Search

In ecotoxicology, evaluation of toxicities and no observed effect concentrations (NOEC) of test compounds in experimental fish is commonly based on molecular-, biochemical- and analytical chemistry analyses of organ/tissue samples and the assessment of (histo-) pathological lesions. Standardization of organ/tissue sampling locations, sample numbers, and sample processing contributes to warrant the reproducibility and inter- and intra-study comparability of analysis results. The present article provides the first comprehensive tissue sampling guidelines specifically adapted to rainbow trout (Oncorhynchus mykiss) as a frequently used fish species in ecotoxicological studies. A broad spectrum of ~40 different organs and tissues is covered. Appropriate sampling locations, sample sizes and sample numbers for subsequent routine histopathological evaluation (all organs/tissue) and for molecular analyses (~30 organs/tissues) are described in detail and illustrated with schematic drawings and representative macroscopic and histological images. These field-proven sampling guidelines were developed based on the pertinent literature and practical experience in ecotoxicological fish studies. They are intended to serve as a standard reference for any routine ecotoxicological study using rainbow trout as a test system. A broad application of the featured tissue sampling procedures will help to improve the reproducibility of analyses and to reduce inter- and intra-study variability induced by sampling bias and (normal) inter-sample morphological variation, and will therefore provide a robust basis for reliable characterization of toxicity and NOEC identification of diverse test substances and aquatic pollutants., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Fiedler et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Immunotherapy has revolutionized the treatment of advanced melanoma. Because the pathways mediating resistance to immunotherapy are largely unknown, we conducted transcriptome profiling of preimmunotherapy tumor biopsies from patients with melanoma that received PD-1 blockade or adoptive cell therapy with tumor-infiltrating lymphocytes. We identified two melanoma-intrinsic, mutually exclusive gene programs, which were controlled by IFNγ and MYC, and the association with immunotherapy outcome. MYC-overexpressing melanoma cells exhibited lower IFNγ responsiveness, which was linked with JAK2 downregulation. Luciferase activity assays, under the control of JAK2 promoter, demonstrated reduced activity in MYC-overexpressing cells, which was partly reversible upon mutagenesis of a MYC E-box binding site in the JAK2 promoter. Moreover, silencing of MYC or its cofactor MAX with siRNA increased JAK2 expression and IFNγ responsiveness of melanomas, while concomitantly enhancing the effector functions of T cells coincubated with MYC-overexpressing cells. Thus, we propose that MYC plays a pivotal role in immunotherapy resistance through downregulation of JAK2., (©2023 American Association for Cancer Research.)

Comprehensive molecular characterization of tumors aims to uncover cancer vulnerabilities, drug resistance mechanisms, and biomarkers. Identification of cancer drivers was suggested as the basis for patient-tailored therapy, and transcriptomic analyses were proposed to reveal the phenotypic outcome of cancer mutations. With the maturation of the proteomic field, studies of protein-RNA discrepancies suggested that RNA analyses are insufficient to predict cellular functions. In this article we discuss the importance of direct mRNA-protein comparisons in clinical cancer studies. We make use of the large amount of data generated by the Clinical Proteomic Tumor Analysis Consortium, which includes protein and mRNA expression analyses from the exact same samples. Analysis of protein-RNA correlations showed marked differences among cancer types, and highlighted the protein-RNA similarities and discrepancies among functional pathways and drug targets. Additionally, unsupervised clustering of the data based on protein or RNA showed substantial differences in tumor classification and the cellular processes that differentiate between clusters. These analyses show the difficulty to predict protein levels from mRNAs, and the critical role of protein analyses for phenotypic tumor characterization., Competing Interests: Conflict of interest G. A. is an employee and T. G. is an advisor of Protai Bio., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Widespread dysregulation of long non-coding RNAs [lncRNAs] including a reduction in GATA6-AS1 was noted in inflammatory bowel disease [IBD]. We previously reported a prominent inhibition of epithelial mitochondrial functions in ulcerative colitis [UC]. However, the connection between reduction of GATA6-AS1 expression and attenuated epithelial mitochondrial functions was not defined., Methods: Mucosal transcriptomics was used to conform GATA6-AS1 reduction in several treatment-naïve independent human cohorts [n=673]. RNA pull-down followed by mass spectrometry was used to determine the GATA6-AS1 interactome. Metabolomics and mitochondrial respiration following GATA6-AS1 silencing in Caco-2 cells were used to elaborate on GATA6-AS1 functions., Results: GATA6-AS1 showed predominant expression in gut epithelia using single cell datasets. GATA6-AS1 levels were reduced in Crohn's disease [CD] ileum and UC rectum in independent cohorts. Reduced GATA6-AS1 lncRNA was further linked to a more severe UC form, and to a less favourable UC course. The GATA6-AS1 interactome showed robust enrichment for mitochondrial proteins, and included TGM2, an autoantigen in coeliac disease that is induced in UC, CD and coeliac disease, in contrast to GATA6-AS1 reduction in these cohorts. GATA6-AS1 silencing resulted in induction of TGM2, and this was coupled with a reduction in mitochondrial membrane potential and mitochondrial respiration, as well as in a reduction of metabolites linked to aerobic respiration relevant to mucosal inflammation. TGM2 knockdown in GATA6-AS1-deficient cells rescued mitochondrial respiration., Conclusions: GATA6-AS1 levels are reduced in UC, CD and coeliac disease, and in more severe UC forms. We highlight GATA6-AS1 as a target regulating epithelial mitochondrial functions, potentially through controlling TGM2 levels., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation.)

As a facultative intracellular pathogen, Salmonella enterica serovar Typhimurium is one of the leading causes of food-borne diseases in humans. With the ingestion of fecal contaminated food or water, S. Typhimurium reaches the intestine. Here, the pathogen efficiently invades intestinal epithelial cells of the mucosal epithelium by the use of multiple virulence factors. Recently, chitinases have been described as emerging virulence factors of S. Typhimurium that contribute to the attachment and invasion of the intestinal epithelium, prevent immune activation, and modulate the host glycome. Here we find that the deletion of chiA leads to diminished adhesion and invasion of polarized intestinal epithelial cells (IEC) compared to wild-type S. Typhimurium. Interestingly, no apparent impact on interaction was detected when using non-polarized IEC or HeLa epithelial cells. In concordance, we demonstrate that chiA gene and ChiA protein expression was solely induced when bacteria gain contact with polarized IEC. The induction of chiA transcripts needs the specific activity of transcriptional regulator ChiR, which is co-localized with chiA in the chitinase operon. Moreover, we established that after chiA is induced, a major portion of the bacterial population expresses chiA, analyzed by flow cytometry. Once expressed, we found ChiA in the bacterial supernatants using Western blot analyses. ChiA secretion was completely abolished when accessory genes within the chitinase operon encoding for a holin and a peptidoglycan hydrolase were deleted. Holins, peptidoglycan hydrolases, and large extracellular enzymes in close proximity have been described as components of the bacterial holin/peptidoglycan hydrolase-dependent protein secretion system or Type 10 Secretion System. Overall, our results confirm that chitinase A is an important virulence factor, tightly regulated by ChiR, that promotes adhesion and invasion upon contact with polarized IEC and is likely secreted by a Type 10 Secretion System (T10SS)., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Krone et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Halogenation of organic molecules causes chemical shifts of C1s core-level binding energies that are commonly used as fingerprints to identify chemical species. Here, we use synchrotron-based X-ray photoelectron spectroscopy and density functional theory calculations to unravel such chemical shifts by examining different partially fluorinated pentacene derivatives. Core-level shifts occur even for carbon atoms distant from the fluorination positions, yielding a continuous shift of about 1.8 eV with increasing degree of fluorination for pentacenes. Since also their LUMO energies shift markedly with the degree of fluorination of the acenes, core-level shifts result in a nearly constant excitation energy of the leading π* resonance as obtained in complementary recorded K-edge X-ray absorption spectra, hence demonstrating that local fluorination affects the entire π-system, including valence and core levels. Our results thus challenge the common picture of characteristic chemical core-level energies as fingerprint signatures of fluorinated π-conjugated molecules.

No current screening methods for high-grade ovarian cancer (HGOC) guarantee effective early detection for high-risk women such as germline BRCA mutation carriers. Therefore, the standard-of-care remains risk-reducing salpingo-oophorectomy (RRSO) around age 40. Proximal liquid biopsy is a promising source of biomarkers, but sensitivity has not yet qualified for clinical implementation. We aimed to develop a proteomic assay based on proximal liquid biopsy, as a decision support tool for monitoring high-risk population. Ninety Israeli BRCA1 or BRCA2 mutation carriers were included in the training set (17 HGOC patients and 73 asymptomatic women), (BEDOCA trial; ClinicalTrials.gov Identifier: NCT03150121). The proteome of the microvesicle fraction of the samples was profiled by mass spectrometry and a classifier was developed using logistic regression. An independent cohort of 98 BRCA mutation carriers was used for validation. Safety information was collected for all women who opted for uterine lavage in a clinic setting. We present a 7-protein diagnostic signature, with AUC >0.97 and a negative predictive value (NPV) of 100% for detecting HGOC. The AUC of the biomarker in the independent validation set was >0.94 and the NPV >99%. The sampling procedure was clinically acceptable, with favorable pain scores and safety. We conclude that the acquisition of Müllerian tract proximal liquid biopsies in women at high-risk for HGOC and the application of the BRCA-specific diagnostic assay demonstrates high sensitivity, specificity, technical feasibility and safety. Similar classifier for an average-risk population is warranted., (© 2022 UICC.)

Global warming is likely to increase the proportion of intense hurricanes in the North Atlantic. Here, we analyze how this may affect economic growth. To this end, we introduce an event-based macroeconomic growth model that temporally resolves how growth depends on the heterogeneity of hurricane shocks. For the United States, we find that economic growth losses scale superlinearly with shock heterogeneity. We explain this by a disproportional increase of indirect losses with the magnitude of direct damage, which can lead to an incomplete recovery of the economy between consecutive intense landfall events. On the basis of two different methods to estimate the future frequency increase of intense hurricanes, we project annual growth losses to increase between 10 and 146% in a 2°C world compared to the period 1980-2014. Our modeling suggests that higher insurance coverage can compensate for this climate change-induced increase in growth losses.

Tumor cells present complex behaviors in their interactions with other cells. This intricate behavior is driving the need to develop new tools to understand these ecosystems. The surge of spatial technologies allows evaluation of the complexity of relationships between cells present in a tumor, giving insights about tumor heterogeneity and the tumor microenvironment while providing clinically relevant metrics for tumor classification. In this review, we describe key results obtained using spatial techniques, present recent advances in methods to uncover spatially relevant biological significance, and summarize their main characteristics. We expect spatial technologies to significantly broaden our understanding of tumor biology and to generate clinically relevant tools that will ultimately impact personalized medicine., Competing Interests: Declaration of interests The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Exercise prevents cancer incidence and recurrence, yet the underlying mechanism behind this relationship remains mostly unknown. Here we report that exercise induces the metabolic reprogramming of internal organs that increases nutrient demand and protects against metastatic colonization by limiting nutrient availability to the tumor, generating an exercise-induced metabolic shield. Proteomic and ex vivo metabolic capacity analyses of murine internal organs revealed that exercise induces catabolic processes, glucose uptake, mitochondrial activity, and GLUT expression. Proteomic analysis of routinely active human subject plasma demonstrated increased carbohydrate utilization following exercise. Epidemiologic data from a 20-year prospective study of a large human cohort of initially cancer-free participants revealed that exercise prior to cancer initiation had a modest impact on cancer incidence in low metastatic stages but significantly reduced the likelihood of highly metastatic cancer. In three models of melanoma in mice, exercise prior to cancer injection significantly protected against metastases in distant organs. The protective effects of exercise were dependent on mTOR activity, and inhibition of the mTOR pathway with rapamycin treatment ex vivo reversed the exercise-induced metabolic shield. Under limited glucose conditions, active stroma consumed significantly more glucose at the expense of the tumor. Collectively, these data suggest a clash between the metabolic plasticity of cancer and exercise-induced metabolic reprogramming of the stroma, raising an opportunity to block metastasis by challenging the metabolic needs of the tumor., Significance: Exercise protects against cancer progression and metastasis by inducing a high nutrient demand in internal organs, indicating that reducing nutrient availability to tumor cells represents a potential strategy to prevent metastasis. See related commentary by Zerhouni and Piskounova, p. 4124., (©2022 The Authors; Published by the American Association for Cancer Research.)

The mTORC1 substrate, S6 Kinase 1 (S6K1), is involved in the regulation of cell growth, ribosome biogenesis, glucose homeostasis, and adipogenesis. Accumulating evidence has suggested a role for mTORC1 signaling in the DNA damage response. This is mostly based on the findings that mTORC1 inhibitors sensitized cells to DNA damage. However, a direct role of the mTORC1-S6K1 signaling pathway in DNA repair and the mechanism by which this signaling pathway regulates DNA repair is unknown. In this study, we discovered a novel role for S6K1 in regulating DNA repair through the coordinated regulation of the cell cycle, homologous recombination (HR) DNA repair (HRR) and mismatch DNA repair (MMR) mechanisms. Here, we show that S6K1 orchestrates DNA repair by phosphorylation of Cdk1 at serine 39, causing G2/M cell cycle arrest enabling homologous recombination and by phosphorylation of MSH6 at serine 309, enhancing MMR. Moreover, breast cancer cells harboring RPS6KB1 gene amplification show increased resistance to several DNA damaging agents and S6K1 expression is associated with poor survival of breast cancer patients treated with chemotherapy. Our findings reveal an unexpected function of S6K1 in the DNA repair pathway, serving as a tumorigenic barrier by safeguarding genomic stability., Competing Interests: AA, VB, AJ, YC, GB, EA, ZS, BM, FH, AS, MA, MB, RW, RA, TG, RK No competing interests declared, (© 2022, Amar-Schwartz, Ben Hur, Jbara et al.)

In the equine clinic of the LMU in Munich, therapeutic vitrectomies have been routinely performed in horses for three decades. The vitreous samples obtained during vitrectomies were usually tested for anti- Leptospira antibodies and for more than 20 years also by PCR for leptospiral DNA. If the indication for surgery was ophthalmologically inconclusive, an aqueous humor was collected preoperatively and examined for evidence of leptospiral infection. In this study, medical records from 2002 to 2017 were analyzed. Records for 1387 eyes affected by equine recurrent uveitis (ERU) and 237 eyes affected by another type of uveitis met the inclusion criteria. A total of 216 samples from healthy eyes were used as controls. In 83% of intraocular samples from ERU eyes, antibody titers of 1:100 or higher were detectable by microscopic agglutination test (MAT). Similarly, 83% of intraocular samples had anti- Leptospira antibodies detected by ELISA. In 72% of the intraocular specimens, leptospiral DNA was detectable by PCR. No antibodies were detectable in the samples from eyes with another type of uveitis or in the samples from healthy eyes. A PCR was positive in only one sample from a healthy eye. These results with a very high number of intraocular specimens demonstrate the great importance of an intraocular leptospiral infection for ERU. It can be concluded that for a reliable diagnosis of intraocular leptospiral infection or to reliably exclude an infection multiple tests should be applied.

Single-cell analyses characterize individual cells, allowing their clustering and characterization in an unsupervised manner. Single-cell genomics and transcriptomics dominate the field of single-cell analysis, however, these often do not accurately reflect cellular functions. In contrast, single-cell protein analyses were until recently only performed using antibody-based approaches. This review aims to highlight the recent developments in mass spectrometry-based single-cell proteomics and discuss the challenges and opportunities. Advances in the field hold the promise to impact biomedical research and contribute to the understanding of complex biological systems., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Anal intraepithelial neoplasia is a precursor to anal carcinoma. The use of anal pap cytology has been accepted as a screening method for anal carcinoma, however sensitivity and specificity vary., Materials and Methods: Retrospective cohort study involving 155 HIV-positive males with abnormal anal cytology and surgical resection., Results: 155 patients met inclusion criteria. 31.6% were diagnosed with atypical cytology, 61.9% with low-grade cytology, and 6.4% with high-grade cytology. At surgery, 19.4% were diagnosed with condylomata, 34.8% with anal intraepithelial neoplasia 1, 17.4% with anal intraepithelial neoplasia 2, 27.1% with anal intraepithelial neoplasia 3 and 1.3% with anal carcinoma. There was a positive correlation between high-grade anal cytology and high-grade histology (r = 0.27; p = 0.0008). Comparison of risk factors showed no significant association., Conclusion: Anal cytology has a significant correlation with surgical histology. There were still instances of high-grade lesions being found after low-grade cytology. This highlights the necessity of patients with low-grade cytology undergoing anoscopic evaluation., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Structural characterization techniques are fundamental to correlate the material macro-, nano-, and molecular-scale structures to their macroscopic properties and to engineer hierarchical materials. Here, we combine X-ray transmission with scanning small- and wide-angle X-ray scattering (sSWAXS) to investigate ultraporous and lightweight biopolymer-based foams using cellulose nanofibrils (CNFs) as building blocks. The power of multimodal sSWAXS for multiscale structural characterization of self-assembled CNFs is demonstrated by spatially resolved maps at the macroscale (foam density and porosity), at the nanoscale (foam structural compactness, CNF orientation in the foam walls, and CNF packing state), and at the molecular scale (cellulose crystallite dimensions). Specifically, we compare the impact of freeze-thawing-drying (FTD) fabrication steps, such as static/stirred freezing and thawing in ethanol/water, on foam structural hierarchy spanning from the molecular to the millimeter scale. As such, we demonstrate the potential of X-ray scattering imaging for hierarchical characterization of biopolymers.

By establishing multi-omics pipelines, we uncover overexpression and gene copy-number alterations of nucleoporin-93 (NUP93), a nuclear pore component, in aggressive human mammary tumors. NUP93 overexpression enhances transendothelial migration and matrix invasion in vitro, along with tumor growth and metastasis in animal models. These findings are supported by analyses of two sets of naturally occurring mutations: rare oncogenic mutations and inactivating familial nephrotic syndrome mutations. Mechanistically, NUP93 binds with importins, boosts nuclear transport of importins' cargoes, such as β-catenin, and activates MYC. Likewise, NUP93 overexpression enhances the ultimate nuclear transport step shared by additional signaling pathways, including TGF-β/SMAD and EGF/ERK. The emerging addiction to nuclear transport exposes vulnerabilities of NUP93-overexpressing tumors. Congruently, myristoylated peptides corresponding to the nuclear translocation signals of SMAD and ERK can inhibit tumor growth and metastasis. Our study sheds light on an emerging hallmark of advanced tumors, which derive benefit from robust nucleocytoplasmic transport., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Dysregulated homeostasis of neural activity has been hypothesized to drive Alzheimer's disease (AD) pathogenesis. AD begins with a decades-long presymptomatic phase, but whether homeostatic mechanisms already begin failing during this silent phase is unknown. We show that before the onset of memory decline and sleep disturbances, familial AD (fAD) model mice display no deficits in CA1 mean firing rate (MFR) during active wakefulness. However, homeostatic down-regulation of CA1 MFR is disrupted during non-rapid eye movement (NREM) sleep and general anesthesia in fAD mouse models. The resultant hyperexcitability is attenuated by the mitochondrial dihydroorotate dehydrogenase (DHODH) enzyme inhibitor, which tunes MFR toward lower set-point values. Ex vivo fAD mutations impair downward MFR homeostasis, resulting in pathological MFR set points in response to anesthetic drug and inhibition blockade. Thus, firing rate dyshomeostasis of hippocampal circuits is masked during active wakefulness but surfaces during low-arousal brain states, representing an early failure of the silent disease stage., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The recognition of the immune system as a key component of the tumor microenvironment (TME) led to promising therapeutics. Because such therapies benefit only subsets of patients, understanding the activities of immune cells in the TME is required. Eosinophils are an integral part of the TME especially in mucosal tumors. Nonetheless, their role in the TME and the environmental cues that direct their activities are largely unknown. We report that breast cancer lung metastases are characterized by resident and recruited eosinophils. Eosinophil recruitment to the metastatic sites in the lung was regulated by G protein-coupled receptor signaling but independent of CCR3. Functionally, eosinophils promoted lymphocyte-mediated antitumor immunity. Transcriptome and proteomic analyses identified the TME rather than intrinsic differences between eosinophil subsets as a key instructing factor directing antitumorigenic eosinophil activities. Specifically, TNFα/IFNγ-activated eosinophils facilitated CD4 + and CD8 + T-cell infiltration and promoted antitumor immunity. Collectively, we identify a mechanism by which the TME trains eosinophils to adopt antitumorigenic properties, which may lead to the development of eosinophil-targeted therapeutics. SIGNIFICANCE: These findings demonstrate antitumor activities of eosinophils in the metastatic tumor microenvironment, suggesting that harnessing eosinophil activity may be a viable clinical strategy in patients with cancer., (©2021 American Association for Cancer Research.)

Equine recurrent uveitis (ERU) is typically caused by chronic intraocular leptospiral infection in warm-blooded horses in central Europe. The most effective therapy for leptospiral-induced ERU is the surgical removal of diseased vitreous (vitrectomy). Since vitrectomy is a highly specialized and invasive surgery, the indication must be determined very carefully. In order to obtain evidence of intraocular leptospiral infection by laboratory diagnostics in questionable leptospiral ERU-cases, sampling of aqueous humor is required, because serum tests using microscopic agglutination test (MAT) are too unspecific. The SNAP Lepto is a cross-species rapid test for the detection of anti-Lipl32 antibodies that has a high sensitivity (0.97) and specificity (1.00) for the detection of anti-leptospiral antibodies using aqueous humor or vitreous samples, which is comparable to MAT. To evaluate sensitivity and specificity of SNAP Lepto using serum, serum samples from 90 horses with confirmed leptospiral ERU and from 103 ocularly healthy horses were tested by both MAT and SNAP Lepto. Sensitivity was similar for both tests (0.82 vs. 0.79), but specificity was lower for MAT (0.52 vs. 0.95). Sensitivity and specificity are therefore lower in serum samples compared to intraocular samples, however, the SNAP Lepto is far superior to MAT and suitable as a screening method using equine serum.

Young generations are severely threatened by climate change.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

In general, mRNAs are assumed to be loaded with ribosomes instantly upon entry into the cytoplasm. To measure ribosome density (RD) on nascent mRNA, we developed nascent Ribo-Seq by combining Ribo-Seq with progressive 4-thiouridine labeling. In mouse macrophages, we determined experimentally the lag between the appearance of nascent mRNA and its association with ribosomes, which was calculated to be 20-22 min for bulk mRNA. In mouse embryonic stem cells, nRibo-Seq revealed an even stronger lag of 35-38 min in ribosome loading. After stimulation of macrophages with lipopolysaccharide, the lag between cytoplasmic and translated mRNA leads to uncoupling between input and ribosome-protected fragments, which gives rise to distorted RD measurements under conditions where mRNA amounts are far from steady-state expression. As a result, we demonstrate that transcriptional changes affect RD in a passive way., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Singlet fission (SF), the photophysical process in which one singlet exciton is transformed into two triplets, depends inter alia on the coupling of electronic states. Here, we use fluorination and the resulting changes in partial charge distribution across the chromophore backbone as a particularly powerful tool to control this parameter in pentacene. We find that the introduction of a permanent dipole moment leads to an enhanced coupling of Frenkel exciton and charge transfer states and to an increased SF rate which we probed using ultrafast transient absorption spectroscopy. These findings are contrasted with H-aggregate formation and a significantly reduced triplet-pair state lifetime in a fluorinated pentacene for which the different partial charge distribution leads to a negligible dipole moment.

Climate change affects precipitation patterns. Here, we investigate whether its signals are already detectable in reported river flood damages. We develop an empirical model to reconstruct observed damages and quantify the contributions of climate and socio-economic drivers to observed trends. We show that, on the level of nine world regions, trends in damages are dominated by increasing exposure and modulated by changes in vulnerability, while climate-induced trends are comparably small and mostly statistically insignificant, with the exception of South & Sub-Saharan Africa and Eastern Asia. However, when disaggregating the world regions into subregions based on river-basins with homogenous historical discharge trends, climate contributions to damages become statistically significant globally, in Asia and Latin America. In most regions, we find monotonous climate-induced damage trends but more years of observations would be needed to distinguish between the impacts of anthropogenic climate forcing and multidecadal oscillations.

Purpose: Treatment of metastatic melanoma has dramatically improved in recent years, thanks to the development of immunotherapy and BRAF-MEK-targeted therapies. However, these developments revealed marked heterogeneity in patient response, which is yet to be fully understood. In this work, we aimed to associate the proteomic profiles of metastatic melanoma with the patient clinical information, to identify protein correlates with metastatic location and prior treatments., Experimental Design: We performed mass spectrometry-based proteomic analysis of 185 metastatic melanoma samples and followed with bioinformatics analysis to examine the association of metastatic location, BRAF status, survival, and immunotherapy response with the tumor molecular profiles., Results: Bioinformatics analysis showed a high degree of functional heterogeneity associated with the site of metastasis. Lung metastases presented higher immune-related proteins, and higher mitochondrial-related processes, which were shown previously to be associated with better immunotherapy response. In agreement, epidemiological analysis of data from the National Cancer Database showed improved response to anti-programmed death 1, mainly in patients with lung metastasis. Focus on lung metastases revealed prognostic and molecular heterogeneity and highlighted potential tissue-specific biomarkers. Analysis of the BRAF mutation status and prior treatments with MAPK inhibitors proposed the molecular basis of the effect on immunotherapy response and suggested coordinated combination of immunotherapy and targeted therapy may increase treatment efficacy., Conclusions: Altogether, the proteomic data provided novel molecular determinants of critical clinical features, including the effects of sequential treatments and metastatic locations. These results can be the basis for development of site-specific treatments toward treatment personalization., (©2021 American Association for Cancer Research.)

Cancer-specific metabolic phenotypes and their vulnerabilities represent a viable area of cancer research. In this study, we explored the association of breast cancer subtypes with different metabolic phenotypes and identified isocitrate dehydrogenase 2 (IDH2) as a key player in triple-negative breast cancer (TNBC) and HER2. Functional assays combined with mass spectrometry-based analyses revealed the oncogenic role of IDH2 in cell proliferation, anchorage-independent growth, glycolysis, mitochondrial respiration, and antioxidant defense. Genome-scale metabolic modeling identified phosphoglycerate dehydrogenase (PHGDH) and phosphoserine aminotransferase (PSAT1) as the synthetic dosage lethal (SDL) partners of IDH2. In agreement, CRISPR-Cas9 knockout of PHGDH and PSAT1 showed the essentiality of serine biosynthesis proteins in IDH2-high cells. The clinical significance of the SDL interaction was supported by patients with IDH2-high/PHGDH-low tumors, who exhibited longer survival than patients with IDH2-high/PHGDH-high tumors. Furthermore, PHGDH inhibitors were effective in treating IDH2-high cells in vitro and in vivo . Altogether, our study creates a new link between two known cancer regulators and emphasizes PHGDH as a promising target for TNBC with IDH2 overexpression. SIGNIFICANCE: These findings highlight the metabolic dependence of IDH2 on the serine biosynthesis pathway, adding an important layer to the connection between TCA cycle and glycolysis, which can be translated into novel targeted therapies., (©2021 American Association for Cancer Research.)

Glioblastoma (GBM) is the most aggressive form of glioma, with poor prognosis exhibited by most patients, and a median survival time of less than 2 years. We assemble a cohort of 87 GBM patients whose survival ranges from less than 3 months and up to 10 years and perform both high-resolution mass spectrometry proteomics and RNA sequencing (RNA-seq). Integrative analysis of protein expression, RNA expression, and patient clinical information enables us to identify specific immune, metabolic, and developmental processes associated with survival as well as determine whether they are shared between expression layers or are layer specific. Our analyses reveal a stronger association between proteomic profiles and survival and identify unique protein-based classification, distinct from the established RNA-based classification. By integrating published single-cell RNA-seq data, we find a connection between subpopulations of GBM tumors and survival. Overall, our findings establish proteomic heterogeneity in GBM as a gateway to understanding poor survival., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Extensive tumour inflammation, which is reflected by high levels of infiltrating T cells and interferon-γ (IFNγ) signalling, improves the response of patients with melanoma to checkpoint immunotherapy 1,2 . Many tumours, however, escape by activating cellular pathways that lead to immunosuppression. One such mechanism is the production of tryptophan metabolites along the kynurenine pathway by the enzyme indoleamine 2,3-dioxygenase 1 (IDO1), which is induced by IFNγ 3-5 . However, clinical trials using inhibition of IDO1 in combination with blockade of the PD1 pathway in patients with melanoma did not improve the efficacy of treatment compared to PD1 pathway blockade alone 6,7 , pointing to an incomplete understanding of the role of IDO1 and the consequent degradation of tryptophan in mRNA translation and cancer progression. Here we used ribosome profiling in melanoma cells to investigate the effects of prolonged IFNγ treatment on mRNA translation. Notably, we observed accumulations of ribosomes downstream of tryptophan codons, along with their expected stalling at the tryptophan codon. This suggested that ribosomes bypass tryptophan codons in the absence of tryptophan. A detailed examination of these tryptophan-associated accumulations of ribosomes-which we term 'W-bumps'-showed that they were characterized by ribosomal frameshifting events. Consistently, reporter assays combined with proteomic and immunopeptidomic analyses demonstrated the induction of ribosomal frameshifting, and the generation and presentation of aberrant trans-frame peptides at the cell surface after treatment with IFNγ. Priming of naive T cells from healthy donors with aberrant peptides induced peptide-specific T cells. Together, our results suggest that IDO1-mediated depletion of tryptophan, which is induced by IFNγ, has a role in the immune recognition of melanoma cells by contributing to diversification of the peptidome landscape.

Identification of early processes leading to complex tissue pathologies, such as inflammatory bowel diseases, ‎poses a major scientific and clinical challenge that is imperative for improved diagnosis and treatment. Most studies of inflammation onset focus on cellular processes and signaling molecules, while overlooking the environment in which they take place, the continuously remodeled extracellular matrix. In this study, we used colitis models for investigating extracellular-matrix dynamics during disease onset, while treating the matrix as a complete and defined entity. Through the analysis of matrix structure, stiffness and composition, we unexpectedly revealed that even prior to the first clinical symptoms, the colon displays its own unique extracellular-matrix signature and found specific markers of clinical potential, which were also validated in human subjects. We also show that the emergence of this pre-symptomatic matrix is mediated by subclinical infiltration of immune cells bearing remodeling enzymes. Remarkably, whether the inflammation is chronic or acute, its matrix signature converges at pre-symptomatic states. We suggest that the existence of a pre-symptomatic extracellular-matrix is general and relevant to a wide range of diseases., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The DNA damage response (DDR) is a complex signaling network that relies on cascades of protein phosphorylation, which are initiated by three protein kinases of the family of PI3-kinase-related protein kinases (PIKKs): ATM, ATR, and DNA-PK. ATM is missing or inactivated in the genome instability syndrome, ataxia-telangiectasia (A-T). The relative shares of these PIKKs in the response to genotoxic stress and the functional relationships among them are central questions in the genome stability field. We conducted a comprehensive phosphoproteomic analysis in human wild-type and A-T cells treated with the double-strand break-inducing chemical, neocarzinostatin, and validated the results with the targeted proteomic technique, selected reaction monitoring. We also matched our results with 34 published screens for DDR factors, creating a valuable resource for identifying strong candidates for novel DDR players. We uncovered fine-tuned dynamics between the PIKKs following genotoxic stress, such as DNA-PK-dependent attenuation of ATM. In A-T cells, partial compensation for ATM absence was provided by ATR and DNA-PK, with distinct roles and kinetics. The results highlight intricate relationships between these PIKKs in the DDR., (© 2020 The Authors.)

The fallopian tube secretory epithelial cells (FTSECs) are the cell-of-origin of most high-grade serous ovarian carcinomas (HGSOC). FTSECs are repeatedly exposed to inflammation induced by follicular fluid (FF) that is released with every ovulation cycle throughout a woman's reproductive years. Uninterrupted ovulation cycles are an established risk factor for HGSOC. Stimuli present in the FF induce an inflammatory environment which may cause DNA damage eventually leading to serous tumorigenesis. With the aim of elucidating possible mechanistic pathways, we established an 'ex vivo persistent ovulation model' mimicking the repeated exposure of human benign fallopian tube epithelium (FTE) to FF. We performed mass spectrometry analysis of the secretome of the ex vivo cultures as well as confirmatory targeted expressional and functional analyses. We demonstrated activation of the NF-κB pathway and upregulation of miR-155 following short-term exposure of FTE to human FF. Increased expression of miR-155 was also detected in primary HGSOC tumors compared with benign primary human FTE and corresponded with changes in the expression of miR-155 target genes. The phenotype of miR-155 overexpression in FTSEC cell line is of increased migratory and altered adhesion capacities. Overall, activation of the NF-κB-miR-155 axis in FTE may represent a possible link between ovulation-induced inflammation, DNA damage, and transcriptional changes that may eventually lead to serious carcinogenesis., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Numerous case studies have reported spontaneous regression of recognized metastases following primary tumor excision, but underlying mechanisms are elusive. Here, we present a model of regression and latency of metastases following primary tumor excision and identify potential underlying mechanisms., Results: Using MDA-MB-231 HM human breast cancer cells that express highly sensitive luciferase, we monitored early development stages of spontaneous metastases in BALB/c nu/nu mice. Removal of the primary tumor caused marked regression of micro-metastases, but not of larger metastases, and in vivo supplementation of tumor secretome diminished this regression, suggesting that primary tumor-secreted factors promote early metastatic growth. Correspondingly, MDA-MB-231 HM -conditioned medium increased in vitro tumor proliferation and adhesion and reduced apoptosis. To identify specific mediating factors, cytokine array and proteomic analysis of MDA-MB-231 HM secretome were conducted. The results identified significant enrichment of angiogenesis, growth factor binding and activity, focal adhesion, and metalloprotease and apoptosis regulation processes. Neutralization of MDA-MB-231 HM -secreted key mediators of these processes, IL-8, PDGF-AA, Serpin E1 (PAI-1), and MIF, each antagonized secretome-induced proliferation. Moreover, their in vivo simultaneous blockade in the presence of the primary tumor arrested the development of micro-metastases. Interestingly, in the METABRIC cohort of breast cancer patients, elevated expression of Serpin E1, IL-8, or the four factors combined predicted poor survival., Conclusions: These results demonstrate regression and latency of micro-metastases following primary tumor excision and a crucial role for primary tumor secretome in promoting early metastatic growth in MDA-MB-231 HM xenografts. If generalized, such findings can suggest novel approaches to control micro-metastases and minimal residual disease.

Tumor cell heterogeneity is primarily dictated by mutational changes, sometimes leading to clones that undergo a metastatic switch. However, little is known about tumor heterogeneity following chemotherapy perturbation. Here we studied the possible involvement of tumor-derived extracellular vesicles, often referred to as tumor-derived microparticles (TMPs), as mediators of the metastatic switch in the tumor microenvironment by hindering cell adhesion properties. Specifically, we show that highly metastatic or chemotherapy-treated breast cancer cells shed an increased number of TMPs compared to their respective controls. We found that these TMPs substantially reduce cell adhesion and disrupt actin filament structure, therefore increasing their biomechanical force pace, further implicating tumor cell dissemination as part of the metastatic cascade. Our results demonstrate that these pro-metastatic effects are mediated in part by CD44 which is highly expressed in TMPs obtained from highly metastatic cells or cells exposed to chemotherapy when compared to cells with low metastatic potential. Consequently, when we inhibited CD44 expression on TMPs by a pharmacological or a genetic approach, increased tumor cell adhesion and re-organized actin filament structure were observed. We also demonstrated that breast cancer patients treated with paclitaxel chemotherapy exhibited increased CD44-expressing TMPs. Overall, our study provides further insights into the role of TMPs in promoting metastasis, an effect which is augmented when tumor cells are exposed to chemotherapy.

We studied the structural and electronic properties of 2,3,9,10-tetrafluoropentacene (F4PEN) on Ag(111) via X-ray standing waves (XSW), low-energy electron diffraction (LEED) as well as ultraviolet and X-ray photoelectron spectroscopy (UPS and XPS). XSW revealed that the adsorption distances of F4PEN in (sub)monolayers on Ag(111) were 3.00 Å for carbon atoms and 3.05 Å for fluorine atoms. The F4PEN monolayer was essentially lying on Ag(111), and multilayers adopted π-stacking. Our study shed light not only on the F4PEN-Ag(111) interface but also on the fundamental adsorption behavior of fluorinated pentacene derivatives on metals in the context of interface energetics and growth mode., (Copyright © 2020, Wang et al.; licensee Beilstein-Institut.)

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry-based proteomic approach and analyzed a breast cancer cohort of 113 formalin-fixed paraffin-embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor-adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post-treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER-positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Endosomes are compositionally dynamic organelles that regulate signaling, nutrient status and organelle quality by specifying whether material entering the cells will be shuttled back to the cell surface or degraded by the lysosome. Recently, membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and endosomes have emerged as important players in endosomal protein sorting, dynamics and motility. Here, we show that PDZD8, a Synaptotagmin-like Mitochondrial lipid-binding Proteins (SMP) domain-containing ER transmembrane protein, utilizes distinct domains to interact with Rab7-GTP and the ER transmembrane protein Protrudin and together these components localize to an ER-late endosome MCS. At these ER-late endosome MCSs, mitochondria are also recruited to form a three-way contact. Thus, our data indicate that PDZD8 is a shared component of two distinct MCSs and suggest a role for SMP-mediated lipid transport in the regulation of endosome function.

In this issue of Cell, articles by Gillette et al., Chen et al., and Xu, et al. collectively provide a deep and comprehensive proteogenomic analysis of lung adenocarcinoma, addressing differences in patient ethnicity and smoking background. They highlight the importance of associating genomics with the functional proteomic outcome., (Copyright © 2020 Elsevier Inc. All rights reserved.)

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

Tumor progression and metastasis are the major causes of death among cancer associated mortality. Metastatic cells acquire features of migration and invasion and usually undergo epithelia-mesenchymal transition (EMT). Acquirement of these various hallmarks rely on different cellular pathways, including TGF-β and Wnt signaling. Recently, we reported that WW domain-containing oxidoreductase (WWOX) acts as a tumor suppressor and has anti-metastatic activities involving regulation of several key microRNAs (miRNAs) in triple-negative breast cancer (TNBC). Here, we report that WWOX restoration in highly metastatic MDA-MB435S cancer cells alters mRNA expression profiles; further, WWOX interacts with various proteins to exert its tumor suppressor function. Careful alignment and analysis of gene and miRNA expression in these cells revealed profound changes in cellular pathways mediating adhesion, invasion and motility. We further demonstrate that WWOX, through regulation of miR-146a levels, regulates SMAD3, which is a member of the TGF-β signaling pathway. Moreover, proteomic analysis of WWOX partners revealed regulation of the Wnt-signaling activation through physical interaction with Disheveled. Altogether, these findings underscore a significant role for WWOX in antagonizing metastasis, further highlighting its role and therapeutic potential in suppressing tumor progression.

Art restoration poses many challenges for scientists and conservators, as any restorative action can lead to lasting modification or damage to the original artefact. Recent interest in gel encapsulation has grown due to the ability to control the cleaning action; yet the restoration of modern paints such as acrylic-based systems still presents issues due to their extremely high sensitivity to most solvents. Herein, the preparation of dual physically and chemically crosslinked hydrogels based on regenerated cellulose and cinnamoyl-modified gelatin is demonstrated. These dual crosslinked hydrogels show increased mechanical strength and enhanced water retention compared to pure physically crosslinked hydrogels. When applied to acrylic-based paint surfaces, the dual crosslinked hydrogels extract a smaller amount of hydrophilic additives (albeit still leading to swelling within the paint film) versus physically crosslinked gels. It is anticipated that this dual crosslinking approach can be broadly applied to prepare gels for conservation of cultural heritage artefacts., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: People living with HIV (PLWH) residing in rural areas experience substantial barriers to HIV care, which may contribute to poor HIV health outcomes, including retention in HIV care and viral suppression. The Health Resources and Services Administration's Ryan White HIV/AIDS Program (HRSA RWHAP) is an important source of HIV medical care and support services in rural areas. The purpose of this analysis was to (1) assess the reach of the RWHAP in rural areas of the United States, (2) compare the characteristics and funded services of RWHAP provider organizations in rural and non-rural areas, and (3) compare the characteristics and clinical outcomes of RWHAP clients accessing medical care and support services in rural and non-rural areas., Methods and Findings: Data for this analysis were abstracted from the 2017 RWHAP Services Report (RSR), the primary source of annual, client-level RWHAP data. Organizations funded to deliver RWHAP any service ("RWHAP providers") were categorized as rural or non-rural according to the HRSA FORHP's definition of modified Rural-Urban Commuting Area (RUCA) codes. RWHAP clients were categorized based on their patterns of RWHAP service use as "visited only rural providers," "visited only non-rural providers," or "visited rural and non-rural providers." In 2017, among the 2,113 providers funded by the RWHAP, 6.2% (n = 132) were located in HRSA-designated rural areas. Rural providers were funded to deliver a greater number of service categories per site than non-rural providers (44.7% funded for ≥5 services vs. 34.1% funded for ≥5 services, respectively). Providers in rural areas served fewer clients than providers in non-rural areas; 47.3% of RWHAP providers in rural areas served 1-99 clients, while 29.6% of non-rural providers served 1-99 clients. Retention in care and viral suppression outcomes did not differ on the basis of whether a client accessed services from rural or non-rural providers., Conclusions: RWHAP providers are a crucial component of HIV care delivery in the rural United States despite evidence of significant barriers to engagement in care for rural PLWH, RWHAP clients who visited rural providers were just as likely to be retained in care and reach viral suppression as their counterparts who visited non-rural providers. The RWHAP, especially in partnership with Rural Health Clinics and federally funded Health Centers, has the infrastructure and expertise necessary to address the HIV epidemic in rural America., Competing Interests: The authors work for the federal agency that implements the Ryan White HIV/AIDS Program which is the subject of this paper.

The properties as well as solid-state structures, singlet fission, and organic field-effect transistor (OFET) performance of three tetrafluoropentacenes (1,4,8,11: 10, 1,4,9,10: 11, 2,3,9,10: 12) are compared herein. The novel compounds 10 and 11 were synthesized in high purity from the corresponding 6,13-etheno-bridged precursors by reaction with dimethyl 1,2,4,5-tetrazine-3,6-dicarboxylate at elevated temperatures. Although most of the molecular properties of the compounds are similar, their chemical reactivity and crystal structures differ considerably. Isomer 10 undergoes the orbital symmetry forbidden thermal [4+4] dimerization, whereas 11 and 12 are much less reactive. The isomers 11 and 12 crystallize in a herringbone motif, but 10 prefers π-π stacking. Although the energy of the first electric dipole-allowed optical transition varies only within 370 cm -1 (0.05 eV) for the neutral compounds, this amounts to roughly 1600 cm -1 (0.20 eV) for radical cations and 1300 cm -1 (0.16 eV) for dications. Transient spectroscopy of films of 11 and 12 reveals singlet-fission time constants (91±11, 73±3 fs, respectively) that are shorter than for pentacene (112±9 fs). OFET devices constructed from 11 and 12 show close to ideal thin-film transistor (TFT) characteristics with electron mobilities of 2×10 -3 and 6×10 -2  cm 2  V -1  s -1 , respectively., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The human KCTD13 gene is located within the 16p11.2 locus and copy number variants of this locus are associated with a high risk for neuropsychiatric diseases including autism spectrum disorder and schizophrenia. Studies in zebrafish point to a role of KCTD13 in proliferation of neural precursor cells which may contribute to macrocephaly in 16p11.2 deletion carriers. KCTD13 is highly expressed in the fetal human brain and in mouse cortical neurons, but its contribution to the development and function of mammalian neurons is not completely understood. In the present study, we deleted the KCTD13 gene in human-induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 nickase. Following neural differentiation of KCTD13 deficient and isogenic control iPSC lines, we detected a moderate but significant inhibition of DNA synthesis and proliferation in KCTD13 deficient human neural precursor cells. KCTD13 deficient cortical neurons derived from iPSCs showed decreased neurite formation and reduced spontaneous network activity. RNA-sequencing and pathway analysis pointed to a role for ERBB signaling in these phenotypic changes. Consistently, activating and inhibiting ERBB kinases rescued and aggravated, respectively, impaired neurite formation. In contrast to findings in non-neuronal human HeLa cells, we did not detect an accumulation of the putative KCTD13/Cullin-3 substrate RhoA, and treatment with inhibitors of RhoA signaling did not rescue decreased neurite formation in human KCTD13 knockout neurons. Taken together, our data provide insight into the role of KCTD13 in neurodevelopmental disorders, and point to ERBB signaling as a potential target for neuropsychiatric disorders associated with KCTD13 deficiency.

Typhoid toxin is a virulence factor for the bacterial pathogen Salmonella Typhi, which causes typhoid fever in humans. After its synthesis by intracellular bacteria, typhoid toxin is secreted into the lumen of the Salmonella -containing vacuole by a secretion mechanism strictly dependent on TtsA, a specific muramidase that facilitates toxin transport through the peptidoglycan layer. Here we show that substrate recognition by TtsA depends on a discrete domain within its carboxy terminus, which targets the enzyme to the bacterial poles to recognize YcbB-edited peptidoglycan. Comparison of the atomic structures of TtsA bound to its substrate and that of a close homolog with different specificity identified specific determinants involved in substrate recognition. Combined with structure-guided mutagenesis and in vitro and in vivo crosslinking experiments, this study provides an unprecedented view of the mechanisms by which a muramidase recognizes its peptidoglycan substrate to facilitate protein secretion., Competing Interests: TG, ML, YX, JG No competing interests declared, (© 2020, Geiger et al.)

MicroRNAs (miR) are small RNA molecules that shape the cell transcriptome and proteome through regulation of mRNA stability and translation. Here, we examined their function as determinants of cell resistance to complement-dependent cytotoxicity (CDC). To achieve this goal, we compared the expression of microRNAs between complement-resistant and -sensitive K562 leukemia, Raji lymphoma, and HCT-116 colorectal carcinoma cells. Global microRNA array analysis identified miR-150, miR-328, and miR-616 as regulators of CDC resistance. Inhibition of miR-150 reduced resistance, whereas inhibition of miR-328 or miR-616 enhanced cell resistance. Treatment of K562 cells with a sublytic dose of complement was shown to rapidly increase miR-150, miR-328, and miR-616 expression. Protein targets of these microRNAs were analyzed in K562 cells by mass spectrometry-based proteomics. Expression of the complement membrane regulatory proteins CD46 and CD59 was significantly enhanced after inhibition of miR-328 and miR-616. Enrichment of proteins of mitochondria, known target organelles in CDC, was observed after miR-150, miR-328, and miR-616 inhibition. In conclusion, miR-150, miR-328, and miR-616 regulate cell resistance to CDC by modifying the expression of the membrane complement regulators CD46 and CD59 and the response of the mitochondria to complement lytic attack. These microRNAs may be considered targets for intervention in complement-associated diseases and in anticancer, complement-based therapy., (©2019 American Association for Cancer Research.)