or references for comparison. There are many reasons to establish a set of standard-based guidelines to evaluate computational protein disorder predictions. This viewpoint explores a handful of these reasons, including standardizing nomenclature to improve communication, rigor and reproducibility, and making it easier for newcomers to enter the field. An approach for reporting predicted disorder in single proteins with respect to whole proteomes is discussed. The suggestions are not intended to be formulaic; they should be viewed as a starting point to establish guidelines for interpreting and reporting computational protein disorder predictions.

QuickMIRSeq: a pipeline for quick and accurate quantification of both known miRNAs and isomiRs by jointly processing multiple samples from microRNA sequencing.

BACKGROUND: Genome-wide miRNA expression data can be used to study miRNA dysregulation comprehensively. Although many open-source tools for microRNA (miRNA)-seq data analyses are available, challenges remain in accurate miRNA quantification from large-scale miRNA-seq dataset. We implemented a pipeline called QuickMIRSeq for accurate quantification of known miRNAs and miRNA isoforms (isomiRs) from multiple samples simultaneously. RESULTS: QuickMIRSeq considers the unique nature of miRNAs and combines many important features into its implementation. First, it takes advantage of high redundancy of miRNA reads and introduces joint mapping of multiple samples to reduce computational time. Second, it incorporates the strand information in the alignment step for more accurate quantification. Third, reads potentially arising from background noise are filtered out to improve the reliability of miRNA detection. Fourth, sequences aligned to miRNAs with mismatches are remapped to a reference genome to further reduce false positives. Finally, QuickMIRSeq generates a rich set of QC metrics and publication-ready plots. CONCLUSIONS: The rich visualization features implemented allow end users to interactively explore the results and gain more insights into miRNA-seq data analyses. The high degree of automation and interactivity in QuickMIRSeq leads to a substantial reduction in the time and effort required for miRNA-seq data analysis.

Loss of the Ubiquitin-conjugating Enzyme UBE2W Results in Susceptibility to Early Postnatal Lethality and Defects in Skin, Immune, and Male Reproductive Systems.

UBE2W ubiquitinates N termini of proteins rather than internal lysine residues, showing a preference for substrates with intrinsically disordered N termini. The in vivo functions of this intriguing E2, however, remain unknown. We generated Ube2w germ line KO mice that proved to be susceptible to early postnatal lethality without obvious developmental abnormalities. Although the basis of early death is uncertain, several organ systems manifest changes in Ube2w KO mice. Newborn Ube2w KO mice often show altered epidermal maturation with reduced expression of differentiation markers. Mirroring higher UBE2W expression levels in testis and thymus, Ube2w KO mice showed a disproportionate decrease in weight of these two organs (~50%), suggesting a functional role for UBE2W in the immune and male reproductive systems. Indeed, Ube2w KO mice displayed sustained neutrophilia accompanied by increased G-CSF signaling and testicular vacuolation associated with decreased fertility. Proteomic analysis of a vulnerable organ, presymptomatic testis, showed a preferential accumulation of disordered proteins in the absence of UBE2W, consistent with the view that UBE2W preferentially targets disordered polypeptides. These mice further allowed us to establish that UBE2W is ubiquitously expressed as a single isoform localized to the cytoplasm and that the absence of UBE2W does not alter cell viability in response to various stressors. Our results establish that UBE2W is an important, albeit not essential, protein for early postnatal survival and normal functioning of multiple organ systems.

Efficacy and safety of an interleukin 6 monoclonal antibody for the treatment of systemic lupus erythematosus: a phase II dose-ranging randomised controlled trial.

OBJECTIVES: This phase II trial evaluated the efficacy and safety of an interleukin (IL) 6 monoclonal antibody for systemic lupus erythematosus (SLE). METHODS: Patients with active disease were randomised to placebo or PF-04236921 10 mg, 50 mg or 200 mg, subcutaneously, every 8 weeks with stable background therapy. SLE Responder Index (SRI-4; primary end point) and British Isles Lupus Assessment Group-based Composite Lupus Assessment (BICLA) were assessed at week 24. Post hoc analysis identified an enriched population based upon planned univariate analyses. RESULTS: 183 patients received treatment (placebo, n=45; 10 mg, n=45; 50 mg, n=47; 200 mg, n=46). The 200 mg dose was discontinued due to safety findings and not included in the primary efficacy analysis. The SRI-4 response rates were not significant for any dose compared with placebo; however, the BICLA response rate was significant for 10 mg (p=0.026). The incidence of severe flares was significantly reduced with 10 mg (n=0) and 50 mg (n=2) combined versus placebo (n=8; p<0.01). In patients with greater baseline disease activity (enriched population), the SRI-4 (p=0.004) and BICLA (p=0.012) response rates were significantly different with 10 mg versus placebo. Four deaths (200 mg, n=3; 10 mg, n=1) occurred. The most frequently reported adverse events included headache, nausea and diarrhoea. CONCLUSIONS: PF-04236921 was not significantly different from placebo for the primary efficacy end point in patients with SLE. Evidence of an effect with 10 mg was seen in a post hoc analysis. Safety was acceptable for doses up to 50 mg as the 200 mg dose was discontinued due to safety findings. TRIAL REGISTRATION NUMBER: NCT01405196; Pre-results.

QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization.

BACKGROUND: RNA sequencing (RNA-seq), a next-generation sequencing technique for transcriptome profiling, is being increasingly used, in part driven by the decreasing cost of sequencing. Nevertheless, the analysis of the massive amounts of data generated by large-scale RNA-seq remains a challenge. Multiple algorithms pertinent to basic analyses have been developed, and there is an increasing need to automate the use of these tools so as to obtain results in an efficient and user friendly manner. Increased automation and improved visualization of the results will help make the results and findings of the analyses readily available to experimental scientists. RESULTS: By combing the best open source tools developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we have implemented QuickRNASeq, a pipeline for large-scale RNA-seq data analyses and visualization. The QuickRNASeq workflow consists of three main steps. In Step #1, each individual sample is processed, including mapping RNA-seq reads to a reference genome, counting the numbers of mapped reads, quality control of the aligned reads, and SNP (single nucleotide polymorphism) calling. Step #1 is computationally intensive, and can be processed in parallel. In Step #2, the results from individual samples are merged, and an integrated and interactive project report is generated. All analyses results in the report are accessible via a single HTML entry webpage. Step #3 is the data interpretation and presentation step. The rich visualization features implemented here allow end users to interactively explore the results of RNA-seq data analyses, and to gain more insights into RNA-seq datasets. In addition, we used a real world dataset to demonstrate the simplicity and efficiency of QuickRNASeq in RNA-seq data analyses and interactive visualizations. The seamless integration of automated capabilites with interactive visualizations in QuickRNASeq is not available in other published RNA-seq pipelines. CONCLUSION: The high degree of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption.