A Genetic Map of the Response to DNA Damage in Human Cells.

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.

Extracellular Vesicle and Particle Biomarkers Define Multiple Human Cancers.

There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

Gut microbial metabolism drives transformation of MSH2-deficient colon epithelial cells.

The etiology of colorectal cancer (CRC) has been linked to deficiencies in mismatch repair and adenomatous polyposis coli (APC) proteins, diet, inflammatory processes, and gut microbiota. However, the mechanism through which the microbiota synergizes with these etiologic factors to promote CRC is not clear. We report that altering the microbiota composition reduces CRC in APC(Min/+)MSH2(-/-) mice, and that a diet reduced in carbohydrates phenocopies this effect. Gut microbes did not induce CRC in these mice through an inflammatory response or the production of DNA mutagens but rather by providing carbohydrate-derived metabolites such as butyrate that fuel hyperproliferation of MSH2(-/-) colon epithelial cells. Further, we provide evidence that the mismatch repair pathway has a role in regulating ß-catenin activity and modulating the differentiation of transit-amplifying cells in the colon. These data thereby provide an explanation for the interaction between microbiota, diet, and mismatch repair deficiency in CRC induction. PAPERCLIP:

FAM72A antagonizes UNG2 to promote mutagenic repair during antibody maturation.

Activation-induced cytidine deaminase (AID) catalyses the deamination of deoxycytidines to deoxyuracils within immunoglobulin genes to induce somatic hypermutation and class-switch recombination1,2. AID-generated deoxyuracils are recognized and processed by subverted base-excision and mismatch repair pathways that ensure a mutagenic outcome in B cells3-6. However, why these DNA repair pathways do not accurately repair AID-induced lesions remains unknown. Here, using a genome-wide CRISPR screen, we show that FAM72A is a major determinant for the error-prone processing of deoxyuracils. Fam72a-deficient CH12F3-2 B cells and primary B cells from Fam72a-/- mice exhibit reduced class-switch recombination and somatic hypermutation frequencies at immunoglobulin and Bcl6 genes, and reduced genome-wide deoxyuracils. The somatic hypermutation spectrum in B cells from Fam72a-/- mice is opposite to that observed in mice deficient in uracil DNA glycosylase 2 (UNG2)7, which suggests that UNG2 is hyperactive in FAM72A-deficient cells. Indeed, FAM72A binds to UNG2, resulting in reduced levels of UNG2 protein in the G1 phase of the cell cycle, coinciding with peak AID activity. FAM72A therefore causes U·G mispairs to persist into S phase, leading to error-prone processing by mismatch repair. By disabling the DNA repair pathways that normally efficiently remove deoxyuracils from DNA, FAM72A enables AID to exert its full effects on antibody maturation. This work has implications in cancer, as the overexpression of FAM72A that is observed in many cancers8 could promote mutagenesis.

Unveiling the Mutational Mechanism of the Bacterial Genotoxin Colibactin in Colorectal Cancer.

In a recent issue of Science, Wilson et al. (2019) provide direct evidence that the bacterial-produced colibactin alkylates DNA in vivo, resulting in DNA adducts, which mediates its genotoxic effect. This work reinforces the role of colibactin-producing bacteria in colon cancer pathogenesis.

Suppression of Class Switch Recombination to IgA by RASA2 and RASA3 through Inhibition of TGF-ß Signaling.

Abs play a pivotal role in adaptive immunity by binding to pathogens and initiating immune responses against infections. Processes such as somatic hypermutation and class switch recombination (CSR) enhance Ab affinity and effector functions. We previously carried out a CRISPR/Cas9 screen in the CH12F3-2 (CH12) lymphoma B cell line to identify novel factors involved in CSR. The screen showed that guide RNAs targeting both Rasa2 and Rasa3 genes were decreased in IgA-negative CH12 B cells, implying that these genes might suppress CSR. Indeed, CSR was increased when either Rasa2 or Rasa3 were knocked out in CH12 cells. Compared to controls, Rasa2-/- and Rasa3-/- CH12 cells had increased expression of activation-induced cytidine deaminase (AID) and Iα transcripts, providing an explanation for the increased CSR. The increased CSR, AID, and Iα expression in Rasa2-/- or Rasa3-/- CH12F3-2 is mediated through TGF-ß stimulation. Indeed, we found that deletion of RASA2 or RASA3 promotes a shift from noncanonical to canonical TGF-ß signaling through SMAD3. These results show that RASA2 and RASA3 are both novel regulators of TGF-ß signaling in B cells, a pathway known to be essential for CSR to IgA.

Mutagenic repair during antibody diversification: emerging insights.

Deoxyuracils (dUs) produced by activation-induced cytidine deaminase (AID) during antibody diversification are processed by base excision repair (BER) and mismatch repair (MMR) pathways that paradoxically expand this lesion within jawed vertebrate immunoglobulin (Ig) genes. We highlight new findings describing mechanisms that allow B cells to carry out mutagenic DNA repair, an essential process for antibody maturation with implications in cancer pathogenesis.

The schizophrenia risk gene C4 induces pathological synaptic loss by impairing AMPAR trafficking.

Neuroimmune interactions play a significant role in regulating synaptic plasticity in both the healthy and diseased brain. The complement pathway, an extracellular proteolytic cascade, exemplifies these interactions. Its activation triggers microglia-dependent synaptic elimination via the complement receptor 3 (CR3). Current models of pathological complement activity in the brain propose that accelerated synaptic loss resulting from overexpression of C4 (C4-OE), a gene associated with schizophrenia, follows this pathway. Here, we report that C4-mediated cortical hypoconnectivity is CR3-independent. Instead, C4-OE triggers impaired GluR1 trafficking through an intracellular mechanism involving the endosomal protein SNX27, resulting in pathological synaptic loss. Moreover, C4 circuit alterations in the prefrontal cortex, a brain region associated with neuropsychiatric disorders, were rescued by increasing neuronal levels of SNX27, which we identify as an interacting partner of this neuroimmune protein. Our results link excessive complement activity to an intracellular endo-lysosomal trafficking pathway altering synaptic plasticity.

Breast adipose tissue-derived extracellular vesicles from obese women alter tumor cell metabolism.

Breast adipose tissue is an important contributor to the obesity-breast cancer link. Extracellular vesicles (EVs) are nanosized particles containing selective cargo, such as miRNAs, that act locally or circulate to distant sites to modulate target cell functions. Here, we find that long-term education of breast cancer cells with EVs obtained from breast adipose tissue of women who are overweight or obese (O-EVs) results in increased proliferation. RNA-seq analysis of O-EV-educated cells demonstrates increased expression of genes involved in oxidative phosphorylation, such as ATP synthase and NADH: ubiquinone oxidoreductase. O-EVs increase respiratory complex protein expression, mitochondrial density, and mitochondrial respiration in tumor cells. The mitochondrial complex I inhibitor metformin reverses O-EV-induced cell proliferation. Several miRNAs-miR-155-5p, miR-10a-3p, and miR-30a-3p-which promote mitochondrial respiration and proliferation, are enriched in O-EVs relative to EVs from lean women. O-EV-induced proliferation and mitochondrial activity are associated with stimulation of the Akt/mTOR/P70S6K pathway, and are reversed upon silencing of P70S6K. This study reveals a new facet of the obesity-breast cancer link with human breast adipose tissue-derived EVs causing metabolic reprogramming of breast cancer cells.

REVEALS: an open-source multi-camera GUI for rodent behavior acquisition.

Deciphering the rich repertoire of mouse behavior is crucial for understanding the functions of both the healthy and diseased brain. However, the current landscape lacks effective, affordable, and accessible methods for acquiring such data, especially when employing multiple cameras simultaneously. We have developed REVEALS (Rodent Behavior Multi-Camera Laboratory Acquisition), a graphical user interface for acquiring rodent behavioral data via commonly used USB3 cameras. REVEALS allows for user-friendly control of recording from one or multiple cameras simultaneously while streamlining the data acquisition process, enabling researchers to collect and analyze large datasets efficiently. We release this software package as a stand-alone, open-source framework for researchers to use and modify according to their needs. We describe the details of the graphical user interface implementation, including the camera control software and the video recording functionality. We validate results demonstrating the graphical user interface's stability, reliability, and accuracy for capturing rodent behavior using DeepLabCut in various behavioral tasks. REVEALS can be incorporated into existing DeepLabCut, MoSeq, or other custom pipelines to analyze complex behavior. In summary, REVEALS offers an interface for collecting behavioral data from single or multiple perspectives, which, when combined with deep learning algorithms, enables the scientific community to identify and characterize complex behavioral phenotypes.

The shieldin complex mediates 53BP1-dependent DNA repair.

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.

"SWALLOWING AND ASPIRATION: HOW TO EVALUATE AND TREAT SWALLOWING DISORDERS ASSOCIATED WITH ASPIRATION PNEUMONIA IN OLDER PERSONS".

BACKGROUND Aspiration pneumonia (AP) is the most severe complication of oropharyngeal dysphagia (OD). It is highly underdiagnosed and undertreated among older patients hospitalized with community-acquired pneumonia (CAP). Our aim is to review the state of the art in the diagnosis and treatment of swallowing disorders associated with AP. METHODOLOGY We performed a narrative review, including our experience with prior studies at Hospital de Mataró, on the diagnosis and treatment of AP. RESULTS AP refers to pneumonia occurring in patients with swallowing disorders, frequently coinciding with poor oral health and vulnerability. Its main risk factors include oropharyngeal aspiration, impaired health status, malnutrition, frailty, immune dysfunction and oral colonization by respiratory pathogens. Incidence is estimated at between 5%-15% of cases of CAP but it is highly underdiagnosed. Diagnostic criteria for AP have not been standardized but should include its main pathophysiological element, oropharyngeal aspiration. Recently, a clinical algorithm was proposed, based on the recommendations of the Japanese Respiratory Society (JRS), that includes aspiration risk factors and clinical evaluation of OD. To facilitate the task for healthcare professionals, new AI-based screening tools for OD combined with validated clinical methods such as the volume-viscosity swallowing test (V-VST) for the detection of AP are being validated. Prevention and treatment of AP require multimodal interventions aimed to cover the main risk factors: textural adaptation of fluids and diets to avoid oropharyngeal aspiration; nutritional support to avoid malnutrition; and oral hygiene to reduce oral bacterial load. CONCLUSIONS The diagnosis of AP must be based on standardized criteria providing evidence on the main etiological factor, oropharyngeal aspiration. Clinical algorithms are valid in the diagnosis of AP and the identification of its main risk factors. Combination of AI-based tools with V-VST can lead to massive screening of OD and save resources and improve efficiency in the detection AP.

Hardware Implementations of a Deep Learning Approach to Optimal Configuration of Reconfigurable Intelligence Surfaces.

Reconfigurable intelligent surfaces (RIS) offer the potential to customize the radio propagation environment for wireless networks, and will be a key element for 6G communications. However, due to the unique constraints in these systems, the optimization problems associated to RIS configuration are challenging to solve. This paper illustrates a new approach to the RIS configuration problem, based on the use of artificial intelligence (AI) and deep learning (DL) algorithms. Concretely, a custom convolutional neural network (CNN) intended for edge computing is presented, and implementations on different representative edge devices are compared, including the use of commercial AI-oriented devices and a field-programmable gate array (FPGA) platform. This FPGA option provides the best performance, with ×20 performance increase over the closest FP32, GPU-accelerated option, and almost ×3 performance advantage when compared with the INT8-quantized, TPU-accelerated implementation. More noticeably, this is achieved even when high-level synthesis (HLS) tools are used and no custom accelerators are developed. At the same time, the inherent reconfigurability of FPGAs opens a new field for their use as enabler hardware in RIS applications.

Correction: Vicencio et al. Transcriptional Signatures and Network-Based Approaches Identified Master Regulators Transcription Factors Involved in Experimental Periodontitis Pathogenesis. Int. J. Mol. Sci. 2023, 24, 14835.

In the original publication [...].

Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma.

OBJECTIVE: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models. DESIGN: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation. RESULTS: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment. CONCLUSION: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.

Mast cells impair melanoma cell homing and metastasis by inhibiting HMGA1 secretion.

Metastatic disease is the major cause of death from cancer. From the primary tumour, cells remotely prepare the environment of the future metastatic sites by secreted factors and extracellular vesicles. During this process, known as pre-metastatic niche formation, immune cells play a crucial role. Mast cells are haematopoietic bone marrow-derived innate immune cells whose function in lung immune response to invading tumours remains to be defined. We found reduced melanoma lung metastasis in mast cell-deficient mouse models (Wsh and MCTP5-Cre-RDTR), supporting a pro-metastatic role for mast cells in vivo. However, due to evidence pointing to their antitumorigenic role, we studied the impact of mast cells in melanoma cell function in vitro. Surprisingly, in vitro co-culture of bone-marrow-derived mast cells with melanoma cells showed that they have an intrinsic anti-metastatic activity. Mass spectrometry analysis of melanoma-mast cell co-cultures secretome showed that HMGA1 secretion by melanoma cells was significantly impaired. Consistently, HMGA1 knockdown in B16-F10 cells reduced their metastatic capacity in vivo. Importantly, analysis of HMGA1 expression in human melanoma tumours showed that metastatic tumours with high HMGA1 expression are associated with reduced overall and disease-free survival. Moreover, we show that HMGA1 is reduced in the nuclei and enriched in the cytoplasm of melanoma metastatic lesions when compared to primary tumours. These data suggest that high HMGA1 expression and secretion from melanoma cells promote metastatic behaviour. Targeting HMGA1 expression intrinsically or extrinsically by mast cells actions reduce melanoma metastasis. Our results pave the way to the use of HMGA1 as anti-metastatic target in melanoma as previously suggested in other cancer types.

Loss of kinesin-8 improves the robustness of the self-assembled spindle in Schizosaccharomyces pombe.

Chromosome segregation in female meiosis in many metazoans is mediated by acentrosomal spindles, the existence of which implies that microtubule spindles self-assemble without the participation of the centrosomes. Although it is thought that acentrosomal meiosis is not conserved in fungi, we recently reported the formation of self-assembled microtubule arrays, which were able to segregate chromosomes, in fission yeast mutants, in which the contribution of the spindle pole body (SPB; the centrosome equivalent in yeast) was specifically blocked during meiosis. Here, we demonstrate that this unexpected microtubule formation represents a bona fide type of acentrosomal spindle. Moreover, a comparative analysis of these self-assembled spindles and the canonical SPB-dependent spindle reveals similarities and differences; for example, both spindles have a similar polarity, but the location of the γ-tubulin complex differs. We also show that the robustness of self-assembled spindles can be reinforced by eliminating kinesin-8 family members, whereas kinesin-8 mutants have an adverse impact on SPB-dependent spindles. Hence, we consider that reinforced self-assembled spindles in yeast will help to clarify the molecular mechanisms behind acentrosomal meiosis, a crucial step towards better understanding gametogenesis.

GPR43 stimulation on TCRαß+ intraepithelial colonic lymphocytes inhibits the recruitment of encephalitogenic T-cells into the central nervous system and attenuates the development of autoimmunity.

INTRODUCTION: Gut microbiota plays a critical role in the regulation of immune homeostasis. Accordingly, several autoimmune disorders have been associated with dysbiosis in the gut microbiota. Notably, the dysbiosis associated with central nervous system (CNS) autoimmunity involves a substantial reduction of bacteria belonging to Clostridia clusters IV and XIVa, which constitute major producers of short-chain fatty acids (SCFAs). Here we addressed the role of the surface receptor-mediated effects of SCFAs on mucosal T-cells in the development of CNS autoimmunity. METHODS: To induce CNS autoimmunity, we used the mouse model of experimental autoimmune encephalomyelitis (EAE) induced by immunization with the myelin oligodendrocyte glycoprotein (MOG)-derived peptide (MOG35-55 peptide). To address the effects of GPR43 stimulation on colonic TCRαß+ T-cells upon CNS autoimmunity, mucosal lymphocytes were isolated and stimulated with a selective GPR43 agonist ex vivo and then transferred into congenic mice undergoing EAE. Several subsets of lymphocytes infiltrating the CNS or those present in the gut epithelium and gut lamina propria were analysed by flow cytometry. In vitro migration assays were conducted with mucosal T-cells using transwells. RESULTS: Our results show a sharp and selective reduction of intestinal propionate at the peak of EAE development, accompanied by increased IFN-γ and decreased IL-22 in the colonic mucosa. Further analyses indicated that GPR43 was the primary SCFAs receptor expressed on T-cells, which was downregulated on colonic TCRαß+ T-cells upon CNS autoimmunity. The pharmacologic stimulation of GPR43 increased the anti-inflammatory function and reduced the pro-inflammatory features in several TCRαß+ T-cell subsets in the colonic mucosa upon EAE development. Furthermore, GPR43 stimulation induced the arrest of CNS-autoreactive T-cells in the colonic lamina propria, thus avoiding their infiltration into the CNS and dampening the disease development. Mechanistic analyses revealed that GPR43-stimulation on mucosal TCRαß+ T-cells inhibits their CXCR3-mediated migration towards CXCL11, which is released from the CNS upon neuroinflammation. CONCLUSIONS: These findings provide a novel mechanism involved in the gut-brain axis by which bacterial-derived products secreted in the gut mucosa might control the CNS tropism of autoreactive T-cells. Moreover, this study shows GPR43 expressed on T-cells as a promising therapeutic target for CNS autoimmunity.

AID in Antibody Diversification: There and Back Again.

Activation-Induced cytidine Deaminase (AID) initiates affinity maturation and isotype switching by deaminating deoxycytidines within immunoglobulin genes, leading to somatic hypermutation (SHM) and class switch recombination (CSR). AID thus potentiates the humoral response to clear pathogens. Marking the 20th anniversary of the discovery of AID, we review the current understanding of AID function. We discuss AID biochemistry and how error-free forms of DNA repair are co-opted to prioritize mutagenesis over accuracy during antibody diversification. We discuss the regulation of DNA double-strand break (DSB) repair pathways during CSR. We describe genomic targeting of AID as a multilayered process involving chromatin architecture, cis- and trans-acting factors, and determining mutagenesis - distinct from AID occupancy at loci that are spared from mutation.

Highly unstable heterogeneous representations in VIP interneurons of the anterior cingulate cortex.

A hallmark of the anterior cingulate cortex (ACC) is its functional heterogeneity. Functional and imaging studies revealed its importance in the encoding of anxiety-related and social stimuli, but it is unknown how microcircuits within the ACC encode these distinct stimuli. One type of inhibitory interneuron, which is positive for vasoactive intestinal peptide (VIP), is known to modulate the activity of pyramidal cells in local microcircuits, but it is unknown whether VIP cells in the ACC (VIPACC) are engaged by particular contexts or stimuli. Additionally, recent studies demonstrated that neuronal representations in other cortical areas can change over time at the level of the individual neuron. However, it is not known whether stimulus representations in the ACC remain stable over time. Using in vivo Ca2+ imaging and miniscopes in freely behaving mice to monitor neuronal activity with cellular resolution, we identified individual VIPACC that preferentially activated to distinct stimuli across diverse tasks. Importantly, although the population-level activity of the VIPACC remained stable across trials, the stimulus-selectivity of individual interneurons changed rapidly. These findings demonstrate marked functional heterogeneity and instability within interneuron populations in the ACC. This work contributes to our understanding of how the cortex encodes information across diverse contexts and provides insight into the complexity of neural processes involved in anxiety and social behavior.