Genomic and Transcriptomic Features of Response to Anti-PD-1 Therapy in Metastatic Melanoma.

PD-1 immune checkpoint blockade provides significant clinical benefits for melanoma patients. We analyzed the somatic mutanomes and transcriptomes of pretreatment melanoma biopsies to identify factors that may influence innate sensitivity or resistance to anti-PD-1 therapy. We find that overall high mutational loads associate with improved survival, and tumors from responding patients are enriched for mutations in the DNA repair gene BRCA2. Innately resistant tumors display a transcriptional signature (referred to as the IPRES, or innate anti-PD-1 resistance), indicating concurrent up-expression of genes involved in the regulation of mesenchymal transition, cell adhesion, extracellular matrix remodeling, angiogenesis, and wound healing. Notably, mitogen-activated protein kinase (MAPK)-targeted therapy (MAPK inhibitor) induces similar signatures in melanoma, suggesting that a non-genomic form of MAPK inhibitor resistance mediates cross-resistance to anti-PD-1 therapy. Validation of the IPRES in other independent tumor cohorts defines a transcriptomic subset across distinct types of advanced cancer. These findings suggest that attenuating the biological processes that underlie IPRES may improve anti-PD-1 response in melanoma and other cancer types.

Cep57 regulates human centrosomes through multivalent interactions.

Human Cep57 is a coiled-coil scaffold at the pericentriolar matrix (PCM), controlling centriole duplication and centrosome maturation for faithful cell division. Genetic truncation mutations of Cep57 are associated with the mosaic-variegated aneuploidy (MVA) syndrome. During interphase, Cep57 forms a complex with Cep63 and Cep152, serving as regulators for centrosome maturation. However, the molecular interplay of Cep57 with these essential scaffolding proteins remains unclear. Here, we demonstrate that Cep57 undergoes liquid-liquid phase separation (LLPS) driven by three critical domains (NTD, CTD, and polybasic LMN). In vitro Cep57 condensates catalyze microtubule nucleation via the LMN motif-mediated tubulin concentration. In cells, the LMN motif is required for centrosomal microtubule aster formation. Moreover, Cep63 restricts Cep57 assembly, expansion, and microtubule polymerization activity. Overexpression of competitive constructs for multivalent interactions, including an MVA mutation, leads to excessive centrosome duplication. In Cep57-depleted cells, self-assembly mutants failed to rescue centriole disengagement and PCM disorganization. Thus, Cep57's multivalent interactions are pivotal for maintaining the accurate structural and functional integrity of human centrosomes.

Spatial transcriptomics and the anatomical pathologist: Molecular meets morphology.

In recent years anatomical pathology has been revolutionised by the incorporation of molecular findings into routine diagnostic practice, and in some diseases the presence of specific molecular alterations are now essential for diagnosis. Spatial transcriptomics describes a group of technologies that provide up to transcriptome-wide expression profiling while preserving the spatial origin of the data, with many of these technologies able to provide these data using a single tissue section. Spatial transcriptomics allows expression profiling of highly specific areas within a tissue section potentially to subcellular resolution, and allows correlation of expression data with morphology, tissue type and location relative to other structures. While largely still research laboratory-based, several spatial transcriptomics methods have now achieved compatibility with formalin-fixed paraffin-embedded tissue (FFPE), allowing their use in diagnostic tissue samples, and with further development potentially leading to their incorporation in routine anatomical pathology practice. This mini review provides an overview of spatial transcriptomics methods, with an emphasis on platforms compatible with FFPE tissue, approaches to assess the data and potential applications in anatomical pathology practice.

Chromosome-scale assembly and gene editing of Solanum americanum genome reveals the basis for thermotolerance and fruit anthocyanin composition.

Solanum americanum serves as a promising source of resistance genes against potato late blight and is considered as a leafy vegetable for complementary food and nutrition. The limited availability of high-quality genome assemblies and gene annotations has hindered the exploration and exploitation of stress-resistance genes in S. americanum. Here, we present a chromosome-level genome assembly of a thermotolerant S. americanum ecotype and identify a crucial heat-inducible transcription factor gene, SaHSF17, essential for heat tolerance. The CRISPR/Cas9 system-mediated knockout of SaHSF17 results in remarkably reduced thermotolerance in S. americanum, exhibiting a significant suppression of multiple HSP gene expressions under heat treatment. Furthermore, our transcriptome analysis and anthocyanin component investigation of fruits indicated that delphinidins are the major anthocyanins accumulated in the mature dark-purple fruits. The accumulation of delphinidins and other pigment components during fruit ripening in S. americanum coincides with the transcriptional regulation of key genes, particularly the F3'5'H and F3'H genes, in the anthocyanin biosynthesis pathway. By integrating existing knowledge, the development of this high-quality reference genome for S. americanum will facilitate the identification and utilization of novel abiotic and biotic stress-resistance genes for improvement of Solanaceae and other crops.

Newly discovered clouting interplay between matrix metalloproteinases structures and novel quaternary Ammonium K21: computational and in-vivo testing.

AIMS AND OBJECTIVES: To analyze anti-MMP mode of action of Quaternary Ammonium Silane (QAS, codenamed as k21) by binding onto specific MMP site using computational molecular simulation and Anti-Sortase A (SrtA) mode of action by binding onto specific site using computational molecular simulation. MATERIALS AND METHODS: In silico Molecular Dynamics (MD) was used to determine the interactions of K21 inside the pocket of the targeted protein (crystal structure of fibroblast collagenase-1 complexed to a diphenyl-ether sulphone based hydroxamic acid; PDB ID: 966C; Crystal structure of MMP-2 active site mutant in complex with APP-derived decapeptide inhibitor. MD simulations were accomplished with the Desmond package in Schrödinger Drug Discovery Suite. Blood samples (~ 0.5 mL) collected into K2EDTA were immediately transferred for further processing using the Litron MicroFlow® PLUS micronucleus analysis kit for mouse blood according to the manufacturer's instructions. Bacterial Reverse Mutation Test of K21 Molecule was performed to evaluate K21 and any possible metabolites for their potential to induce point mutations in amino acid-requiring strains of Escherichia coli (E. coli) (WP2 uvrA (tryptophan-deficient)). RESULTS: Molecular Simulation depicted that K21 has a specific pocket binding on various MMPs and SrtA surfaces producing a classical clouting effect. K21 did not induce micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus, in the peripheral blood reticulocytes of male and female CD-1 mice when administered by oral gavage up to the maximum recommended dose of 2000 mg/kg. The test item, K21, was not mutagenic to Salmonella typhimurium (S. typhimurium) strains TA98, TA100, TA1535 and TA1537 and E. coli strain WP2 uvrA in the absence and presence of metabolic activation when tested up to the limit of cytotoxicity or solubility under the conditions of the test. CONCLUSION: K21 could serve as a potent protease inhibitor maintaining the physical and biochemical properties of dental structures.

In Situ Transformation of an Amorphous Supramolecular Coating to a Hydrogen-Bonded Organic Framework Membrane to Trigger Selective Gas Permeation.

We introduce a "solution-processing-transformation" strategy, deploying solvent vapor as scaffolds, to fabricate high-quality hydrogen-bonded organic framework (HOF) membranes. This strategy can overcome the mismatch in processing conditions and crystal growth thermodynamics faced during the facile solution processing of the membrane. The procedure includes the vapor-trigged in situ transformation of dense amorphous supramolecules to crystalline HOF-16, with HOF-11 as the transient state. The mechanism involves a vapor-activated dissolution-precipitation equilibrium shifting and hydrogen bonding-guided molecule rearrangement, elucidated through combined experimental and theoretical analysis. Upon removal of the molecular scaffolds, the resulting HOF-16 membranes showcase significant improvement in hydrogen separation performance over their amorphous counterparts and previously reported HOF membranes. The method's broad applicability is evidenced by successfully extending it to other substrates and HOF structures. This study provides a fundamental understanding of guest-induced ordered supramolecular assembly and paves the way for the advanced manufacture of high-performance HOF membranes for gas separation processes.

Design, synthesis, and evaluation of 2,2'-bipyridyl derivatives as bifunctional agents against Alzheimer's disease.

Alzheimer's disease (AD) is a complex multifactorial neurodegenerative disease. Metal ion dyshomeostasis and Aß aggregation have been proposed to contribute to AD progression. Metal ions can bind to Aß and promote Aß aggregation, and ultimately lead to neuronal death. Bifunctional (metal chelation and Aß interaction) compounds are showing promise against AD. In this work, eleven new 3,3'-diamino-2,2'-bipyridine derivatives 4a-4k were synthesized, and evaluated as bifunctional agents for AD treatment. In vitro Aß aggregation inhibition assay confirmed that most of the synthesized compounds exhibited significant self-induced Aß1-42 aggregation inhibition. Among them, compound 4d displayed the best inhibitory potency of self-induced Aß1-42 aggregation with IC50 value of 9.4 µM, and it could selectively chelate with Cu2+ and exhibited 66.2% inhibition of Cu2+-induced Aß1-42 aggregation. Meanwhile, compound 4d showed strong neuroprotective activity against Aß1-42 and Cu2+-treated Aß1-42 induced cell damage. Moreover, compound 4d in high dose significantly reversed Aß-induced memory impairment in mice.

The Sm core protein SmEb regulates salt stress responses through maintaining proper splicing of RCD1 pre-mRNA in Arabidopsis.

Salt stress adversely impacts crop production. Several spliceosome components have been implicated in regulating salt stress responses in plants, however, the underlying molecular basis is still unclear. Here we report that the spliceosomal core protein SmEb is essential to salt tolerance in Arabidopsis. Transcriptome analysis showed that SmEb modulates alternative splicing of hundreds of pre-mRNAs in plant response to salt stress. Further study revealed that SmEb is crucial in maintaining proper ratio of two RCD1 splicing variants (RCD1.1/RCD1.2) important for salt stress response. In addition, RCD1.1 but not RCD1.2 is able to interact with the stress regulators and attenuates salt-sensitivity by decreasing salt-induced cell death in smeb-1 mutant. Together, our findings uncovered the essential role of SmEb in the regulation of alternative pre-mRNA splicing in salt stress response.

Investigating the relationship between sleep and migraine in a global sample: a Bayesian cross-sectional approach.

BACKGROUND: There is a bidirectional link between sleep and migraine, however causality is difficult to determine. This study aimed to investigate this relationship using data collected from a smartphone application. METHODS: Self-reported data from 11,166 global users (aged 18-81 years, mean: 41.21, standard deviation: 11.49) were collected from the Migraine Buddy application (Healint Pte. Ltd.). Measures included: start and end times of sleep and migraine attacks, and pain intensity. Bayesian regression models were used to predict occurrence of a migraine attack the next day based on users' deviations from average sleep, number of sleep interruptions, and hours slept the night before in those reporting ≥ 8 and < 25 migraine attacks on average per month. Conversely, we modelled whether attack occurrence and pain intensity predicted hours slept that night. RESULTS: There were 724 users (129 males, 412 females, 183 unknown, mean age = 41.88 years, SD = 11.63), with a mean monthly attack frequency of 9.94. More sleep interruptions (95% Highest Density Interval (95%HDI [0.11 - 0.21]) and deviation from a user's mean sleep (95%HDI [0.04 - 0.08]) were significant predictors of a next day attack. Total hours slept was not a significant predictor (95%HDI [-0.04 - 0.04]). Pain intensity, but not attack occurrence was a positive predictor of hours slept. CONCLUSIONS: Sleep fragmentation and deviation from typical sleep are the main drivers of the relationship between sleep and migraine. Having a migraine attack does not predict sleep duration, yet the pain associated with it does. This study highlights sleep as crucial in migraine management.

Modulation of plant development and chilling stress responses by alternative splicing events under control of the spliceosome protein SmEb in Arabidopsis.

Cold stress resulting from chilling and freezing temperatures substantially inhibits plant growth and reduces crop production worldwide. Tremendous research efforts have been focused on elucidating the molecular mechanisms of freezing tolerance in plants. However, little is known about the molecular nature of chilling stress responses in plants. Here we found that two allelic mutants in a spliceosome component gene SmEb (smeb-1 and smeb-2) are defective in development and responses to chilling stress. RNA-seq analysis revealed that SmEb controls the splicing of many pre-messenger RNAs (mRNAs) under chilling stress. Our results suggest that SmEb is important to maintain proper ratio of the two COP1 splicing variants (COP1a/COP1b) to fine tune the level of HY5. In addition, the transcription factor BES1 shows a dramatic defect in pre-mRNA splicing in the smeb mutants. Ectopic expression of the two BES1 splicing variants enhances the chilling sensitivity of the smeb-1 mutant. Furthermore, biochemical and genetic analysis showed that CBFs act as negative upstream regulators of SmEb by directly suppressing its transcription. Together, our results demonstrate that proper alternative splicing of pre-mRNAs controlled by the spliceosome component SmEb is critical for plant development and chilling stress responses.

Polysulfide Anions as Visible Light Photoredox Catalysts for Aryl Cross-Couplings.

Polysulfide anions are endowed with unique redox properties, attracting considerable attentions for their applications in alkali metals-sulfur batteries. However, the employment of these anionic species in redox catalysis for small molecule synthesis remains underdeveloped due to their moderate-poor electrochemical potential in the ground state, whereas some of them are characterized by photoabsorptions in visible spectral regions. Herein, we disclose the use of polysulfide anions as visible light photoredox catalysts for aryl cross-coupling reactions. The reaction design enables single-electron reduction of aryl halides upon the photoexcitation of tetrasulfide dianions (S42-). The resulting aryl radicals are engaged in (hetero)biaryl cross-coupling, borylation, and hydrogenation in a redox catalytic regime involving S4• -/S42- and S3• -/S32- redox couples.

A network pharmacology study with molecular docking to investigate the possibility of licorice against posttraumatic stress disorder.

Post traumatic stress disorder (PTSD) is a mental health condition that has a debilitating effect on a person's quality of life and leads to a high socioeconomic burden. Licorice has been demonstrated to have neuroprotective and antidepressant-like effects, but little is known about its effects for the treatment of PTSD. The present study aimed to explore the potential of licorice for PTSD therapy using a network pharmacology approach with molecular docking studies. The compounds of licorice were obtained from databases with screening by absorption, distribution, metabolism and excretion (ADME) evaluation. Genes associated with compounds or PTSD were obtained from public databases, and the genes overlapping between licorice compounds and PTSD were compared by Venn diagram. A network of medicine-ingredients-targets-disease was constructed, visualized, and analyzed using cytoscape software. Protein-protein interactions, gene ontology, pathway enrichment and molecular docking were performed to evaluate the effect of licorice for the treatment of PTSD. 69 potential compounds were screened after ADME evaluation. A total of 81 compound-related genes and 566 PTSD-related genes were identified in the databases with 27 overlapping genes. Licorice compounds (e.g., medicarpin, 7-methoxy-2-methyl isoflavone, shinpterocarpin, formononetin, licochalcone a) and target proteins (e.g., ESR1, PTGS2, NOS2, and ADRB2) with high degree in the network were involved in G protein-coupled receptor signaling pathways at the postsynaptic/synaptic membrane. Moreover, neuroactive ligand-receptor interactions, calcium signaling, cholinergic synapse, serotonergic synapse and adrenergic signaling in cardiomyocytes may play important roles in the treatment of PTSD by licorice. This study provides molecular evidence of the beneficial effects of licorice for the treatment of PTSD.

Genetic analysis implicates a molecular chaperone complex in regulating epigenetic silencing of methylated genomic regions.

DNA cytosine methylation confers stable epigenetic silencing in plants and many animals. However, the mechanisms underlying DNA methylation-mediated genomic silencing are not fully understood. We conducted a forward genetic screen for cellular factors required for the silencing of a heavily methylated p35S:NPTII transgene in the Arabidopsis thaliana rdm1-1 mutant background, which led to the identification of a Hsp20 family protein, RDS1 (rdm1-1 suppressor 1). Loss-of-function mutations in RDS1 released the silencing of the p35S::NPTII transgene in rdm1-1 mutant plants, without changing the DNA methylation state of the transgene. Protein interaction analyses suggest that RDS1 exists in a protein complex consisting of the methyl-DNA binding domain proteins MBD5 and MBD6, two other Hsp20 family proteins, RDS2 and IDM3, a Hsp40/DNAJ family protein, and a Hsp70 family protein. Like rds1 mutations, mutations in RDS2, MBD5, or MBD6 release the silencing of the transgene in the rdm1 mutant background. Our results suggest that Hsp20, Hsp40, and Hsp70 proteins may form a complex that is recruited to some genomic regions with DNA methylation by methyl-DNA binding proteins to regulate the state of silencing of these regions.

Hydromagnesiation of 1,3-Enynes by Magnesium Hydride for Synthesis of Tri- and Tetra-substituted Allenes.

A protocol for regio-controlled hydromagnesiation of 1,3-enynes was developed using magnesium hydride that is generated in situ by solvothermal treatment of sodium hydride (NaH) and magnesium iodide (MgI2 ) in THF. The resulting allenylmagnesium species could be converted into tri- and tetra-substituted allenes by subsequent treatment with various carbon- and silicon-based electrophiles with the aid of CuCN as a catalyst.

Atypical ductal hyperplasia is a multipotent precursor of breast carcinoma.

The current model for breast cancer progression proposes independent 'low grade (LG)-like' and 'high grade (HG)-like' pathways but lacks a known precursor to HG cancer. We applied low-coverage whole-genome sequencing to atypical ductal hyperplasia (ADH) with and without carcinoma to shed light on breast cancer progression. Fourteen out of twenty isolated ADH cases harboured at least one copy number alteration (CNA), but had fewer aberrations than LG or HG ductal carcinoma in situ (DCIS). ADH carried more HG-like CNA than LG DCIS (e.g. 8q gain). Correspondingly, 64% (7/11) of ADH cases with synchronous HG carcinoma were clonally related, similar to LG carcinoma (67%, 6/9). This study represents a significant shift in our understanding of breast cancer progression, with ADH as a common precursor lesion to the independent 'low grade-like' and 'high grade-like' pathways. These data suggest that ADH can be a precursor of HG breast cancer and that LG and HG carcinomas can evolve from a similar ancestor lesion. We propose that although LG DCIS may be committed to a LG molecular pathway, ADH may remain multipotent, progressing to either LG or HG carcinoma. This multipotent nature suggests that some ADH cases could be more clinically significant than LG DCIS, requiring biomarkers for personalising management. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Molecular comparison of interval and screen-detected breast cancers.

Breast cancer (BC) diagnosed after a negative mammogram but prior to the next screening episode is termed an 'interval BC' (IBC). Understanding the molecular differences between IBC and screen-detected BCs (SDBC) could improve mammographic screening and management options. Therefore, we assessed both germline and somatic genomic aberrations in a prospective cohort. Utilising the Lifepool cohort of >54 000 women attending mammographic screening programs, 930 BC cases with screening status were identified (726 SDBC and 204 IBC). Clinico-pathological and family history information were recorded. Germline and tumour DNA were collected where available and sequenced for BC predisposition and driver gene mutations. Compared to SDBC, IBCs were significantly associated with a younger age at diagnosis and tumour characteristics associated with worse prognosis. Germline DNA assessment of BC cases that developed post-enrolment (276 SDBCs and 77 IBCs) for pathogenic mutations in 12 hereditary BC predisposition genes identified 8 carriers (2.27%). The germline mutation frequency was higher in IBC versus SDBC, although not statistically significant (3.90% versus 1.81%, p = 0.174). Comparing somatic genetic features of IBC and SDBC matched for grade, histological subtype and hormone receptor revealed no significant differences, with the exception of higher homologous recombination deficiency scores in IBC, and copy number changes on chromosome Xq in triple negative SDBCs. Our data demonstrates that while IBCs are clinically more aggressive than SDBC, when matched for confounding clinico-pathological features they do not represent a unique molecular class of invasive BC, but could be a consequence of timing of tumour initiation and mammographic screening. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cross-Linking between Sodalite Nanoparticles and Graphene Oxide in Composite Membranes to Trigger High Gas Permeance, Selectivity, and Stability in Hydrogen Separation.

Thin membranes (900 nm) were prepared by direct transformation of infiltrated amorphous precursor nanoparticles, impregnated in a graphene oxide (GO) matrix, into hydroxy sodalite (SOD) nanocrystals. The amorphous precursor particles rich in silanols (Si-OH) enhanced the interactions with the GO, thus leading to the formation of highly adhesive and stable SOD/GO membranes via strong bonding. The cross-linking of SOD nanoparticles with the GO in the membranes promoted both the high gas permeance and enhanced selectivity towards H2 from a mixture containing CO2 and H2 O. The SOD/GO membranes are moisture resistance and exhibit steady separation performance (H2 permeance of about 4900 GPU and H2 /CO2 selectivity of 56, with no degradation in performance during the test of 50 h) at high temperature (200 °C) under water vapor (4 mol %).

Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma.

BACKGROUND: Approximately 75% of objective responses to anti-programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti-PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor-associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.).

Concerted Nucleophilic Aromatic Substitution Reactions.

Recent developments in experimental and computational chemistry have identified a rapidly growing class of nucleophilic aromatic substitutions that proceed by concerted (cSN Ar) rather than classical, two-step, SN Ar mechanisms. Whereas traditional SN Ar reactions require substantial activation of the aromatic ring by electron-withdrawing substituents, such activating groups are not mandatory in the concerted pathways.