Longitudinal molecular profiling elucidates immunometabolism dynamics in breast cancer.

Although metabolic reprogramming within tumor cells and tumor microenvironment (TME) is well described in breast cancer, little is known about how the interplay of immune state and cancer metabolism evolves during treatment. Here, we characterize the immunometabolic profiles of tumor tissue samples longitudinally collected from individuals with breast cancer before, during and after neoadjuvant chemotherapy (NAC) using proteomics, genomics and histopathology. We show that the pre-, on-treatment and dynamic changes of the immune state, tumor metabolic proteins and tumor cell gene expression profiling-based metabolic phenotype are associated with treatment response. Single-cell/nucleus RNA sequencing revealed distinct tumor and immune cell states in metabolism between cold and hot tumors. Potential drivers of NAC based on above analyses were validated in vitro. In summary, the study shows that the interaction of tumor-intrinsic metabolic states and TME is associated with treatment outcome, supporting the concept of targeting tumor metabolism for immunoregulation.

GARD™skin and GARD™potency: A proof-of-concept study investigating applicability domain for agrochemical formulations.

Several New Approach Methodologies (NAMs) for hazard assessment of skin sensitisers have been formally validated. However, data regarding their applicability on certain product classes are limited. The purpose of this project was to provide initial evidence on the applicability domain of GARD™skin and GARD™potency for the product class of agrochemical formulations. For this proof of concept, 30 liquid and 12 solid agrochemical formulations were tested in GARDskin for hazard predictions. Formulations predicted as sensitisers were further evaluated in the GARDpotency assay to determine GHS skin sensitisation category. The selected formulations were of product types, efficacy groups and sensitisation hazard classes representative of the industry's products. The performance of GARDskin was estimated by comparing results to existing in vivo animal data. The overall accuracy, sensitivity, and specificity were 76.2% (32/42), 85.0% (17/20), and 68.2% (15/22), respectively, with the predictivity for liquid formulations being slightly higher compared to the solid formulations. GARDpotency correctly subcategorized 14 out of the 17 correctly predicted sensitisers. Lack of concordance was justifiable by compositional or borderline response analysis. In conclusion, GARDskin and GARDpotency showed satisfactory performance in this initial proof-of-concept study, which supports consideration of agrochemical formulations being within the applicability domain of the test methods.

Evaluation of the applicability of GARDskin to predict skin sensitizers in extracts from medical device materials.

Biocompatibility testing of medical devices is governed by the ISO 10993 series of standards and includes evaluation of skin sensitization potential of the final product. A majority of all medical devices are tested using in vivo methods, largely due to the lack of in vitro methods validated within the applicability domain of solid materials. The GARDskin method for assessment of chemical skin sensitizers is a validated method included in the OECD Test Guideline 442E, based on evaluation of transcriptional patterns of an endpoint-specific genomic biomarker signature in a dendritic cell-like cell, following test chemical exposure. The current study aimed to evaluate the applicability of GARDskin for the purpose of testing solid materials by incorporation of extraction procedures described in ISO 10993-12:2021, as well as to demonstrate the functionality of the proposed protocols, by testing of custom-made materials spiked with sensitizing agents. It was shown that GARDskin is compatible with both polar and non-polar extraction vehicles frequently used for the purpose of medical device biological testing. Further, exploring three different material types spiked with up to four different sensitizing agents, as well as three unspiked control materials and commercial reference products, it was shown that the method correctly classified all evaluated test materials. Taken together, the data presented suggest that GARDskin may constitute a valid alternative to in vivo experimentation for the purpose of skin sensitization assessment of medical devices.

GARDskin dose-response assay and its application in conducting Quantitative Risk Assessment (QRA) for fragrance materials using a Next Generation Risk Assessment (NGRA) framework.

Development of New Approach Methodologies (NAMs) capable of providing a No Expected Sensitization Induction Level (NESIL) value remains a high priority for the fragrance industry for conducting a Quantitative Risk Assesment (QRA) to evaluate dermal sensitization. The in vitro GARDskin assay was recently adopted by the OECD (TG 442E) for the hazard identification of skin sensitizers. Continuous potency predictions are derived using a modified protocol that incorporates dose-response measurements. Linear regression models have been developed to predict human NESIL values. The aim of the study was to evaluate the precision and reproducibility of the continuous potency predictions from the GARDskin Dose-Response (DR) assay and its application in conducting QRA for fragrance materials using a Next Generation Risk Assessment (NGRA) framework. Results indicated that the GARDskin Dose-Response model predicted human NESIL values with a good degree of concordance with published NESIL values, which were also reproducible in 3 separate experiments. Using Isocyclocitral as an example, a QRA was conducted to determine its safe use levels in different consumer product types using a NGRA framework. This study represents a major step towards the establishment of the assay to derive NESIL values for conducting QRA evaluations for fragrance materials using a NGRA framework.

SOX11 is a novel binding partner and endogenous inhibitor of SAMHD1 ara-CTPase activity in mantle cell lymphoma.

ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.

Kerr Black Holes from Massive Higher-Spin Gauge Symmetry.

We propose that the dynamics of Kerr black holes is strongly constrained by the principle of gauge symmetry. We initiate the construction of effective field theories for Kerr black holes of any integer quantum spin s using Stückelberg fields, and show that the known three-point Kerr amplitudes are uniquely predicted using massive higher-spin gauge symmetry. This symmetry is argued to be connected to an enhanced range of validity for the Kerr effective field theories. We consider the closely related root-Kerr electromagnetic solution in parallel, for which the dynamical interactions with photons are also constrained by massive higher-spin gauge symmetry. Finally, the spin-s Compton amplitudes are analyzed, and we discuss contact-term constraints at s=2 from Ward identities.

FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress.

Fanconi anemia (FA) signaling, a key genomic maintenance pathway, is activated in response to replication stress. Here, we report that phosphorylation of the pivotal pathway protein FANCD2 by CHK1 triggers its FBXL12-dependent proteasomal degradation, facilitating FANCD2 clearance at stalled replication forks. This promotes efficient DNA replication under conditions of CYCLIN E- and drug-induced replication stress. Reconstituting FANCD2-deficient fibroblasts with phosphodegron mutants failed to re-establish fork progression. In the absence of FBXL12, FANCD2 becomes trapped on chromatin, leading to replication stress and excessive DNA damage. In human cancers, FBXL12, CYCLIN E, and FA signaling are positively correlated, and FBXL12 upregulation is linked to reduced survival in patients with high CYCLIN E-expressing breast tumors. Finally, depletion of FBXL12 exacerbated oncogene-induced replication stress and sensitized cancer cells to drug-induced replication stress by WEE1 inhibition. Collectively, our results indicate that FBXL12 constitutes a vulnerability and a potential therapeutic target in CYCLIN E-overexpressing cancers.

A longitudinal study of exercise-induced bronchoconstriction and laryngeal obstruction in high school athletes.

BACKGROUND: Exercise-induced bronchoconstriction (EIB) and exercise-induced laryngeal obstruction (EILO) are common in elite athletes. Knowledge of which factors are related to incident EIB and EILO is limited. The aim of this study was to explore the course of EIB and EILO in adolescent athletes over a 2 years period and baseline characteristics related to incident EIB. METHODS: Questionnaire data on respiratory symptoms, asthma, and aeroallergy and results of objective EIB and EILO tests were collected from 58 participants (27 tested for EILO) at baseline and after 2 years (follow-up). Associations between incident EIB and baseline asthma-like symptoms, exercise-induced symptoms, fractional exhaled nitric oxide (FeNO), aeroallergy, and sex were assessed using logistic regression models. RESULTS: Ten participants had incident EIB, and eight participants had persistent EIB. Five were EIB positive at baseline but negative at follow-up, while 35 participants were EIB negative at both time points. Having incident EIB was associated with reporting waking up with chest tightness (OR = 4.38; 95% CI: 1.06, 22.09). Reporting an increased number of asthma-like symptoms increased the likelihood of incident EIB (OR = 2.78; 95% CI: 1.16, 6.58). No associations were found between exercise-induced symptoms, FeNO, aeroallergy, or sex and incident EIB. Incident EILO was found in three and persistent EILO in two of the 27 participants tested. CONCLUSION: Two in nine had incident EIB and one eighth had incident EILO, suggesting that recurrent testing for EIB and EILO may be relevant in young athletes. Particularly, EIB-negative athletes reporting multiple asthma-like symptoms could benefit from recurrent EIB testing.

The GARD™skin assay: Investigation of the applicability domain for metals.

New approach methodologies (NAMs) for hazard identification of skin sensitizing chemicals were adopted as test guidelines by the OECD during the last decade. These alternatives to animal models align to individual key events (KE) in the adverse outcome pathway (AOP) for skin sensitization for which the molecular initiating event (MIE) is covalent binding to proteins. As it currently stands, the AOP does not include mechanistic events of sensitization by metals, and limited information is available on whether NAMs accurately predict the sensitization potential of such molecules, which have been proposed to act via alternative mechanisms to organic chemicals. Methods for assessing the sensitization potential of metals would be valuable tools to support risk management within, e.g., occupational settings during production of new metal salts or within the medical device industry to evaluate leachables from metal alloys. This paper describes a systematic evaluation of the applicability domain of the GARD™skin assay for the assessment of metals. Hazard classifications were supplemented with an extended analysis of gene expression profiles induced by metal sensitizers to compare the induction of toxicity pathways between metals and organic sensitizers. Based on the results of this study, the accuracy, sensitivity, and specificity of GAR­D™skin for the prediction of skin sensitizing hazard were 92% (12/13), 100% (7/7), and 83% (5/6), respectively. Thus, the performance of GARD™skin for the assessment of metals was found to be similar to that observed for conventional organic substances, providing support for inclusion of metals within the applicability domain of the test method.

Exploration of the GARD™skin applicability domain: Indirectly acting haptens, hydrophobic substances and UVCBs.

Hazard assessments of skin sensitizers are increasingly performed using new approach methodologies (NAMs), with several in chemico, in vitro, and most recently, also defined approaches accepted for regulatory use. However, keeping track of potential limitations of each method to define applicability domains remains a crucial component to ensure adequate predictivity and to facilitate the appropriate selection of method(s) for each hazard assessment task. The objective of this report is to share test results generated with the GARD™skin assay on chemicals that have traditionally been considered difficult to test in some of the conventional in vitro and in chemico OECD Test Guidelines for skin sensitization. Such compounds may include, for example, indirectly acting haptens, hydrophobic substances, and substances of unknown or variable composition, complex reaction products or biological substances (UVCBs). Based on the results of this study, the sensitivity for prediction of skin sensitizing hazard of indirectly acting haptens was 92.4% and 87.5% when compared with local lymph node assay (LLNA) (n = 25) and human data (n = 8), respectively. Similarly, the sensitivity for prediction of skin sensitizing hazard of hydrophobic substances was 85.1% and 100% when compared with LLNA (n = 24) and human data (n = 9), respectively. Lastly, a case study involving assessment of a set of hydrophobic UVCBs (n = 7) resulted in a sensitivity of 100% compared to available reference data. These data provide support for the inclusion of such chemistries in the GARD™skin applicability domain without an increased risk of false negative classifications.

Different chest HRCT scan protocols change the extent of ground glass opacities.

BACKGROUND: Ground glass opacity (GGO) is the main HRCT feature representing alveolitis in systemic sclerosis-associated interstitial lung disease (SSc-ILD), but may also represent other conditions such as atelectasis or edema. It is unclear how much this is affected by the HRCT scan protocol used. We aimed to compare the performance of three different HRCT protocols to evaluate the degree of SSc-ILD related changes. METHODS: Eleven patients with SSc underwent chest HRCT scan by three different protocols: First, a supine scan after lying down for 15 minutes, then two scans in alternating order: A prone position scan, and a supine position scan after performing 10 deep breaths using a positive expiratory pressure (PEP) device. The HRCT scans were evaluated by the Warrick score system for ILD-related findings. RESULTS: The three HRCT protocols were compared and resulted in different mean (95% CI) Warrick scores: 9.4 (5.3-13.4) in supine after rest; 7.5 (95% CI 3.8-11.1) in prone and 7.6 (95% CI 4.2-11.1) in supine after PEP. When comparing supine after rest to prone and supine after PEP, the latter two scans had a significantly lower score (p = 0.001 for both comparisons). In all cases, only sub-scores for ground glass opacities differed, while sub-scores for fibrosis-related changes did not change. CONCLUSIONS: Different HRCT scan protocols significantly altered the Warrick severity score for SSc-ILD findings, primarily because of changes in ground glass opacities. These differences may be clinically meaningful.

Arabidopsis B-box transcription factors BBX20-22 promote UVR8 photoreceptor-mediated UV-B responses.

Plants undergo photomorphogenic development in the presence of light. Photomorphogenesis is repressed by the E3 ubiquitin ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1), which binds to substrates through their valine-proline (VP) motifs. The UV RESISTANCE LOCUS 8 (UVR8) photoreceptor senses UV-B and inhibits COP1 through the cooperative binding of its own VP motif and photosensing core to COP1, thereby preventing COP1 binding to substrates, including the basic leucine zipper (bZIP) transcriptional regulator ELONGATED HYPOCOTYL 5 (HY5). As a key promoter of visible light and UV-B photomorphogenesis, HY5 requires coregulators for its function. The B-box family transcription factors BBX20-BBX22 were recently described as HY5 rate-limiting coactivators under red light, but their role in UVR8 signaling was unknown. Here we describe a hypermorphic bbx21-3D mutant with enhanced photomorphogenesis, carrying a proline-to-leucine mutation at position 314 in the VP motif that impairs the interaction with and regulation by COP1. We show that BBX21 and BBX22 are UVR8-dependently stabilized after UV-B exposure, which is counteracted by a repressor induced by HY5/BBX activity. bbx20 bbx21 bbx22 mutants under UV-B are impaired in hypocotyl growth inhibition, photoprotective pigment accumulation and the expression of several HY5-dependent genes under continuous UV-B, but the immediate induction of marker genes after exposure to UV-B remains surprisingly rather unaffected. We conclude that BBX20-BBX22 contribute to HY5 activity in a subset of UV-B responses, but that additional, presently unknown, coactivators for HY5 are functional in early UVR8 signaling.

Kinematic Hopf Algebra for Bern-Carrasco-Johansson Numerators in Heavy-Mass Effective Field Theory and Yang-Mills Theory.

We present a closed formula for all Bern-Carrasco-Johansson (BCJ) numerators describing D-dimensional tree-level scattering amplitudes in a heavy-mass effective field theory with two massive particles and an arbitrary number of gluons. The corresponding gravitational amplitudes obtained via the double copy directly enter the computation of black-hole scattering and gravitational-wave emission. Our construction is based on finding a kinematic algebra for the numerators, which we relate to a quasishuffle Hopf algebra. The BCJ numerators thus obtained have a compact form and intriguing features: gauge invariance is manifest, locality is respected for massless exchange, and they contain poles corresponding to massive exchange. Counting the number of terms in a BCJ numerator for n-2 gluons gives the Fubini numbers F_{n-3}, reflecting the underlying quasishuffle Hopf algebra structure. Finally, by considering an appropriate factorization limit, the massive particles decouple, and we thus obtain a kinematic algebra and all tree-level BCJ numerators for D-dimensional pure Yang-Mills theory.

Proteome Analysis of Pancreatic Tumors Implicates Extracellular Matrix in Patient Outcome.

Pancreatic cancer remains a disease with unmet clinical needs and inadequate diagnostic, prognostic, and predictive biomarkers. In-depth characterization of the disease proteome is limited. This study thus aims to define and describe protein networks underlying pancreatic cancer and identify protein centric subtypes with clinical relevance. Mass spectrometry-based proteomics was used to identify and quantify the proteome in tumor tissue, tumor-adjacent tissue, and patient-derived xenografts (PDX)-derived cell lines from patients with pancreatic cancer, and tissues from patients with chronic pancreatitis. We identified, quantified, and characterized 11,634 proteins from 72 pancreatic tissue samples. Network focused analysis of the proteomics data led to identification of a tumor epithelium-specific module and an extracellular matrix (ECM)-associated module that discriminated pancreatic tumor tissue from both tumor adjacent tissue and pancreatitis tissue. On the basis of the ECM module, we defined an ECM-high and an ECM-low subgroup, where the ECM-high subgroup was associated with poor prognosis (median survival months: 15.3 vs. 22.9 months; log-rank test, P = 0.02). The ECM-high tumors were characterized by elevated epithelial-mesenchymal transition and glycolytic activities, and low oxidative phosphorylation, E2F, and DNA repair pathway activities. This study offers novel insights into the protein network underlying pancreatic cancer opening up for proteome precision medicine development. Significance: Pancreatic cancer lacks reliable biomarkers for prognostication and treatment of patients. We analyzed the proteome of pancreatic tumors, nonmalignant tissues of the pancreas and PDX-derived cell lines, and identified proteins that discriminate between patients with good and poor survival. The proteomics data also unraveled potential novel drug targets.

Reliability of the Swedish version of the multidimensional outcome expectations for exercise scale (MOEES-SW) in a cardiac population.

PURPOSE: Translate and adapt the Multidimensional Outcome Expectations for Exercise Scale (MOEES) into Swedish language and to explore psychometric properties, in terms of test-retest reliability, internal consistency as well as factor structure and floor and ceiling effects, of the Swedish version of MOEES in cardiac patients. MATERIALS AND METHODS: This prospective psychometrical study set in a Swedish cardiac rehabilitation outpatient clinic included 74 patients; age 318 years after acute coronary syndrome or that had undergone cardiac surgery. Translation and adaptation procedure of the MOEES followed established international guidelines. To evaluate test-retest reliability, relative (intra-class correlation coefficient ICC, 2.1)and absolute reliability (standard error of measurement (SEM) standard error of measurement % (SEM%), percentage of absolute agreement and Kappa coefficient for each item were calculated. Internal consistency was assessed with Cronbach´s alpha. The original 3-factor structure was tested with a confirmatory factor analysis. Floor and ceiling effects were calculated. RESULTS: In total, 60 patients, mean age 65 years, were included in the study. The test-retest showed for the three outcome expectations subscales (Physical, Self-evaluative and Social) ICC-values of 0.40 (CI 95% 0.20-0.58), 0.57 (0.39-0.71) and 0.72 (0.57-0.83), respectively. In general, the Kappa coefficients were low and varied between 0.11 and 0.44. Two questions had low loadings in the confirmatory factor analysis (<0.5) , contributing to a weak fit of the model. There was no floor effect, but the subscales physical and self-evaluative outcome expectation showed ceiling effects. CONCLUSION: This is the first study to analyse test-retest reliability of the translated version of MOEES into Swedish in cardiac patients and shows need for further development of the instrument before use in clinical practice and research.

Self-reported exercise-induced dyspnea and airways obstruction assessed by oscillometry and spirometry in adolescents.

BACKGROUND: Self-reported exercise-induced dyspnea (EID) is common among adolescents. Possible underlying pathologies are exercise-induced bronchoconstriction (EIB) and laryngeal obstruction (EILO). The forced oscillation technique (FOT) may evaluate exercise-induced changes in airway caliber. AIM: To investigate in adolescents the relationship between EID, EIB (post-exercise fall in forced expiratory volume in 1s (FEV1 )≥10%), EILO, and post-exercise challenge changes in FOT parameters. METHODS: One hundred and forty-three subjects (97 with EID) of 13-15 years old underwent a standardized exercise challenge with FOT measurement and spirometry repeatedly performed between 2 and 30 min post-exercise. EILO was studied in a subset of 123 adolescents. Subjects showing greater changes than the healthy subgroup in the modulus of the inspiratory impedance were considered FOT responders. RESULTS: EID-nonEIB subjects presented similar post-exercise changes in all FOT parameters to nonEID-nonEIB adolescents. Changes in all FOT parameters correlated with FEV1 fall. 45 of 97 EID subjects responded neither by FEV1 nor FOT to exercise. 19 and 18 subjects responded only by FEV1 (onlyFEV1 responders) or FOT (onlyFOTresponders), respectively. Only a lower baseline forced vital capacity (FVC)%predicted and a higher FEV1 /FVC distinguished the onlyFEV1 responders from onlyFOTresponders. FOT parameters did not present specific post-exercise patterns in EILO subjects. CONCLUSION: FOT can be used to identify post-exercise changes in lower airway function. However, EID has a modest relation with both FEV1 and FOT responses, highlighting the need for objective testing. More research is needed to understand whether onlyFEV1 responders and onlyFOTresponders represent different endotypes.

Proteogenomics of non-small cell lung cancer reveals molecular subtypes associated with specific therapeutic targets and immune evasion mechanisms.

Despite major advancements in lung cancer treatment, long-term survival is still rare, and a deeper understanding of molecular phenotypes would allow the identification of specific cancer dependencies and immune evasion mechanisms. Here we performed in-depth mass spectrometry (MS)-based proteogenomic analysis of 141 tumors representing all major histologies of non-small cell lung cancer (NSCLC). We identified six distinct proteome subtypes with striking differences in immune cell composition and subtype-specific expression of immune checkpoints. Unexpectedly, high neoantigen burden was linked to global hypomethylation and complex neoantigens mapped to genomic regions, such as endogenous retroviral elements and introns, in immune-cold subtypes. Further, we linked immune evasion with LAG3 via STK11 mutation-dependent HNF1A activation and FGL1 expression. Finally, we develop a data-independent acquisition MS-based NSCLC subtype classification method, validate it in an independent cohort of 208 NSCLC cases and demonstrate its clinical utility by analyzing an additional cohort of 84 late-stage NSCLC biopsy samples.

Reprogrammed transsulfuration promotes basal-like breast tumor progression via realigning cellular cysteine persulfidation.

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast tumors with poor prognosis and limited molecular-targeted therapy options. We show that BLBC cells have a high Cys demand and reprogrammed Cys metabolism. Patient-derived BLBC tumors from four different cohorts exhibited elevated expression of the transsulfuration enzyme cystathione ß-synthetase (CBS). CBS silencing (shCBS) made BLBC cells less invasive, proliferate slower, more vulnerable to oxidative stress and cystine (CySSCy) deprivation, prone to ferroptosis, and less responsive to HIF1-α activation under hypoxia. shCBS xenograft tumors grew slower than controls and exhibited impaired angiogenesis and larger necrotic areas. Sulfur metabolite profiling suggested that realigned sulfide/persulfide-inducing functions of CBS are important in BLBC tumor progression. Supporting this, the exclusion of serine, a substrate of CBS for producing Cys but not for producing sulfide/persulfide, did not exacerbate CySSCy deprivation-induced ferroptosis in shCBS BLBC cells. Impaired Tyr phosphorylation was detected in shCBS cells and xenografts, likely due to persulfidation-inhibited phosphatase functions. Overexpression of cystathione γ-lyase (CSE), which can also contribute to cellular sulfide/persulfide production, compensated for the loss of CBS activities, and treatment of shCBS xenografts with a CSE inhibitor further blocked tumor growth. Glutathione and protein-Cys levels were not diminished in shCBS cells or xenografts, but levels of Cys persulfidation and the persulfide-catabolizing enzyme ETHE1 were suppressed. Finally, expression of enzymes of the oxidizing Cys catabolism pathway was diminished, but expression of the persulfide-producing CARS2 was elevated in human BLBC tumors. Hence, the persulfide-producing pathways are major targetable determinants of BLBC pathology that could be therapeutically exploited.

Fine Tuning of Phosphorothioate Inclusion in 2'-O-Methyl Oligonucleotides Contributes to Specific Cell Targeting for Splice-Switching Modulation.

Splice-switching antisense oligonucleotide- (SSO-) mediated correction of framedisrupting mutation-containing premessenger RNA (mRNA) transcripts using exon skipping is a highly promising treatment method for muscular diseases such as Duchenne muscular dystrophy (DMD). Phosphorothioate (PS) chemistry, a commonly used oligonucleotide modification, has been shown to increase the stability of and improve the pharmacokinetics of SSOs. However, the effect of PS inclusion in 2'-O-methyl SSOs (2OMe) on cellular uptake and splice switching is less well-understood. At present, we demonstrate that the modification of PS facilitates the uptake of 2OMe in H2k-mdx myoblasts. Furthermore, we found a dependency of SSO nuclear accumulation and high splice-switching activity on PS inclusion in 2OMe (2OMePS), as tested in various reporter cell lines carrying pLuc/705. Increased exon-inclusion activity was observed in muscle, neuronal, liver, and bone cell lineages via both the gymnotic uptake and lipofection of 2OMePS. Using the photoactivatable ribonucleoside-enhanced crosslinking and a subsequent proteomic approach, we identified several 2OMePS-binding proteins, which are likely to play a role in the trafficking of 2OMePS to the nucleus. Ablation of one of them, Ncl by small-interfering RNA (siRNA) enhanced 2OMePS uptake in C2C12 myoblasts and upregulated luciferase RNA splicing in the HeLa Luc/705 reporter cell line. Overall, we demonstrate that PS inclusion increases nuclear delivery and splice switching in muscle, neuronal, liver, and bone cell lineages and that the modulation of 2OMePS-binding partners may improve SSO delivery.

Quantitative assessment of sensitizing potency using a dose-response adaptation of GARDskin.

Hundreds of chemicals have been identified as skin sensitizers. These are chemicals that possess the ability to induce hypersensitivity reactions in humans, giving rise to a condition termed allergic contact dermatitis. The capacity to limit hazardous exposure to such chemicals depends upon the ability to accurately identify and characterize their skin sensitizing potency. This has traditionally been accomplished using animal models, but their widespread use offers challenges from both an ethical and a scientific perspective. Comprehensive efforts have been made by the scientific community to develop new approach methodologies (NAMs) capable of replacing in vivo assays, which have successfully yielded several methods that can identify skin sensitizers. However, there is still a lack of new approaches that can effectively measure skin sensitizing potency. We present a novel methodology for quantitative assessment of skin sensitizing potency, which is founded on the already established protocols of the GARDskin assay. This approach analyses dose-response relationships in the GARDskin assay to identify chemical-specific concentrations that are sufficient to induce a positive response in the assay. We here compare results for 22 skin sensitizers analyzed using this method with both human and LLNA potency reference data and show that the results correlate strongly and significantly with both metrics (rLLNA = 0.81, p = 9.1 × 10-5; rHuman = 0.74, p = 1.5 × 10-3). In conclusion, the results suggest that the proposed GARDskin dose-response methodology provides a novel non-animal approach for quantitative potency assessment, which could represent an important step towards reducing the need for in vivo experiments.