NeoSplice: a bioinformatics method for prediction of splice variant neoantigens.

Motivation: Splice variant neoantigens are a potential source of tumor-specific antigen (TSA) that are shared between patients in a variety of cancers, including acute myeloid leukemia. Current tools for genomic prediction of splice variant neoantigens demonstrate promise. However, many tools have not been well validated with simulated and/or wet lab approaches, with no studies published that have presented a targeted immunopeptidome mass spectrometry approach designed specifically for identification of predicted splice variant neoantigens. Results: In this study, we describe NeoSplice, a novel computational method for splice variant neoantigen prediction based on (i) prediction of tumor-specific k-mers from RNA-seq data, (ii) alignment of differentially expressed k-mers to the splice graph and (iii) inference of the variant transcript with MHC binding prediction. NeoSplice demonstrates high sensitivity and precision (>80% on average across all splice variant classes) through in silico simulated RNA-seq data. Through mass spectrometry analysis of the immunopeptidome of the K562.A2 cell line compared against a synthetic peptide reference of predicted splice variant neoantigens, we validated 4 of 37 predicted antigens corresponding to 3 of 17 unique splice junctions. Lastly, we provide a comparison of NeoSplice against other splice variant prediction tools described in the literature. NeoSplice provides a well-validated platform for prediction of TSA vaccine targets for future cancer antigen vaccine studies to evaluate the clinical efficacy of splice variant neoantigens. Availability and implementation: https://github.com/Benjamin-Vincent-Lab/NeoSplice. Supplementary information: Supplementary data are available at Bioinformatics Advances online.

Climate-change-driven growth decline of European beech forests.

The growth of past, present, and future forests was, is and will be affected by climate variability. This multifaceted relationship has been assessed in several regional studies, but spatially resolved, large-scale analyses are largely missing so far. Here we estimate recent changes in growth of 5800 beech trees (Fagus sylvatica L.) from 324 sites, representing the full geographic and climatic range of species. Future growth trends were predicted considering state-of-the-art climate scenarios. The validated models indicate growth declines across large region of the distribution in recent decades, and project severe future growth declines ranging from -20% to more than -50% by 2090, depending on the region and climate change scenario (i.e. CMIP6 SSP1-2.6 and SSP5-8.5). Forecasted forest productivity losses are most striking towards the southern distribution limit of Fagus sylvatica, in regions where persisting atmospheric high-pressure systems are expected to increase drought severity. The projected 21st century growth changes across Europe indicate serious ecological and economic consequences that require immediate forest adaptation.

Entinostat induces antitumor immune responses through immune editing of tumor neoantigens.

Although immune-checkpoint inhibitors (ICIs) have been a remarkable advancement in bladder cancer treatment, the response rate to single-agent ICIs remains suboptimal. There has been substantial interest in the use of epigenetic agents to enhance ICI efficacy, although precisely how these agents potentiate ICI response has not been fully elucidated. We identified entinostat, a selective HDAC1/3 inhibitor, as a potent antitumor agent in our immune-competent bladder cancer mouse models (BBN963 and BBN966). We demonstrate that entinostat selectively promoted immune editing of tumor neoantigens, effectively remodeling the tumor immune microenvironment, resulting in a robust antitumor response that was cell autonomous, dependent upon antigen presentation, and associated with increased numbers of neoantigen-specific T cells. Finally, combination treatment with anti-PD-1 and entinostat led to complete responses and conferred long-term immunologic memory. Our work defines a tumor cell-autonomous mechanism of action for entinostat and a strong preclinical rationale for the combined use of entinostat and PD-1 blockade in bladder cancer.

Landscape and selection of vaccine epitopes in SARS-CoV-2.

BACKGROUND: Early in the pandemic, we designed a SARS-CoV-2 peptide vaccine containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation. The rationale for this design was to drive both humoral and cellular immunity with high specificity while avoiding undesired effects such as antibody-dependent enhancement (ADE). METHODS: We explored the set of computationally predicted SARS-CoV-2 HLA-I and HLA-II ligands, examining protein source, concurrent human/murine coverage, and population coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, sequence conservation, source protein abundance, and coverage of high frequency HLA alleles. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering for surface accessibility, sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. RESULTS: From 58 initial candidates, three B cell epitope regions were identified. From 3730 (MHC-I) and 5045 (MHC-II) candidate ligands, 292 CD8+ and 284 CD4+ T cell epitopes were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we proposed a set of 22 SARS-CoV-2 vaccine peptides for use in subsequent murine studies. We curated a dataset of ~ 1000 observed T cell epitopes from convalescent COVID-19 patients across eight studies, showing 8/15 recurrent epitope regions to overlap with at least one of our candidate peptides. Of the 22 candidate vaccine peptides, 16 (n = 10 T cell epitope optimized; n = 6 B cell epitope optimized) were manually selected to decrease their degree of sequence overlap and then synthesized. The immunogenicity of the synthesized vaccine peptides was validated using ELISpot and ELISA following murine vaccination. Strong T cell responses were observed in 7/10 T cell epitope optimized peptides following vaccination. Humoral responses were deficient, likely due to the unrestricted conformational space inhabited by linear vaccine peptides. CONCLUSIONS: Overall, we find our selection process and vaccine formulation to be appropriate for identifying T cell epitopes and eliciting T cell responses against those epitopes. Further studies are needed to optimize prediction and induction of B cell responses, as well as study the protective capacity of predicted T and B cell epitopes.

Landscape and Selection of Vaccine Epitopes in SARS-CoV-2.

There is an urgent need for a vaccine with efficacy against SARS-CoV-2. We hypothesize that peptide vaccines containing epitope regions optimized for concurrent B cell, CD4+ T cell, and CD8+ T cell stimulation would drive both humoral and cellular immunity with high specificity, potentially avoiding undesired effects such as antibody-dependent enhancement (ADE). Additionally, such vaccines can be rapidly manufactured in a distributed manner. In this study, we combine computational prediction of T cell epitopes, recently published B cell epitope mapping studies, and epitope accessibility to select candidate peptide vaccines for SARS-CoV-2. We begin with an exploration of the space of possible T cell epitopes in SARS-CoV-2 with interrogation of predicted HLA-I and HLA-II ligands, overlap between predicted ligands, protein source, as well as concurrent human/murine coverage. Beyond MHC affinity, T cell vaccine candidates were further refined by predicted immunogenicity, viral source protein abundance, sequence conservation, coverage of high frequency HLA alleles and co-localization of CD4+ and CD8+ T cell epitopes. B cell epitope regions were chosen from linear epitope mapping studies of convalescent patient serum, followed by filtering to select regions with surface accessibility, high sequence conservation, spatial localization near functional domains of the spike glycoprotein, and avoidance of glycosylation sites. From 58 initial candidates, three B cell epitope regions were identified. By combining these B cell and T cell analyses, as well as a manufacturability heuristic, we propose a set of SARS-CoV-2 vaccine peptides for use in subsequent murine studies and clinical trials.

Atmospheric depositions affect the growth patterns of Scots pines (Pinus sylvestris L.)-a long-term cause-effect monitoring study using biomarkers.

Recording the causes, effects, and effect mechanisms of vegetation health is crucial to understand process-pattern interactions in ecosystem processes. NOX and SOX in the form of air pollution are both triggers and sources of vegetation health that can have an effect on the local or the global level and whose impacts need to be monitored. In this study, the growth patterns in Scots pines (Pinus sylvestris L.) were studied in the context of changing atmospheric depositions in the lowlands of north-eastern Germany. Under the influence of atmospheric sulfur (S) and nitrogen (N) depositions, pine stands showed temporal variations in their normal growth behavior. In such cases, the patterns of normal growth can be suppressed or accelerated. Pine stands which were influenced by high S deposition up until 1990 changed from suppressed growth to accelerated growth by decreasing S, but increasing N depositions between 1990 and 2003. The cause of these changes in pine growth patterns was imbalances in S and N nutrition, in particular, enrichments of sulfate, non-protein nitrogen or arginine, and finally, also imbalances and deficiencies in phosphorus, glucose, and adenosine triphosphate in the needles. Our long-term monitoring study shows that biochemical markers (traits) are crucial bioindicators for the qualitative and quantitative assessment of tree vitality and growth patterns in Scots pines. Furthermore, we were able to show that NOX and SOX depositions need to be monitored locally to be able to assess the local effects of biomolecular markers on the growth patterns in Scots pine stands.

Metal complex SERMs (selective oestrogen receptor modulators). The influence of different metal units on breast cancer cell antiproliferative effects.

The selective oestrogen receptor modulator tamoxifen is a leading agent in the adjuvant treatment of breast cancer. Several organometallic moieties have been vectorised with tamoxifen, in order to improve on the latter's antiproliferative properties by the addition of a potentially cytotoxic moiety, and have been evaluated versus both oestrogen receptor positive (MCF7) and oestrogen receptor negative (MDA-MB231) breast cancer cells. For tamoxifen analogues with ((R,R)-trans-1,2-diaminocyclohexane)platinum(II), cyclopentadienyl rhenium tricarbonyl, and ruthenocene tethers, there was no enhancement of the antiproliferative effect on oestrogen receptor positive cells, nor any cytotoxic effect on oestrogen receptor negative cells, while those containing cyclopentadienyl titanium dichloride showed an oestrogenic effect. However, compounds where ferrocene replaces tamoxifen's phenyl ring were strongly cytotoxic against both cell lines. The synthesis and biological results of these compounds is reviewed and placed in the historic context of inorganic compounds in therapy.

The chemistry of the fac-[Re(CO)2(NO)]2+ fragment in aqueous solution.

The anions [ReX3(CO)2(NO)]- (with X = Cl, 1; X = Br, 2) have been prepared with different counterions. Complex 1 was found to lose its chloride ligands in water within 24 h. The [Re(H2O)3(CO)2(NO)]2+ cation obtained after hydrolysis is a strong acid, which consequently undergoes a slow condensation reaction in water to form the very stable [Re(mu3-O)(CO)2(NO)]4 cluster 4 at pH > 2, that precipitates from the aqueous solution and is insoluble also in organic solvents. Fast deprotonation of [Re(H2O)3(CO)2(NO)]2+ did not lead to 4 but rather to the mononuclear species [Re(OH)(H2O)2(CO)2(NO)]+. Subsequent attack of OH- at a CO group resulted in the formation of a rhenacarboxylic acid and its carboxylate anion. For solutions of even higher pH, IR spectroscopy provided evidence for the formation of a Re(C(O)ON(O)) species. These processes were found to be reversible on lowering the pH. Starting from cluster 4 it was possible to obtain complexes of the types [ReX(CO)2(NO)L2] or [Re(CO)2(NO)L3](L2 = 2-picolinate, 2,2'-bipyridine, L-phenylalanate; L3 = tris(pyrazolyl)methane, 1,4,7-trithiacyclononane) in the presence of an acid in protic solvents, but only in low yields. In further synthetic studies, complexes 1 and 2 were found to be superior starting materials for substitution reactions to form [ReX(CO)2(NO)L2] or [Re(CO)2(NO)L3] complexes.

Alpha-amino acids with metallocenyl side chains.

A straightforward method for the synthesis of enantiomerically pure bis(valine)metallocenes is presented. Derivatives of lithium cyclopentadienylvaline 1a, b were obtained by addition of the (R)- or (S)-Schöllkopf reagents to 6,6-dimethylfulvene as single enantiomers and gave with FeCl2 or [RuCl2(dmso)4] the chiral metallocenes [Fe[C5H4-CMe2-[C4H2N2(OMe)2iPr]]2] (2a, b) and [Ru[C5H4-CMe2-[C4H2N2(OMe)2iPr]]2] (3a, b). Complex 2b was hydrolyzed to the ferrocenylene-bis(valine-methylester) [[Fe[C5H4-CMe2-CH(NH3+)COOMe]2]2+(Cl-)2] (7) without racemization. Complex 7 could be used as ligand and was treated with [[Cp*IrCl2]2] to afford [Fe[C5H4-CMe2-CH(COOMe)(NH2-IrCp*Cl2)]2] (10). The reactions of 1 with CoCl2, [Re(CO)5Br], [[(cod)RhCl2]2] (cod= 1,5-cyclooctadiene) or [Cp*MCl3] (M= Ti, Zr) gave the cyclopentadienyl complexes [[Co[C5H4-CMe2-[C4H2N2(OMe)2iPr]]2]+ I-] (11) and [Re[C5H4-CMe2-[C4H2N2(OMe)2iPr]](CO)3] (13), [(C8H12)Rh[C5H4-CMe2-[C4H2N2(OMe)2(iPr)]]] (14). [[Rh[C5H4-CMe2-[C4H2N2(OMe)2(iPr)]]I]2(mu-I)2] (15), [Cp*Cl2Ti-[C5H4-CMe2-[C4H2N2(OMe)2(iPr)]]] (16), and [Cp*Cl2Zr[C5H4-CMe2-[C4H2N2(OMe)2(iPr)]]] (17), with chiral valine derivatives as substituents on the cyclopentadienyl ring and with excellent diastereoselectivities. Also the Seebach reagent (Boc-BMI) or O'Donnell reagent could be added to 6,6-dimethylfulvene to give the lithium cyclopentadienides Li[C5H4-CMe2-[C3H2(tBu)(N-Boc)(NMe)O]] (18) and Li[C5H4-CMe2-CH(NCPh2)(COOEt)] (21), which formed the ferrocene derivatives [Fe[C5H4-CMe2-[C3H2(tBu)(N-Boc)(NMe)O]]2] (19) and [Fe[C5H4-CMe2-CH(NCPh2)(COOEt)]2] (22). The stable cobaltocinium cation in 11 and the complex 19 could be hydrolyzed to the metallocenes 12 and [Fe(C5H4-CMe2-CH(NH3+)(COO-)]2] (20) with two valines in the 1,1'-position. The structures of 2a, b, 11, 15, and 16 were determined by X-ray diffraction and confirm the diastereomeric purity of the compounds.

Facile Synthesis of Cyclic Tetrapeptides from Nonactivated Peptide Esters on Metal Centers.

The cyclization of dipeptide esters of α-, ß-, γ-, and δ-amino acids can be achieved by using NiII , PdII , or CuII templates. The structure of one of the complexes (1) obtained, which was determined by X-ray crystallography, reveals that the anions form layers and are linked to water molecules by hydrogen bonds.

Bioorganometallic Chemistry - Transition Metal Complexes with α-Amino Acids and Peptides.

A new, interdisciplinary research area has emerged known as bioorganometallic chemistry. It focuses on the introduction of organometallic fragments into biomolecules (see, for example, structure on the right). "Classical" α-amino acid and peptide ligands have proven particularly versatile, and provide access to compounds that display interesting stereochemistry. α-Amino acids and peptides can be synthesized, labeled, stabilized, or activated by organometallic fragments.

Synthesis, Structure, and Magnetic Properties of Transition Metal Complexes of the Nitroxide 2,5-Dihydro-4,5,5-trimethyl-2,2-bis(2-pyridyl)imidazole-1-oxyl.

With the radical 2,5-dihydro-4,5,5-trimethyl-2,2-bis(2-pyridyl)imidazole-1-oxyl (L) a series of transition metal complexes have been prepared: [ML(2)](SbF(6))(2) with M(2+) = Mn(2+) (1), Fe(2+) (2), Co(2+) (3), Ni(2+) (4), Cu(2+) (5), and Zn(2+) (6), Cu(L)(Cl)(2)(MeOH) (7), and Cu(L)SO(4).H(2)O (8). The structures of 1, 3, and 6 were determined by X-ray structural analyses. In these compounds the tridendate L is coordinated to the metal ion by the two pyridine nitrogen donors and by the oxygen atom of the nitroxide group. The N-O bond distances are 1.25(2) Å (1), 1.267(13) Å (3), and 1.260(11) Å (6). The M-O-N angles are 117.0(10) degrees (1), 114.5(8) degrees (3), and 114.2(7) degrees (6). Crystal data: space group P2(1)/n, for 1, 3, and 6; compound 1, a = 10.806(3) Å, b = 14.101(6) Å, c = 14.253(4) Å, beta = 108.82(2) degrees, V = 2055.7(12) Å(3), Z = 2, R(1) = 0.0677, wR(2) = 0.1512. Compound 3, a = 10.761(4) Å, b = 14.253(6) Å, c = 14.108(5) Å, beta = 111.16(3) degrees, V = 2017.9(13) Å(3), Z = 2, R(1) = 0.0702, wR(2) = 0.1460; compound 6, a = 10.788(2) Å, b = 14.147(3) Å, c = 14.196(3) Å, beta = 109.93(3) degrees, V = 2036.8(7) Å(3), Z = 2, R(1) = 0.0573, wR(2) = 0.1194. Magnetic measurements of 1, 2, 5, and 8 show strong antiferromagnetic interaction between the spin of the metal ion and the spin of the radical which increases at lower temperatures. For 6 the magnetic moment corresponds to two noninteracting spins in the temperature range 60-300 K.