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Osteosarcoma (OS) mortality stems from lung metastases. Matrix metalloproteinases (MMPs) facilitate metastatic dissemination by degrading extracellular matrix components. Herein we studied the impact of targeted MMP downregulation on OS metastasis. Differential gene expression analysis of human OS cell lines revealed high MMP9 expression in the majority of OS cell lines. Furthermore, 143B, a metastatic OS cell line, exhibited increased MMP1 and MMP9 mRNA levels. Gene set enrichment analysis on metastatic and non-metastatic OS patient specimens indicated epithelial-mesenchymal transition as the most enriched gene set, with MMP9 displaying strong association to genes in this network. Using the same dataset, Kaplan-Meier analysis revealed a correlation between MMP1 expression and dismal patient survival. Hence, we undertook targeted suppression of MMP1 and MMP9 gene expression in OS cell lines. The ability of OS cells to migrate and form colonies was markedly reduced upon MMP1 and MMP9 downregulation, whereas their cell proliferation capacity remained intact. MMP9 downregulation decreased tumor growth and lung metastases area in an orthotopic mouse OS model. Consistently, human OS lung metastasis specimens displayed marked MMP9 protein expression. Our findings highlight the role of MMP1 and MMP9 in OS metastasis, warranting further exploration of simultaneous inhibition of MMPs for future OS therapeutics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The proteasome, the catalytic arm of the ubiquitin system, is regulated via its dynamic compartmentation between the nucleus and the cytoplasm, among other mechanisms. Under amino acid shortage, the proteolytic complex is translocated to the cytoplasm, where it stimulates proteolysis to supplement recycled amino acids for essential protein synthesis. This response is mediated via the mTOR pathway and the lack of the three aromatic amino acids Tyr, Trp, and Phe (YWF). mTOR activation by supplementation of the triad inhibits proteasome translocation, leading to cell death. We now show that tumoral inherent stress conditions result in translocation of the proteasome from the nucleus to the cytosol. We further show that the modulation of the signaling cascade governed by YWF is applicable also to non-starved cells by using higher concentration of the triad to achieve a surplus relative to all other amino acids. Based on these two phenomena, we found that the modulation of stress signals via the administration of YWF leads to nuclear proteasome sequestration and inhibition of growth of xenograft, spontaneous, and metastatic mouse tumor models. In correlation with the observed effect of YWF on tumors, we found - using transcriptomic and proteomic analyses - that the triad affects various cellular processes related to cell proliferation, migration, and death. In addition, Sestrin3-a mediator of YWF sensing upstream of mTOR-is essential for proteasome translocation, and therefore plays a pro-tumorigenic role, positioning it as a potential oncogene. This newly identified approach for hijacking the cellular "satiety center" carries therefore potential therapeutic implications for cancer., (© 2024. The Author(s).)

In T-cell acute lymphoblastic leukemia (T-ALL), more than 50% of cases display autoactivation of Notch1 signaling, leading to oncogenic transformation. We have previously identified a specific chemovar of Cannabis that induces apoptosis by preventing Notch1 maturation in leukemia cells. Here, we isolated three cannabinoids from this chemovar that synergistically mimic the effects of the whole extract. Two were previously known, cannabidiol (CBD) and cannabidivarin (CBDV), whereas the third cannabinoid, which we termed 331-18A, was identified and fully characterized in this study. We demonstrated that these cannabinoids act through cannabinoid receptor type 2 and TRPV1 to activate the integrated stress response pathway by depleting intracellular Ca 2+ . This is followed by increased mRNA and protein expression of ATF4, CHOP, and CHAC1, which is hindered by inhibiting the upstream initiation factor eIF2α. The increased abundance of CHAC1 prevents Notch1 maturation, thereby reducing the levels of the active Notch1 intracellular domain, and consequently decreasing cell viability and increasing apoptosis. Treatment with the three isolated molecules resulted in reduced tumor size and weight in vivo and slowed leukemia progression in mice models. Altogether, this study elucidated the mechanism of action of three distinct cannabinoids in modulating the Notch1 pathway, and constitutes an important step in the establishment of a new therapy for treating NOTCH1 -mutated diseases and cancers such as T-ALL., Competing Interests: EB, AG, SP, PB No competing interests declared, DM A patent pertaining to the results of the paper has been applied for the use of cannabinoids in the treatment of Notch-related diseases. This patent PCT/IL2021/051352 was licensed to Cavnox Ltd. to test the combination of the molecules in human clinical trials, (© 2023, Besser et al.)

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by ADAR enzymes, is a prevalent and conserved RNA modification. While A-to-I RNA editing is essential in mammals, in Caenorhabditis elegans, it is not, making them invaluable for RNA editing research. In C. elegans, ADR-2 is the sole catalytic A-to-I editing enzyme, and ADR-1 is an RNA editing regulator. ADAR localization is well-studied in humans but not well-established in C. elegans. In this study, we examine the cellular and tissue-specific localization of ADR-2. We show that while ADR-2 is present in most cells in the embryo, at later developmental stages, its expression is both tissue- and cell-type-specific. Additionally, both ADARs are mainly in the nucleus. ADR-2 is adjacent to the chromosomes during the cell cycle. We show that the nuclear localization of endogenous ADR-2 depends on ADBP-1, not ADR-1. In adbp-1 mutant worms, ADR-2 is mislocalized, while ADR-1 is not, leading to decreased editing levels and de-novo editing, mostly in exons, suggesting that ADR-2 is also functional in the cytoplasm. Besides, mutated ADBP-1 affects gene expression. Furthermore, we show that ADR-2 targets adenosines with different surrounding nucleotides in exons and introns. Our findings indicate that ADR-2 cellular localization is highly regulated and affects its function., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Developmental patterns of behavior are variably organized in time and among different individuals. However, long-term behavioral diversity was previously studied using pre-defined behavioral parameters, representing a limited fraction of the full individuality structure. Here, we continuously extract ∼1.2 billion body postures of ∼2,200 single C. elegans individuals throughout their full development time to create a complete developmental atlas of stereotyped and individual-unique behavioral spaces. Unsupervised inference of low-dimensional movement modes of each single individual identifies a dynamic developmental trajectory of stereotyped behavioral spaces and exposes unique behavioral trajectories of individuals that deviate from the stereotyped patterns. Moreover, classification of behavioral spaces within tens of neuromodulatory and environmentally perturbed populations shows plasticity in the temporal structures of stereotyped behavior and individuality. These results present a comprehensive atlas of continuous behavioral dynamics across development time and a general framework for unsupervised dissection of shared and unique developmental signatures of behavior., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Genome-wide studies have demonstrated regulatory roles for diverse non-coding elements, but their precise and interrelated functions have often remained enigmatic. Addressing the need for mechanistic insight, we studied their roles in expression of Lhb which encodes the pituitary gonadotropic hormone that controls reproduction. We identified a bi-directional enhancer in gonadotrope-specific open chromatin, whose functional eRNA (eRNA2) supports permissive chromatin at the Lhb locus. The central untranscribed region of the enhancer contains an iMotif (iM), and is bound by Hmgb2 which stabilizes the iM and directs transcription specifically towards the functional eRNA2. A distinct downstream lncRNA, associated with an inducible G-quadruplex (G4) and iM, also facilitates Lhb expression, following its splicing in situ. GnRH activates Lhb transcription and increased levels of all three RNAs, eRNA2 showing the highest response, while estradiol, which inhibits Lhb, repressed levels of eRNA2 and the lncRNA. The levels of these regulatory RNAs and Lhb mRNA correlate highly in female mice, though strikingly not in males, suggesting a female-specific function. Our findings, which shed new light on the workings of non-coding elements and non-canonical DNA structures, reveal novel mechanisms regulating transcription which have implications not only in the central control of reproduction but also for other inducible genes., (© 2024. The Author(s).)

Proteasome activity is crucial for cellular integrity, but how tissues adjust proteasome content in response to catabolic stimuli is uncertain. Here, we demonstrate that transcriptional coordination by multiple transcription factors is required to increase proteasome content and activate proteolysis in catabolic states. Using denervated mouse muscle as a model system for accelerated proteolysis in vivo, we reveal that a two-phase transcriptional program activates genes encoding proteasome subunits and assembly chaperones to boost an increase in proteasome content. Initially, gene induction is necessary to maintain basal proteasome levels, and in a more delayed phase (7-10 days after denervation), it stimulates proteasome assembly to meet cellular demand for excessive proteolysis. Intriguingly, the transcription factors PAX4 and α-PALNRF-1 control the expression of proteasome among other genes in a combinatorial manner, driving cellular adaptation to muscle denervation. Consequently, PAX4 and α-PALNRF-1 represent new therapeutic targets to inhibit proteolysis in catabolic diseases (e.g., type-2 diabetes, cancer)., (© 2024 Gilda et al.)

Cancer is a significant global health concern associated with multiple distinct factors, including microbial and viral infections. Numerous studies have elucidated the role of microorganisms, such as Helicobacter pylori (H. pylori), as well as viruses for example human papillomavirus (HPV), hepatitis B virus (HBV), and hepatitis C virus (HCV), in the development of human malignancies. Substantial attention has been focused on the treatment of these microorganism- and virus-associated cancers, with promising outcomes observed in studies employing peptide-based therapies. The current paper provides an overview of microbe- and virus-induced cancers and their underlying molecular mechanisms. We discuss an assortment of peptide-based therapies which are currently being developed, including tumor-targeting peptides and microbial/viral peptide-based vaccines. We describe the major technological advancements that have been made in the design, screening, and delivery of peptides as anticancer agents. The primary focus of the current review is to provide insight into the latest research and development in this field and to provide a realistic glimpse into the future of peptide-based therapies for microbe- and virus-induced neoplasms., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Fluoropyrimidines (FLs) [5-Fluorouracil, Capecitabine] are used in the treatment of several solid tumors. Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme for FL detoxification, and its deficiency could lead to severe, life-threatening or fatal toxicity after FL administration. Testing with a pharmacogenetic panel of four deleterious variants in the dihydropyrimidine dehydrogenase gene (DPYD) ( DPYD*2A, DPYD*13 , c.2846A > T, c.1129-5923C > G) prior to FL treatment, is recommended by scientific consortia (e.g., CPIC, DPWG) and drug regulatory agencies (e.g., EMA). However, this panel identifies < 20% of patients at risk of severe FL-related toxicity. Cumulative recent evidence highlights the potential clinical value of rare (minor allele frequency < 1%) and novel DPYD genetic variants for identifying an additional fraction of DPD-deficient patients at increased risk of severe FL-related toxicity. In this review, we aimed to comprehensively describe the available evidence regarding the potential clinical predictive role of novel and rare DPYD variants as toxicity markers in FL-treated patients, and to discuss the challenges and opportunities in tailoring FL treatment based upon clinical application of such markers. Although we must overcome existing barriers to the clinical implementation, the available data support that comprehensive assessment of the DPYD sequence, including rare and novel genetic variants, may significantly enhance the pre-emptive identification of at-risk patients, compared to the current targeted approach., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

The role of the endocannabinoid system (ECS) in depression and suicidality has recently emerged. The purpose of the study was to identify changes in plasma endocannabinoid concentrations of several endocannabinoids and correlate them with depressive symptoms and suicidality in patients with severe major depression undergoing electroconvulsive therapy (ECT). The study included 17 patients that were evaluated in four visits at different stages of therapy. At each visit depression, anxiety and suicidality symptoms were assessed and blood samples collected. Several endocannabinoid concentrations increased following six sessions of ECT, as 2-AG (p < 0.05) and LEA (p < 0.01), and following twelve sessions of ECT, as 2-AG (p < 0.05), AEA (p < 0.05), LEA (p < 0.05) and DH-Gly (p < 0.05). Endocannabinoids also correlated with symptoms of depression, anxiety and suicidality at baseline and at the sixth ECT session. Finally, we found one endocannabinoid, l-Gly, that differentiated between remitted and not-remitted patients at the seventh and thirteenth ECT sessions (p < 0.05). Our findings suggest that depression is markedly related to imbalance of the endocannabinoid system, and further regulated by ECT. Plasma endocannabinoids could be promising biomarkers for detection of depression response and remission., Competing Interests: Declaration of competing interest None of the authors reports any financial or other conflict of interest with regard to this study. The authors furthermore confirm that this manuscript or parts thereof have not been published and are not under consideration for publication elsewhere. They also confirm that all of them contributed significantly to the study. They furthermore, confirm giving their approval to all statements and declarations included in this submission., (Copyright © 2024. Published by Elsevier B.V.)

The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive laboratory evolution. However, in the case of the beta-lactam ampicillin, bacteria evolved in laboratory settings do not recapitulate clinical-like resistance levels, hindering efforts to identify major evolutionary paths and their dependency on genetic background. Here, we used the Microbial Evolution and Growth Arena (MEGA) plate to select ampicillin-resistant Escherichia coli mutants with varying degrees of resistance. Whole-genome sequencing of resistant isolates revealed that ampicillin resistance was acquired via a combination of single-point mutations and amplification of the gene encoding beta-lactamase AmpC. However, blocking AmpC-mediated resistance revealed latent adaptive pathways: strains deleted for ampC were able to adapt through combinations of changes in genes involved in multidrug resistance encoding efflux pumps, transcriptional regulators, and porins. Our results reveal that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance to ampicillin even independently of beta-lactamases., (© 2024. The Author(s).)

Proper distribution of organelles can play an important role in a moving cell's performance. During C. elegans gonad morphogenesis, the nucleus of the leading distal tip cell (DTC) is always found at the front, yet the significance of this localization is unknown. Here, we identified the molecular mechanism that keeps the nucleus at the front, despite a frictional force that pushes it backward. The Klarsicht/ANC-1/Syne homology (KASH) domain protein UNC-83 links the nucleus to the motor protein kinesin-1 that moves along a polarized acentrosomal microtubule network. Interestingly, disrupting nuclear positioning on its own did not affect gonad morphogenesis. However, reducing actomyosin contractility on top of nuclear mispositioning led to a dramatic phenotype: DTC splitting and gonad bifurcation. Long-term live imaging of the double knockdown revealed that, while the gonad attempted to perform a planned U-turn, the DTC was stretched due to the lagging nucleus until it fragmented into a nucleated cell and an enucleated cytoplast, each leading an independent gonadal arm. Remarkably, the enucleated cytoplast had polarity and invaded, but it could only temporarily support germ cell proliferation. Based on a qualitative biophysical model, we conclude that the leader cell employs two complementary mechanical approaches to preserve its integrity and ensure proper organ morphogenesis while navigating through a complex 3D environment: active nuclear positioning by microtubule motors and actomyosin-driven cortical contractility., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Acute inflammatory responses often involve the production of reactive oxygen and nitrogen species by innate immune cells, particularly macrophages. How activated macrophages protect themselves in the face of oxidative-inflammatory stress remains a long-standing question. Recent evidence implicates reactive sulfur species (RSS) in inflammatory responses; however, how endogenous RSS affect macrophage function and response to oxidative and inflammatory insults remains poorly understood. In this study, we investigated the endogenous pathways of RSS biogenesis and clearance in macrophages, with a particular focus on exploring how hydrogen sulfide (H 2 S)-mediated S-persulfidation influences macrophage responses to oxidative-inflammatory stress. We show that classical activation of mouse or human macrophages using lipopolysaccharide and interferon-γ (LPS/IFN-γ) triggers substantial production of H 2 S/RSS, leading to widespread protein persulfidation. Biochemical and proteomic analyses revealed that this surge in cellular S-persulfidation engaged ∼2% of total thiols and modified over 800 functionally diverse proteins. S-persulfidation was found to be largely dependent on the cystine importer xCT and the H 2 S-generating enzyme cystathionine γ-lyase and was independent of changes in the global proteome. We further investigated the role of the sulfide-oxidizing enzyme sulfide quinone oxidoreductase (SQOR), and found that it acts as a negative regulator of S-persulfidation. Elevated S-persulfidation following LPS/IFN-γ stimulation or SQOR inhibition was associated with increased resistance to oxidative stress. Upregulation of persulfides also inhibited the activation of the macrophage NLRP3 inflammasome and provided protection against inflammatory cell death. Collectively, our findings shed light on the metabolism and effects of RSS in macrophages and highlight the crucial role of persulfides in enabling macrophages to withstand and alleviate oxidative-inflammatory stress., Competing Interests: Declaration of competing interest All authors declare that they have no financial interests that may be relevant to the submitted work., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Positional proteomics methodologies have transformed protease research, and have brought mass spectrometry (MS)-based degradomics studies to the forefront of protease characterization and system-wide interrogation of protease signaling. Considerable advancements in both sensitivity and throughput of liquid chromatography (LC)-MS/MS instrumentation enable the generation of enormous positional proteomics datasets of natural and protein termini and neo-termini of cleaved protease substrates. However, concomitant progress has not been observed to the same extent in data analysis and post-processing steps, arguably constituting the largest bottleneck in positional proteomics workflows. Here, we present a computational tool, CLIPPER 2.0, that builds on prior algorithms developed for MS-based protein termini analysis, facilitating peptide-level annotation and data analysis. CLIPPER 2.0 can be used with several sample preparation workflows and proteomics search algorithms and enables fast and automated database information retrieval, statistical and network analysis, as well as visualization of terminomic datasets. We demonstrate the applicability of our tool by analyzing GluC and MMP9 cleavages in HeLa lysates. CLIPPER 2.0 is available at https://github.com/UadKLab/CLIPPER-2.0., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Plants host a large array of commensal bacteria that interact with the host. The growth of both bacteria and plants is often dependent on nutrients derived from the cognate partners, and the bacteria fine-tune host immunity against pathogens. This ancient interaction is common in all studied land plants and is critical for proper plant health and development. We hypothesized that the spatial vicinity and the long-term relationships between plants and their microbiota may promote cross-kingdom horizontal gene transfer (HGT), a phenomenon that is relatively rare in nature. To test this hypothesis, we analyzed the Arabidopsis thaliana genome and its extensively sequenced microbiome to detect events of horizontal transfer of full-length genes that transferred between plants and bacteria. Interestingly, we detected 75 unique genes that were horizontally transferred between plants and bacteria. Plants and bacteria exchange in both directions genes that are enriched in carbohydrate metabolism functions, and bacteria transferred to plants genes that are enriched in auxin biosynthesis genes. Next, we provided a proof of concept for the functional similarity between a horizontally transferred bacterial gene and its Arabidopsis homologue in planta . The Arabidopsis DET2 gene is essential for biosynthesis of the brassinosteroid phytohormones, and loss of function of the gene leads to dwarfism. We found that expression of the DET2 homologue from Leifsonia bacteria of the Actinobacteria phylum in the Arabidopsis det2 background complements the mutant and leads to normal plant growth. Together, these data suggest that cross-kingdom HGT events shape the metabolic capabilities and interactions between plants and bacteria., Competing Interests: None., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

The developing mouse pancreas is surrounded by mesoderm compartments providing signals that induce pancreas formation. Most pancreatic organoid protocols lack this mesoderm niche and only partially capture the pancreatic cell repertoire. This work aims to generate pancreatic aggregates by differentiating mouse embryonic stem cells (mESCs) into mesoderm progenitors (MPs) and pancreas progenitors (PPs), without using Matrigel. First, mESCs were differentiated into epiblast stem cells (EpiSCs) to enhance the PP differentiation rate. Next, PPs and MPs aggregated together giving rise to various pancreatic cell types, including endocrine, acinar, and ductal cells, and to endothelial cells. Single-cell RNA sequencing analysis revealed a larger endocrine population within the PP + MP aggregates, as compared to PPs alone or PPs in Matrigel aggregates. The PP + MP aggregate gene expression signatures and its endocrine population percentage closely resembled those of the endocrine population found in the mouse embryonic pancreas, which holds promise for studying pancreas development., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

Competing Interests: Conflict of interest statement. None declared.

Double-strand DNA breaks are the severest type of genomic damage, requiring rapid response to ensure survival. RecBCD helicase in prokaryotes initiates processive and rapid DNA unzipping, essential for break repair. The energetics of RecBCD during translocation along the DNA track are quantitatively not defined. Specifically, it's essential to understand the mechanism by which RecBCD switches between its binding states to enable its translocation. Here, we determine, by systematic affinity measurements, the degree of coupling between DNA and nucleotide binding to RecBCD. In the presence of ADP, RecBCD binds weakly to DNA that harbors a double overhang mimicking an unwinding intermediate. Consistently, RecBCD binds weakly to ADP in the presence of the same DNA. We did not observe coupling between DNA and nucleotide binding for DNA molecules having only a single overhang, suggesting that RecBCD subunits must both bind DNA to 'sense' the nucleotide state. On the contrary, AMPpNp shows weak coupling as RecBCD remains strongly bound to DNA in its presence. Detailed thermodynamic analysis of the RecBCD reaction mechanism suggests an 'energetic compensation' between RecB and RecD, which may be essential for rapid unwinding. Our findings provide the basis for a plausible stepping mechanism' during the processive translocation of RecBCD., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Objective: Deep-learning techniques, particularly the Transformer model, have shown great potential in enhancing the prediction performance of longitudinal health records. Previous methods focused on fixed-time risk prediction, however, time-to-event prediction is often more appropriate for clinical scenarios. Here, we present STRAFE, a generalizable survival analysis Transformer-based architecture for electronic health records., Materials and Methods: The input for STRAFE is a sequence of visits with SNOMED-CT codes in OMOP-CDM format. A Transformer-based architecture was developed to calculate probabilities of the occurrence of the event in each of 48 months. Performance was evaluated using a real-world claims dataset of over 130 000 individuals with stage 3 chronic kidney disease (CKD)., Results: STRAFE showed improved mean absolute error (MAE) compared to other time-to-event algorithms in predicting the time to deterioration to stage 5 CKD. Additionally, STRAFE showed an improved area under the receiver operating curve compared to binary outcome algorithms. We show that STRAFE predictions can improve the positive predictive value of high-risk patients by 3-fold. Finally, we suggest a novel visualization approach to predictions on a per-patient basis., Discussion: Time-to-event predictions are the most appropriate approach for clinical predictions. Our deep-learning algorithm outperformed not only other time-to-event prediction algorithms but also fixed-time algorithms, possibly due to its ability to train on censored data. We demonstrated possible clinical usage by identifying the highest-risk patients., Conclusions: The ability to accurately identify patients at high risk and prioritize their needs can result in improved health outcomes, reduced costs, and more efficient use of resources., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Medical Informatics Association. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The phosphorylation of photosystem II (PSII) and its antenna (LHCII) proteins has been studied, and its involvement in state transitions and PSII repair is known. Yet, little is known about the phosphorylation of photosystem I (PSI) and its antenna (LHCI) proteins. Here, we applied proteomics analysis to generate a map of the phosphorylation sites of the PSI-LHCI proteins in Chlorella ohadii cells that were grown under low or extreme high-light intensities (LL and HL). Furthermore, we analyzed the content of oxidized tryptophans and PSI-LHCI protein degradation products in these cells, to estimate the light-induced damage to PSI-LHCI. Our work revealed the phosphorylation of 17 of 22 PSI-LHCI subunits. The analyses detected the extensive phosphorylation of the LHCI subunits Lhca6 and Lhca7, which is modulated by growth light intensity. Other PSI-LHCI subunits were phosphorylated to a lesser extent, including PsaE, where molecular dynamic simulation proposed that a phosphoserine stabilizes ferredoxin binding. Additionally, we show that HL-grown cells accumulate less oxidative damage and degradation products of PSI-LHCI proteins, compared with LL-grown cells. The significant phosphorylation of Lhca6 and Lhca7 at the interface with other LHCI subunits suggests a physiological role during photosynthesis, possibly by altering light-harvesting characteristics and binding of other subunits., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

Parasitic protozoa of the genus Leishmania cycle between the phagolysosome of mammalian macrophages, where they reside as rounded intracellular amastigotes, and the midgut of female sand flies, which they colonize as elongated extracellular promastigotes. Previous studies indicated that protein kinase A (PKA) plays an important role in the initial steps of promastigote differentiation into amastigotes. Here, we describe a novel regulatory subunit of PKA (which we have named PKAR3) that is unique to Leishmania and most (but not all) other Kinetoplastidae. PKAR3 is localized to subpellicular microtubules (SPMT) in the cell cortex, where it recruits a specific catalytic subunit (PKAC3). Promastigotes of pkar3 or pkac3 null mutants lose their elongated shape and become rounded but remain flagellated. Truncation of an N-terminal formin homology (FH)-like domain of PKAR3 results in its detachment from the SPMT, also leading to rounded promastigotes. Thus, the tethering of PKAC3 via PKAR3 at the cell cortex is essential for maintenance of the elongated shape of promastigotes. This role of PKAR3 is reminiscent of PKARIβ and PKARIIβ binding to microtubules of mammalian neurons, which is essential for the elongation of dendrites and axons, respectively. Interestingly, PKAR3 binds nucleoside analogs, but not cAMP, with a high affinity similar to the PKAR1 isoform of Trypanosoma. We propose that these early-diverged protists have re-purposed PKA for a novel signaling pathway that spatiotemporally controls microtubule remodeling and cell shape., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Fischer Weinberger 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.)

Protein-protein interactions are at the heart of all cellular processes, with the ribosome emerging as a platform, orchestrating the nascent-chain interplay dynamics. Here, to study the characteristics governing co-translational protein folding and complex assembly, we combine selective ribosome profiling, imaging, and N-terminomics with all-atoms molecular dynamics. Focusing on conserved N-terminal acetyltransferases (NATs), we uncover diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We find that only a few residues serve as "hotspots," initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harboring hotspots are highly thermolabile, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complexation, leading to aggregation. Accordingly, conservation analysis reveals that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include phosphofructokinase, anthranilate synthase, and nucleoporin subcomplex, we employ AlphaFold-Multimer to model the complexes' complete structures. Computing MD-derived interface energy profiles, we find similar trends. Here, we propose a model based on the distribution of interface energy as a strong predictor of co-translational assembly., (© 2024. The Author(s).)

Photosynthesis fuels primary production at the base of marine food webs. Yet, in many surface ocean ecosystems, diel-driven primary production is tightly coupled to daily loss. This tight coupling raises the question: which top-down drivers predominate in maintaining persistently stable picocyanobacterial populations over longer time scales? Motivated by high-frequency surface water measurements taken in the North Pacific Subtropical Gyre (NPSG), we developed multitrophic models to investigate bottom-up and top-down mechanisms underlying the balanced control of Prochlorococcus populations. We find that incorporating photosynthetic growth with viral- and predator-induced mortality is sufficient to recapitulate daily oscillations of Prochlorococcus abundances with baseline community abundances. In doing so, we infer that grazers in this environment function as the predominant top-down factor despite high standing viral particle densities. The model-data fits also reveal the ecological relevance of light-dependent viral traits and non-canonical factors to cellular loss. Finally, we leverage sensitivity analyses to demonstrate how variation in life history traits across distinct oceanic contexts, including variation in viral adsorption and grazer clearance rates, can transform the quantitative and even qualitative importance of top-down controls in shaping Prochlorococcus population dynamics., (© 2024. The Author(s).)

Proteasomes are multisubunit, multicatalytic protein complexes present in eukaryotic cells that degrade misfolded, damaged, or unstructured proteins. In this study, we used an activity-guided proteomic methodology based on a fluorogenic peptide substrate to characterize the composition of proteasome complexes in WT yeast and the changes these complexes undergo upon the deletion of Pre9 (Δα3) or of Sem1 (ΔSem1). A comparison of whole-cell proteomic analysis to activity-guided proteasome profiling indicates that the amounts of proteasomal proteins and proteasome interacting proteins in the assembled active proteasomes differ significantly from their total amounts in the cell as a whole. Using this activity-guided profiling approach, we characterized the changes in the abundance of subunits of various active proteasome species in different strains, quantified the relative abundance of active proteasomes across these strains, and charted the overall distribution of different proteasome species within each strain. The distributions obtained by our mass spectrometry-based quantification were markedly higher for some proteasome species than those obtained by activity-based quantification alone, suggesting that the activity of some of these species is impaired. The impaired activity appeared mostly among 20S Blm10 proteasome species which account for 20% of the active proteasomes in WT. To identify the factors behind this impaired activity, we mapped and quantified known proteasome-interacting proteins. Our results suggested that some of the reduced activity might be due to the association of the proteasome inhibitor Fub1. Additionally, we provide novel evidence for the presence of nonmature and therefore inactive proteasomal protease subunits β2 and β5 in the fully assembled proteasomes., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr-Ala-Thr (TAT) motif in the third transmembrane helix. Because of a low pK a value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TAT HIMB ) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TAT HIMB , they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pK a , oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TAT HIMB and contributes to further studies on mechanisms of unique properties of TwRs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Although GqPCR activation often leads to cell survival by activating the PI3K/AKT pathway, it was previously shown that in several cell types AKT activity is reduced and leads to JNK activation and apoptosis. The mechanism of AKT inactivation in these cells involves an IGBP1-coupled PP2Ac switch that induces the dephosphorylation and inactivation of both PI3K and AKT. However, the machinery involved in the initiation of PP2A switch is not known., Methods: We used phospho-mass spectrometry to identify the phosphorylation site of PP2Ac, and raised specific antibodies to follow the regulation of this phosphorylation. Other phosphorylations were monitored by commercial antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by a TUNEL assay as well as PARP1 cleavage using SDS-PAGE and Western blotting., Results: We identified Ser24 as a phosphorylation site in PP2Ac. The phosphorylation is mediated mainly by classical PKCs (PKCα and PKCβ) but not by novel PKCs (PKCδ and PKCε). By replacing the phosphorylated residue with either unphosphorylatable or phosphomimetic residues (S24A and S24E), we found that this phosphorylation event is necessary and sufficient to mediate the PP2A switch, which ultimately induces AKT inactivation, and a robust JNK-dependent apoptosis., Conclusion: Our results show that the PP2A switch is induced by PKC-mediated phosphorylation of Ser24-PP2Ac and that this phosphorylation leads to apoptosis upon GqPCR induction of various cells. We propose that this mechanism may provide an unexpected way to treat some cancer types or problems in the endocrine machinery., (© 2024. The Author(s).)

Congenital cytomegalovirus (cCMV) is the most common intrauterine infection, leading to infant neurodevelopmental disabilities. An improved knowledge of correlates of protection against cCMV is needed to guide prevention strategies. Here, we employ an ex vivo model of human CMV (HCMV) infection in decidual tissues of women with and without preconception immunity against CMV, recapitulating nonprimary vs. primary infection at the authentic maternofetal transmission site. We show that decidual tissues of women with preconception immunity against CMV exhibit intrinsic resistance to HCMV, mounting a rapid activation of tissue-resident memory CD8 + and CD4 + T cells upon HCMV reinfection. We further reveal the role of HCMV-specific decidual-tissue-resident CD8 + T cells in local protection against nonprimary HCMV infection. The findings could inform the development of a vaccine against cCMV and provide insights for further studies of the integrity of immune defense against HCMV and other pathogens at the human maternal-fetal interface., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In all organisms, the innate immune system defends against pathogens through basal expression of molecules that provide critical barriers to invasion and inducible expression of effectors that combat infection. The adenosine deaminase that act on RNA (ADAR) family of RNA-binding proteins has been reported to influence innate immunity in metazoans. However, studies on the susceptibility of ADAR mutant animals to infection are largely lacking., Results: Here, by analyzing adr-1 and adr-2 null mutants in well-established slow-killing assays, we find that both Caenorhabditis elegans ADARs are important for organismal survival to gram-negative and gram-positive bacteria, all of which are pathogenic to humans. Furthermore, our high-throughput sequencing and genetic analysis reveal that ADR-1 and ADR-2 function in the same pathway to regulate collagen expression. Consistent with this finding, our scanning electron microscopy studies indicate adr-1;adr-2 mutant animals also have altered cuticle morphology prior to pathogen exposure., Conclusions: Our data uncover a critical role of the C. elegans ADAR family of RNA-binding proteins in promoting cuticular collagen expression, which represents a new post-transcriptional regulatory node that influences the extracellular matrix. In addition, we provide the first evidence that ADAR mutant animals have altered susceptibility to infection with several opportunistic human pathogens, suggesting a broader role of ADARs in altering physical barriers to infection to influence innate immunity., (© 2024. The Author(s).)

Synthesis of all proteins in eukaryotic cells, apart from a few organellar proteins, is done by cytosolic ribosomes. Many of these ribosomes are localized in the vicinity of the functional site of their encoded protein, enabling local protein synthesis. Studies in various organisms and tissues revealed that such locally translating ribosomes are also present near mitochondria. Here, we provide a brief summary of evidence for localized translation near mitochondria, then present data suggesting that these localized ribosomes may enable local translational regulatory processes in response to mitochondria needs. Finally, we describe the involvement of such localized ribosomes in the quality control of protein synthesis and mitochondria. These emerging views suggest that ribosomes localized near mitochondria are a hub for a variety of activities with diverse implications on mitochondria physiology., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Background: Erythropoietin-producing hepatocellular (EPH) receptors are the largest known family of receptor tyrosine kinases characterized in humans. These proteins are involved in tissue organization, synaptic plasticity, vascular development and the progression of various diseases including cancer. The Erythropoietin-producing hepatocellular receptor tyrosine kinase member EphB6 is a pseudokinase which has not attracted an equivalent amount of interest as its enzymatically-active counterparts. The aim of this study was to assess the expression of EphB6 in pituitary tumors., Methods and Results: Human normal pituitaries and pituitary tumors were examined for EphB6 mRNA expression using real-time PCR and for EphB6 protein by immunohistochemistry and Western blotting. EphB6 was highly expressed in non-functioning pituitary neuroendocrine tumors (NF-PitNETs) versus the normal pituitary and GH-secreting PitNETs. EphB6 mRNA expression was correlated with tumor size., Conclusions: Our results suggest EphB6 aberrant expression in NF-PitNETs. Future studies are warranted to determine the role and significance of EphB6 in NF-PitNETs tumorigenesis., (© 2024. The Author(s).)

Introduction: As the medical use of Cannabis is evolving there is a greater demand for high-quality products for patients. One of the main steps in the manufacturing process of medical Cannabis is drying. Most current drying methods in the Cannabis industry are relatively slow and inefficient processes. Materials and Methods: This article presents a drying method based on solid-state microwave (MW) that provides fast and uniform drying, and examines its efficiency for drying Cannabis inflorescences compared with the traditional drying method. We assessed 67 cannabinoids and 36 terpenoids in the plant in a range of drying temperatures (40°C, 50°C, 60°C, and 80°C). The identification and quantification of these secondary metabolites were done by chromatography methods. Results: This method resulted in a considerable reduction of drying time, from several days to a few hours. The multiple frequency-phase combination states of the system allowed control and prediction of moisture levels during drying, thus preventing overdrying. A drying temperature of 50°C provided the most effective results in terms of both short drying time and preservation of the composition of the secondary metabolites compared with traditional drying. At 50°C, the chemical profile of phytocannabinoids and terpenoids was best kept to that of the original plant before drying, suggesting less degradation by chemical reactions such as decarboxylation. The fast-drying time also reduced the susceptibility of the plant to microbial contamination. Conclusion: Our results support solid-state MW drying as an effective postharvest step to quickly dry the plant material for improved downstream processing with a minimal negative impact on product quality.

Background: Organisms from many distinct evolutionary lineages acquired the capacity to enter a dormant state in response to environmental conditions incompatible with maintaining normal life activities. Most studied organisms exhibit seasonal or annual episodes of dormancy, but numerous less studied organisms enter long-term dormancy, lasting decades or even centuries. Intriguingly, many planktonic animals produce encased embryos known as resting eggs or cysts that, like plant seeds, may remain dormant for decades. Herein, we studied a rotifer Brachionus plicatilis as a model planktonic species that forms encased dormant embryos via sexual reproduction and non-dormant embryos via asexual reproduction and raised the following questions: Which genes are expressed at which time points during embryogenesis? How do temporal transcript abundance profiles differ between the two types of embryos? When does the cell cycle arrest? How do dormant embryos manage energy?, Results: As the molecular developmental kinetics of encased embryos remain unknown, we employed single embryo RNA sequencing (CEL-seq) of samples collected during dormant and non-dormant embryogenesis. We identified comprehensive and temporal transcript abundance patterns of genes and their associated enriched functional pathways. Striking differences were uncovered between dormant and non-dormant embryos. In early development, the cell cycle-associated pathways were enriched in both embryo types but terminated with fewer nuclei in dormant embryos. As development progressed, the gene transcript abundance profiles became increasingly divergent between dormant and non-dormant embryos. Organogenesis was suspended in dormant embryos, concomitant with low transcript abundance of homeobox genes, and was replaced with an ATP-poor preparatory phase characterized by very high transcript abundance of genes encoding for hallmark dormancy proteins (e.g., LEA proteins, sHSP, and anti-ROS proteins, also found in plant seeds) and proteins involved in dormancy exit. Surprisingly, this period appeared analogous to the late maturation phase of plant seeds., Conclusions: The study highlights novel divergent temporal transcript abundance patterns between dormant and non-dormant embryos. Remarkably, several convergent functional solutions appear during the development of resting eggs and plant seeds, suggesting a similar preparatory phase for long-term dormancy. This study accentuated the broad novel molecular features of long-term dormancy in encased animal embryos that behave like "animal seeds"., (© 2024. The Author(s).)

We aimed to elucidate the effect of Medical Cannabis (MC) on appetite and nutritional status among patients with inflammatory bowel disease (IBD). A case series of patients with IBD were initiating treatment with MC for disease-related symptoms, at the IBD clinic of a tertiary referral medical center. Patients' demographics, anthropometrics, medical history and treatment and MC use were systematically recorded. An appetite and food frequency questionnaire (SNAQ and FFQ) were filled before, and at 3 and 6 months of treatment. Patients with IBD initiating MC were enrolled (n = 149, age 39.0 ± 14.1 years, 42.3% female), and 33.6% (n = 50) were treated for improvement of nutritional status. A modest increase in appetite after 3 months was detected among all patients enrolled (Pv = 0.08), but there were no significant differences in energy or macronutrient intake, and in patients' body mass index (BMI). A significant appetite improvement after 3 months was detected among 34.0% (n = 17) of patients, but this was not associated with increased caloric intake or BMI at 3 or 6 months. Among patients without increased appetite after 3 months of MC therapy, BMI decreased at 6 months (24.1 ± 3.7 vs. 23.4 ± 3.6, Pv = 0.010). MC may be a potential strategy to improve appetite among some patients with IBD, but not caloric intake or BMI.

Migration from one location to another often comes with a change in environmental conditions. Here, we analysed features of DNA methylation in young, adult British-Bangladeshi women who experienced different environments during their childhoods: a) migrants, who grew up in Bangladesh with exposure to comparatively higher pathogen loads and poorer health care, and b) second-generation British-Bangladeshis, born to Bangladeshi parents, who grew up in the UK. We used buccal DNA to estimate DNA methylation-based age (DNAm age) from 14 migrants and 11 second-generation migrants, aged 18-35 years. 'AgeAccel,' a measure of DNAm age, independent of chronological age, showed that the group of women who spent their childhood in Bangladesh had higher AgeAccel (P = 0.028), compared to their UK peers. Since epigenetic clocks have been proposed to be associated with maintenance processes of epigenetic systems, we evaluated the preference for concordant DNA methylation at the luteinizing hormone/choriogonadotropin receptor ( LHCGR/LHR ) locus, which harbours one of the CpGs contributing to Horvath's epigenetic clock. Measurements on both strands of individual, double-stranded DNA molecules indicate higher stability of DNA methylation states at this LHCGR/LHR locus in samples of women who grew up in Bangladesh. Together, our two independent analytical approaches imply that childhood environments may induce subtle changes that are detectable long after exposure occurred, which might reflect altered activity of the epigenetic maintenance system or a difference in the proportion of cell types in buccal tissue. This exploratory work supports our earlier findings that adverse childhood environments lead to phenotypic life history trade-offs.

Since adult stem cells are responsible for replenishing tissues throughout life, it is vital to understand how failure to undergo apoptosis can dictate stem cell behavior both intrinsically and non-autonomously. Here, we report that depletion of pro-apoptotic Bax protein bestows hair follicle stem cells with the capacity to eliminate viable neighboring cells by sequestration of TNFα in their membrane. This in turn induces apoptosis in "loser" cells in a contact-dependent manner. Examining the underlying mechanism, we find that Bax loss-of-function competitive phenotype is mediated by the intrinsic activation of NFκB. Notably, winner stem cells differentially respond to TNFα, owing to their elevated expression of TNFR2. Finally, we report that in vivo depletion of Bax results in an increased stem cell pool, accelerating wound-repair and de novo hair follicle regeneration. Collectively, we establish a mechanism of mammalian cell competition, which can have broad therapeutic implications for tissue regeneration and tumorigenesis., (© 2023. The Author(s).)

The marine tetraflagellate Cymbomonas tetramitiformis has drawn attention as an early diverging green alga that uses a phago-mixotrophic mode of nutrition (i.e., the ability to derive nourishment from both photosynthesis and bacterial prey). The Cymbomonas nuclear genome was sequenced previously, but due to the exclusive use of short-read (Illumina) data, the assembly suffered from missing a large proportion of the genome's repeat regions. For this study, we generated Oxford Nanopore long-read and additional short-read Illumina data and performed a hybrid assembly that significantly improved the total assembly size and contiguity. Numerous endogenous viral elements were identified in the repeat regions of the new assembly. These include the complete genome of a giant Algavirales virus along with many genomes of integrated Polinton-like viruses (PLVs) from two groups: Gezel-like PLVs and a novel group of prasinophyte-specific PLVs. The integrated ∼400 kb genome of the giant Algavirales virus is the first account of the association of the uncultured viral family AG&#95;03 with green algae. The complete PLV genomes from C. tetramitiformis ranged between 15 and 25 kb in length and showed a diverse gene content. In addition, heliorhodopsin gene-containing repeat elements of putative mirusvirus origin were identified. These results illustrate past (and possibly ongoing) multiple alga-virus interactions that accompanied the genome evolution of C. tetramitiformis., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Protein structure, both at the global and local level, dictates function. Proteins fold from chains of amino acids, forming secondary structures, α-helices and β-strands, that, at least for globular proteins, subsequently fold into a three-dimensional structure. Here, we show that a Ramachandran-type plot focusing on the two dihedral angles separated by the peptide bond, and entirely contained within an amino acid pair, defines a local structural unit. We further demonstrate the usefulness of this cross-peptide-bond Ramachandran plot by showing that it captures β-turn conformations in coil regions, that traditional Ramachandran plot outliers fall into occupied regions of our plot, and that thermophilic proteins prefer specific amino acid pair conformations. Further, we demonstrate experimentally that the effect of a point mutation on backbone conformation and protein stability depends on the amino acid pair context, i.e., the identity of the adjacent amino acid, in a manner predictable by our method.

Background: Clinical studies on medical cannabis (MC) treatment have shown sex-related differences, including higher susceptibility to adverse events among women and greater analgesia among men. Here, we used the Syqe metered-dose inhaler (MDI) and a single chemovar to analyze sex differences., Methods: A total of 1249 Israeli chronic pain patients were assessed for pain intensity, sleep and adverse events (AEs) over 240 days., Results: Following the first two weeks, no significant sex differences were found in the effectiveness or safety of MC treatment ( p > 0.05). Inhaled Δ 9 -THC doses did not vary significantly between sexes ( p > 0.05) except in the first month of treatment. Pain reduction and sleep improvement were similar for both sexes ( p > 0.05). The overall rate of AEs was equal and relatively low at 10% (n = 65, 10% of women and n = 60, 10% of men; χ 2 (1) = 0.05, p = 0.820). A secondary analysis of pharmacokinetic data showed no significant differences between sexes in Δ 9 -THC and its metabolite pharmacokinetics, cardiovascular measures, or AE severity ( p > 0.05)., Conclusions: Uniform MC treatment via the Syqe MDI showed no sex differences in short-term effectiveness, safety and pharmacokinetics, nor in long-term effects, under "real-life" conditions. These findings provide insights into MC treatment which may inform clinical practice and policy-making in the field.

Objectives: Diagnosis of light chain amyloidosis (AL) requires demonstration of amyloid deposits in a tissue biopsy followed by appropriate typing. Previous studies demonstrated increased dimerization of monoclonal serum free light chains (FLCs) as a pathological feature of AL. To further examine the pathogenicity of FLC, we aimed at testing amino acid sequence homology between circulating and deposited light chains (LCs)., Methods: Matched tissue biopsy and serum of 10 AL patients were subjected to tissue proteomic amyloid typing and nephelometric FLC assay, respectively. Serum FLC monomers (M) and dimers (D) were analyzed by Western blotting (WB) and mass spectrometry (MS)., Results: WB of serum FLCs showed predominance of either κ or λ type, in agreement with the nephelometric assay data. Abnormal FLC M-D patterns typical of AL amyloidosis were demonstrated in 8 AL-λ patients and in one of two AL-κ patients: increased levels of monoclonal FLC dimers, high D/M ratio values of involved FLCs, and high ratios of involved to uninvolved dimeric FLCs. MS of serum FLC dimers showed predominant constant domain sequences, in concordance with the tissue proteomic amyloid typing. Most importantly, variable domain sequence homology between circulating and deposited LC species was demonstrated, mainly in AL-λ cases., Conclusions: This is the first study to demonstrate homology between circulating FLCs and tissue-deposited LCs in AL-λ amyloidosis. The applied methodology can facilitate studying the pathogenicity of circulating FLC dimers in AL amyloidosis. The study also highlights the potential of FLC monomer and dimer analysis as a non-invasive screening tool for this disease., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

Seaweeds of the intertidal zone are subjected to diverse stresses due to environmental changes in radiation, salinity, water quality, herbivore communities, etc. Thus, marine seaweeds developed various unique compounds to deal with environmental fluctuations. Therefore, they are a good source of unique novel compounds. Here, we explored the seasonal metabolomic changes in Jania rubens and found notable changes between extracts of different seasons in the metabolomic profile and in their anticancer activity. The most bioactive extract was from samples collected during the Fall season, which demonstrated an LC50 of 178.39 (± 10.02 SD) µg/ml toward Non Small Cell Lung Cancer (NSCLC) followed by the Winter season extract. The Fall and Winter extracts also displayed more resemblance in their metabolic profile relative to Spring and Summer extracts. The Fall extract was fractionated and tested for cytotoxic activity toward an array of cancer cell lines. Eventually, using a bio-guided assay and multiple fractionation steps, we isolated and identified the essential fatty acid, eicosapentaenoic acid, as the active anticancer agent, showing an LC50 of 5.23 (± 0.07 SD) µg/ml toward NSCLC. Our results emphasize the potential use of J. rubens as a source of beneficial fatty acids and stress the importance of environmental effects on metabolic constitutes., (© 2023. Springer Nature Limited.)

5α-reductase-1 catalyzes production of various steroids, including neurosteroids. We reported previously that expression of its encoding gene, Srd5a1, drops in murine ovaries and hypothalamic preoptic area (POA) after early-life immune stress, seemingly contributing to delayed puberty and ovarian follicle depletion, and in the ovaries the first intron was more methylated at two CpGs. Here, we hypothesized that this CpG-containing locus comprises a methylation-sensitive transcriptional enhancer for Srd5a1 . We found that ovarian Srd5a1 mRNA increased 8-fold and methylation of the same two CpGs decreased up to 75% between postnatal days 10 and 30. Estradiol (E 2 ) levels rise during this prepubertal stage, and exposure of ovarian cells to E 2 increased Srd5a1 expression. Chromatin immunoprecipitation in an ovarian cell line confirmed ESR1 binding to this differentially methylated genomic region and enrichment of the enhancer modification, H3K4me1. Targeting dCas9-DNMT3 to this locus increased CpG2 methylation 2.5-fold and abolished the Srd5a1 response to E 2 . In the POA, Srd5a1 mRNA levels decreased 70% between postnatal days 7 and 10 and then remained constant without correlation to CpG methylation levels. Srd5a1 mRNA levels did not respond to E 2 in hypothalamic GT1-7 cells, even after dCas9-TET1 reduced CpG1 methylation by 50%. The neonatal drop in POA Srd5a1 expression occurs at a time of increasing glucocorticoids, and treatment of GT1-7 cells with dexamethasone reduced Srd5a1 mRNA levels; chromatin immunoprecipitation confirmed glucocorticoid receptor binding at the enhancer. Our findings on the tissue-specific regulation of Srd5a1 and its methylation-sensitive control by E 2 in the ovaries illuminate epigenetic mechanisms underlying reproductive phenotypic variation that impact life-long health., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Endocrine Society.)

The p38 members of the mitogen-activated protein kinases (MAPKs) family mediate various cellular responses to stress conditions, inflammatory signals, and differentiation factors. They are constitutively active in chronic inflammatory diseases and some cancers. The differences between their transient effects in response to signals and the chronic effect in diseases are not known. The family is composed of four isoforms, of which p38α seems to be abnormally activated in diseases. p38α and p38β are almost identical in sequence, structure, and biochemical and pharmacological properties, and the specific unique effects of each of them, if any, have not yet been revealed. This study aimed to reveal the specific effects induced by p38α and p38β, both when transiently activated in response to stress and when chronically active. This was achieved via large-scale proteomics and phosphoproteomics analyses using stable isotope labeling of two experimental systems: one, mouse embryonic fibroblasts (MEFs) deficient in each of these p38 kinases and harboring either an empty vector or vectors expressing p38α WT , p38β WT , or intrinsically active variants of these MAPKs; second, induction of transient stress by exposure of MEFs, p38α -/- , and p38β -/- MEFs to anisomycin. Significant differences in the repertoire of the proteome and phosphoproteome between cells expressing active p38α and p38β suggest distinct roles for each kinase. Interestingly, in both cases, the constitutive activation induced adaptations of the cells to the chronic activity so that known substrates of p38 were downregulated. Within the dramatic effect of p38s on the proteome and phosphoproteome, some interesting affected phosphorylation sites were those found in cancer-associated p53 and Hspb1 (HSP27) proteins and in cytoskeleton-associated proteins. Among these, was the stronger direct phosphorylation by p38α of p53-Ser309, which was validated on the Ser315 in human p53. In summary, this study sheds new light on the differences between chronic and transient p38α and p38β signaling and on the specific targets of these two kinases.

Inorganic phosphate (Pi) is a necessary macronutrient for basic biological processes. Plants modulate their root system architecture (RSA) and cellular processes to adapt to Pi deprivation albeit with a growth penalty. Excess application of Pi fertilizer, on the contrary, leads to eutrophication and has a negative environmental impact. We compared RSA, root hair elongation, acid phosphatase activity, metal ion accumulation, and brassinosteroid hormone levels of Solanum lycopersicum (tomato) and Solanum pennellii, which is a wild relative of tomato, under Pi sufficiency and deficiency conditions to understand the molecular mechanism of Pi deprivation response in tomato. We showed that S. pennellii is partially insensitive to phosphate deprivation. Furthermore, it mounts a constitutive response under phosphate sufficiency. We demonstrate that activated brassinosteroid signaling through a tomato BZR1 ortholog gives rise to the same constitutive phosphate deficiency response, which is dependent on zinc overaccumulation. Collectively, these results reveal an additional strategy by which plants can adapt to phosphate starvation., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)