Intratumoral follicular regulatory T cells curtail anti-PD-1 treatment efficacy.

Immune-checkpoint blockade (ICB) has shown remarkable clinical success in boosting antitumor immunity. However, the breadth of its cellular targets and specific mode of action remain elusive. We find that tumor-infiltrating follicular regulatory T (TFR) cells are prevalent in tumor tissues of several cancer types. They are primarily located within tertiary lymphoid structures and exhibit superior suppressive capacity and in vivo persistence as compared with regulatory T cells, with which they share a clonal and developmental relationship. In syngeneic tumor models, anti-PD-1 treatment increases the number of tumor-infiltrating TFR cells. Both TFR cell deficiency and the depletion of TFR cells with anti-CTLA-4 before anti-PD-1 treatment improve tumor control in mice. Notably, in a cohort of 271 patients with melanoma, treatment with anti-CTLA-4 followed by anti-PD-1 at progression was associated with better a survival outcome than monotherapy with anti-PD-1 or anti-CTLA-4, anti-PD-1 followed by anti-CTLA-4 at progression or concomitant combination therapy.

Probing single electrons across 300-mm spin qubit wafers.

Building a fault-tolerant quantum computer will require vast numbers of physical qubits. For qubit technologies based on solid-state electronic devices1-3, integrating millions of qubits in a single processor will require device fabrication to reach a scale comparable to that of the modern complementary metal-oxide-semiconductor (CMOS) industry. Equally important, the scale of cryogenic device testing must keep pace to enable efficient device screening and to improve statistical metrics such as qubit yield and voltage variation. Spin qubits1,4,5 based on electrons in Si have shown impressive control fidelities6-9 but have historically been challenged by yield and process variation10-12. Here we present a testing process using a cryogenic 300-mm wafer prober13 to collect high-volume data on the performance of hundreds of industry-manufactured spin qubit devices at 1.6 K. This testing method provides fast feedback to enable optimization of the CMOS-compatible fabrication process, leading to high yield and low process variation. Using this system, we automate measurements of the operating point of spin qubits and investigate the transitions of single electrons across full wafers. We analyse the random variation in single-electron operating voltages and find that the optimized fabrication process leads to low levels of disorder at the 300-mm scale. Together, these results demonstrate the advances that can be achieved through the application of CMOS-industry techniques to the fabrication and measurement of spin qubit devices.

Tissue-resident memory features are linked to the magnitude of cytotoxic T cell responses in human lung cancer.

Therapies that boost the anti-tumor responses of cytotoxic T lymphocytes (CTLs) have shown promise; however, clinical responses to the immunotherapeutic agents currently available vary considerably, and the molecular basis of this is unclear. We performed transcriptomic profiling of tumor-infiltrating CTLs from treatment-naive patients with lung cancer to define the molecular features associated with the robustness of anti-tumor immune responses. We observed considerable heterogeneity in the expression of molecules associated with activation of the T cell antigen receptor (TCR) and of immunological-checkpoint molecules such as 4-1BB, PD-1 and TIM-3. Tumors with a high density of CTLs showed enrichment for transcripts linked to tissue-resident memory cells (TRM cells), such as CD103, and CTLs from CD103hi tumors displayed features of enhanced cytotoxicity. A greater density of TRM cells in tumors was predictive of a better survival outcome in lung cancer, and this effect was independent of that conferred by CTL density. Here we define the 'molecular fingerprint' of tumor-infiltrating CTLs and identify potentially new targets for immunotherapy.

Resetting transcription factor control circuitry toward ground-state pluripotency in human.

Current human pluripotent stem cells lack the transcription factor circuitry that governs the ground state of mouse embryonic stem cells (ESC). Here, we report that short-term expression of two components, NANOG and KLF2, is sufficient to ignite other elements of the network and reset the human pluripotent state. Inhibition of ERK and protein kinase C sustains a transgene-independent rewired state. Reset cells self-renew continuously without ERK signaling, are phenotypically stable, and are karyotypically intact. They differentiate in vitro and form teratomas in vivo. Metabolism is reprogrammed with activation of mitochondrial respiration as in ESC. DNA methylation is dramatically reduced and transcriptome state is globally realigned across multiple cell lines. Depletion of ground-state transcription factors, TFCP2L1 or KLF4, has marginal impact on conventional human pluripotent stem cells but collapses the reset state. These findings demonstrate feasibility of installing and propagating functional control circuitry for ground-state pluripotency in human cells.

Determining the Impact of Roller Compaction Processing Conditions on Granulate and API Properties: Impact of Formulation API Load.

Previous work demonstrated that roller compaction of a 40%w/w theophylline-loaded formulation resulted in granulate consisting of un-compacted fractions which were shown to constitute between 34 and 48%v/v of the granulate dependent on processing conditions. The active pharmaceutical ingredient (API) primary particle size within the un-compacted fraction was also shown to have undergone notable size reduction. The aim of the current work was to test the hypothesis that the observations may be more indicative of the relative compactability of the API due to the formulation being above the percolation threshold. This was done by assessing the impact of varied API loads in the formulation on the non-granulated fraction of the final granulate and the extent of attrition of API particles within the non-granulated fraction. The influence of processing conditions for all formulations was also investigated. The results verify that the observations, both of this study and the previous work, are not a consequence of exceeding the percolation threshold. The volume of un-compacted material within the granulate samples was observed to range between 34.7 and 65.5% depending on the API load and roll pressure, whilst the API attrition was equivalent across all conditions.

Phase 1 study of GSK3368715, a type I PRMT inhibitor, in patients with advanced solid tumors.

BACKGROUND: GSK3368715, a first-in-class, reversible inhibitor of type I protein methyltransferases (PRMTs) demonstrated anticancer activity in preclinical studies. This Phase 1 study (NCT03666988) evaluated safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of GSK3368715 in adults with advanced-stage solid tumors. METHODS: In part 1, escalating doses of oral once-daily GSK3368715 (50, 100, and 200 mg) were evaluated. Enrollment was paused at 200 mg following a higher-than-expected incidence of thromboembolic events (TEEs) among the first 19 participants, resuming under a protocol amendment starting at 100 mg. Part 2 (to evaluate preliminary efficacy) was not initiated. RESULTS: Dose-limiting toxicities were reported in 3/12 (25%) patients at 200 mg. Nine of 31 (29%) patients across dose groups experienced 12 TEEs (8 grade 3 events and 1 grade 5 pulmonary embolism). Best response achieved was stable disease, occurring in 9/31 (29%) patients. Following single and repeat dosing, GSK3368715 maximum plasma concentration was reached within 1 h post dosing. Target engagement was observed in the blood, but was modest and variable in tumor biopsies at 100 mg. CONCLUSION: Based on higher-than-expected incidence of TEEs, limited target engagement at lower doses, and lack of observed clinical efficacy, a risk/benefit analysis led to early study termination. TRIAL REGISTRATION NUMBER: NCT03666988.

Optimal Management of Asymptomatic Carotid Artery Stenosis: A Systematic Review and Network Meta-Analysis.

OBJECTIVE: Management of asymptomatic carotid artery stenosis (ACAS), including carotid endarterectomy (CEA), carotid artery stenting (CAS), and best medical treatment (BMT), remains inconsistent in current practice. Early studies reported a benefit of CEA vs. BMT; however, the current risk-benefit profile of invasive therapy lacks consensus. By evaluating the effects of modern BMT vs. invasive intervention on patient outcomes, this study aimed to influence the future management of ACAS. METHODS: A systematic review and series of network meta-analyses were performed assessing peri-operative (within 30 days) and long term (30 days - 5 years) stroke and mortality risk between ACAS interventions. Total stroke, major, minor, ipsilateral, and contralateral stroke subtypes were assessed independently. Traditional (pre-2000) and modern (post-2000) BMT were compared to assess clinical improvements in medical therapy over the previous two decades. Risks of myocardial infarction (MI) and cranial nerve injury (CNI) were also assessed. RESULTS: Seventeen reports of 14 310 patients with > 50% ACAS were included. CEA reduced the odds of a peri-operative stroke event occurring vs. CAS (odds ratio [OR] 1.6, 95% confidence interval [CI] 1.1 - 2.2 [0 - 20 fewer/1 000]). CEA and CAS reduced the long term odds of minor strokes (OR 0.35, 95% CI 0.21 - 0.59 [20 fewer/1 000]) and ipsilateral strokes (OR 0.27, 95% CI 0.19 - 0.39 [30 fewer/1 000]) vs. all BMT. CEA reduced the odds of major strokes and combined stroke and mortality vs. traditional BMT; however, no difference was found between CEA and modern BMT. CAS reduced the odds of peri-operative MI (OR 0.49, 95% CI 0. 26 - 0.91) and CNI (OR 0.07, 95% CI 0.01 - 0.42) vs. CEA. CONCLUSION: Modern BMT demonstrates similar reductions in major stroke, combined stroke, and mortality to CEA. The overall risk reductions are low and data were unavailable to assess subgroups which may benefit from intervention. However, BMT carries the potential to reduce the requirement for surgical intervention in patients with ACAS.

Social anxiety, behavioural activation and depression risk in older men: protection through Men's Shed membership.

Reducing rates of depressive symptoms in older adults is a public health priority. Men's Sheds are a community organisation that may protect against depressive symptoms in older men. It is currently unclear how social anxiety and behavioural activation may relate to depressive symptoms for Men's Shed members. We employed a cross-sectional design to explore whether the relationships between social anxiety, behavioural activation and depressive symptoms were contingent upon Shed social network quality in a sample of 164 Men's Shed members. Conditional effects analysis found social anxiety (B = -0.08, p < 0.01) and behavioural activation's (B = 0.02, p < 0.001) relationships with depression to be contingent on Shed social network quality. Additionally, we found evidence for a conditional effect of social anxiety on the relationship between behavioural activation and depression (B = -0.03, p < 0.01) such that this relationship was stronger for those with higher levels of social anxiety. Our findings suggest that a strong social network within a Men's Shed weakens the association between social anxiety and depression, that the relationship between behavioural activation and depression is stronger in those with poorer Shed social networks, and that the relationship between behavioural activation and depression may be stronger for those with higher levels of social anxiety. We suggest that our findings contribute to increasing quantitative support for the mental health benefits of Men's Shed membership, highlight the potential importance of Shed social network quality and explore how social anxiety may affect the mental health outcomes for members.

Mechanistic Modeling of In Vitro Skin Permeation and Extrapolation to In Vivo for Topically Applied Metronidazole Drug Products Using a Physiologically Based Pharmacokinetic Model.

Physiologically based pharmacokinetic (PBPK) modeling has increasingly been employed in dermal drug development and regulatory assessment, providing a framework to integrate relevant information including drug and drug product attributes, skin physiology parameters, and population variability. The current study aimed to develop a stepwise modeling workflow with knowledge gained from modeling in vitro skin permeation testing (IVPT) to describe in vivo exposure of metronidazole locally in the stratum corneum following topical application of complex semisolid drug products. The initial PBPK model of metronidazole in vitro skin permeation was developed using infinite and finite dose aqueous metronidazole solution. Parameters such as stratum corneum lipid-water partition coefficient (Ksclip/water) and stratum corneum lipid diffusion coefficient (Dsclip) of metronidazole were optimized using IVPT data from simple aqueous solutions (infinite) and MetroGel (10 mg/cm2 dose application), respectively. The optimized model, when parameterized with physical and structural characteristics of the drug products, was able to accurately predict the mean cumulative amount permeated (cm2/h) and flux (µg/cm2/h) profiles of metronidazole following application of different doses of MetroGel and MetroCream. Thus, the model was able to capture the impact of differences in drug product microstructure and metamorphosis of the dosage form on in vitro metronidazole permeation. The PBPK model informed by IVPT study data was able to predict the metronidazole amount in the stratum corneum as reported in clinical studies. In summary, the proposed model provides an enhanced understanding of the potential impact of drug product attributes in influencing in vitro skin permeation of metronidazole. Key kinetic parameters derived from modeling the metronidazole IVPT data improved the predictions of the developed PBPK model of in vivo local metronidazole concentrations in the stratum corneum. Overall, this work improves our confidence in the proposed workflow that accounts for drug product attributes and utilizes IVPT data toward improving predictions from advanced modeling and simulation tools.

Highly parallel direct RNA sequencing on an array of nanopores.

Sequencing the RNA in a biological sample can unlock a wealth of information, including the identity of bacteria and viruses, the nuances of alternative splicing or the transcriptional state of organisms. However, current methods have limitations due to short read lengths and reverse transcription or amplification biases. Here we demonstrate nanopore direct RNA-seq, a highly parallel, real-time, single-molecule method that circumvents reverse transcription or amplification steps. This method yields full-length, strand-specific RNA sequences and enables the direct detection of nucleotide analogs in RNA.

Epigenetic resetting of human pluripotency.

Much attention has focussed on the conversion of human pluripotent stem cells (PSCs) to a more naïve developmental status. Here we provide a method for resetting via transient histone deacetylase inhibition. The protocol is effective across multiple PSC lines and can proceed without karyotype change. Reset cells can be expanded without feeders with a doubling time of around 24 h. WNT inhibition stabilises the resetting process. The transcriptome of reset cells diverges markedly from that of primed PSCs and shares features with human inner cell mass (ICM). Reset cells activate expression of primate-specific transposable elements. DNA methylation is globally reduced to a level equivalent to that in the ICM and is non-random, with gain of methylation at specific loci. Methylation imprints are mostly lost, however. Reset cells can be re-primed to undergo tri-lineage differentiation and germline specification. In female reset cells, appearance of biallelic X-linked gene transcription indicates reactivation of the silenced X chromosome. On reconversion to primed status, XIST-induced silencing restores monoallelic gene expression. The facile and robust conversion routine with accompanying data resources will enable widespread utilisation, interrogation, and refinement of candidate naïve cells.

Effect of Quantum Hall Edge Strips on Valley Splitting in Silicon Quantum Wells.

We determine the energy splitting of the conduction-band valleys in two-dimensional electrons confined to low-disorder Si quantum wells. We probe the valley splitting dependence on both perpendicular magnetic field B and Hall density by performing activation energy measurements in the quantum Hall regime over a large range of filling factors. The mobility gap of the valley-split levels increases linearly with B and is strikingly independent of Hall density. The data are consistent with a transport model in which valley splitting depends on the incremental changes in density eB/h across quantum Hall edge strips, rather than the bulk density. Based on these results, we estimate that the valley splitting increases with density at a rate of 116 µeV/10^{11} cm^{-2}, which is consistent with theoretical predictions for near-perfect quantum well top interfaces.

Proteomics Profiling of CLL Versus Healthy B-cells Identifies Putative Therapeutic Targets and a Subtype-independent Signature of Spliceosome Dysregulation.

Chronic lymphocytic leukemia (CLL) is a heterogeneous B-cell cancer exhibiting a wide spectrum of disease courses and treatment responses. Molecular characterization of RNA and DNA from CLL cases has led to the identification of important driver mutations and disease subtypes, but the precise mechanisms of disease progression remain elusive. To further our understanding of CLL biology we performed isobaric labeling and mass spectrometry proteomics on 14 CLL samples, comparing them with B-cells from healthy donors (HDB). Of 8694 identified proteins, ∼6000 were relatively quantitated between all samples (q<0.01). A clear CLL signature, independent of subtype, of 544 significantly overexpressed proteins relative to HDB was identified, highlighting established hallmarks of CLL (e.g. CD5, BCL2, ROR1 and CD23 overexpression). Previously unrecognized surface markers demonstrated overexpression (e.g. CKAP4, PIGR, TMCC3 and CD75) and three of these (LAX1, CLEC17A and ATP2B4) were implicated in B-cell receptor signaling, which plays an important role in CLL pathogenesis. Several other proteins (e.g. Wee1, HMOX1/2, HDAC7 and INPP5F) were identified with significant overexpression that also represent potential targets. Western blotting confirmed overexpression of a selection of these proteins in an independent cohort. mRNA processing machinery were broadly upregulated across the CLL samples. Spliceosome components demonstrated consistent overexpression (p = 1.3 × 10-21) suggesting dysregulation in CLL, independent of SF3B1 mutations. This study highlights the potential of proteomics in the identification of putative CLL therapeutic targets and reveals a subtype-independent protein expression signature in CLL.

Determining the Impact of Roller Compaction Processing Conditions on Granule and API Properties.

The attrition of drug particles during the process of dry granulation, which may (or may not) be incorporated into granules, could be an important factor in determining the subsequent performance of that granulation, including key factors such as sticking to punches and bio-performance of the dosage form. It has previously been demonstrated that such attrition occurs in one common dry granulation process train; however, the fate of these comminuted particles in granules was not determined. An understanding of the phenomena of attrition and incorporation into granule will improve our ability to understand the performance of granulated systems, ultimately leading to an improvement in our ability to optimize and model the process. Unique feeding mechanisms, geometry, and milling systems of roller compaction equipment mean that attrition could be more or less substantial for any given equipment train. In this work, we examined attrition of API particles and their incorporation into granule in an equipment train from Gerteis, a commonly used equipment train for dry granulation. The results demonstrate that comminuted drug particles can exist free in post-milling blends of roller compaction equipment trains. This information can help better understand the performance of the granulations, and be incorporated into mechanistic models to optimize such processes.

Dermal Absorption of Pesticide Residues.

Current guidance for dermal exposure assessment of plant protection products typically uses in vitro skin penetration data for the active ingredient when applied as both the concentrated product and relevant spray dilutions thereof. However, typical re-entry scenarios involve potential skin exposure to a "dried residue" of the spray dilution, from which the absorption of a pesticide may be quite different. The research reported in this paper has shown: (1) The method to assess the transfer of dried pesticide residues from a surface to the skin is reproducible for four active ingredients of diverse physicochemical properties, after their application in commercially relevant formulations. (2) Skin absorption of all four pesticides examined was significantly less from a dried residue than from a spray dilution; the difference, in general, was of the order of a factor of 2. (3) Decontamination experiments with one of the active ingredients tested (trinexapac-ethyl) showed that, post-exposure to a spray dilution, skin surface cleaning must be performed within 1 h to significantly reduce potential systemic exposure (relative to continual contact for 24 h); in contrast, after contact with a dried residue, the sooner decontamination was performed, the greater the decrease in exposure achieved, even when the time of contact was as long as 8 h.

Evaluating the effect of immune cells on the outcome of patients with mesothelioma.

BACKGROUND: We systematically assessed the prognostic and predictive value of infiltrating adaptive and innate immune cells in a large cohort of patients with advanced mesothelioma. METHODS: A tissue microarray from 302 samples was constructed. Markers of adaptive immune response in T-cells (CD8+, FOXP3+, CD4+, CD45RO+, CD3+) and B-cells (CD20+), and of innate immune response; neutrophils (NP57+), natural killer cells (CD56+) and macrophages (CD68+) were evaluated. RESULTS: We found that in the epithelioid tumours, high CD4+ and CD20+ counts, and low FOXP3+, CD68+ and NP57+ counts linked to better outcome. In the non-epithelioid group low CD8+ and low FOXP3+ counts were beneficial.On multivariate analysis low FOXP3+ remained independently associated with survival in both groups. In the epithelioid group additionally high CD4+, high CD20+, and low NP57+ counts were prognostic. CONCLUSIONS: Our data demonstrate for the first time, in predominately advanced disease, the association of key markers of adaptive and innate immunity with survival and the differential effect of histology. A better understanding of the immunological drivers of the different subtypes of mesothelioma will assist prognostication and disease-specific clinical decision-making.

Epimeric Face-Selective Oxidations and Diastereodivergent Transannular Oxonium Ion Formation Fragmentations: Computational Modeling and Total Syntheses of 12-Epoxyobtusallene IV, 12-Epoxyobtusallene II, Obtusallene X, Marilzabicycloallene C, and Marilzabicycloallene D.

The total syntheses of 12-epoxyobtusallene IV, 12-epoxyobtusallene II, obtusallene X, marilzabicycloallene C, and marilzabicycloallene D as halogenated C15-acetogenin 12-membered bicyclic and tricyclic ether bromoallene-containing marine metabolites from Laurencia species are described. Two enantiomerically pure C4-epimeric dioxabicyclo[8.2.1]tridecenes were synthesized by E-selective ring-closing metathesis where their absolute stereochemistry was previously set via catalytic asymmetric homoallylic epoxidation and elaborated via regioselective epoxide-ring opening and diastereoselective bromoetherification. Epimeric face-selective oxidation of their Δ12,13 olefins followed by bromoallene installation allowed access to the oppositely configured 12,13-epoxides of 12-epoxyobtusallene II and 12-epoxyobtusallene IV. Subsequent exploration of their putative biomimetic oxonium ion formation-fragmentations reactions revealed diastereodivergent pathways giving marilzabicycloallene C and obtusallene X, respectively. The original configurations of the substrates evidently control oxonium ion formation and their subsequent preferred mode of fragmentation by nucleophilic attack at C9 or C12. Quantum modeling of this stereoselectivity at the ωB97X-D/Def2-TZVPPD/SCRF = methanol level revealed that in addition to direction resulting from hydrogen bonding, the dipole moment of the ion-pair transition state is an important factor. Marilzabicycloallene D as a pentahalogenated 12-membered bicyclic ether bromoallene was synthesized by a face-selective chloronium ion initiated oxonium ion formation-fragmentation process followed by subsequent bromoallene installation.