Early-onset cancers: Biological bases and clinical implications.

Since the nineties, the incidence of sporadic early-onset (EO) cancers has been rising worldwide. The underlying reasons are still unknown. However, identifying them is vital for advancing both prevention and intervention. Here, we exploit available knowledge derived from clinical observations to formulate testable hypotheses aimed at defining the causal factors of this epidemic and discuss how to experimentally test them. We explore the potential impact of exposome changes from the millennials to contemporary young generations, considering both environmental exposures and enhanced susceptibilities to EO-cancer development. We emphasize how establishing the time required for an EO cancer to develop is relevant to defining future screening strategies. Finally, we discuss the importance of integrating multi-dimensional data from international collaborations to generate comprehensive knowledge and translate these findings back into clinical practice.

RAD51 protects abasic sites to prevent replication fork breakage.

Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.

A high-throughput screening identifies MCM chromatin loading inhibitors targeting cells with increased replication origins.

Replication origin assembly is a pivotal step in chromosomal DNA replication. In this process, the ORC complex binds DNA and, together with the CDC6 and CDT1, promotes the loading of the MCM helicase. Chemicals targeting origin assembly might be useful to sensitize highly proliferative cancer cells. However, identifying such compounds is challenging due to the multistage nature of this process. Here, using Xenopus laevis egg extract we set up a high-throughput screening to isolate MCM chromatin loading inhibitors, which led to the identification of NSC-95397 as a powerful inhibitor of replication origin assembly that targets CDC6 protein and promotes its degradation. Using systems developed to test selective drug-induced lethality we show that NSC-95397 triggers cell death both in human cells and Xenopus embryos that have higher proliferative ability. These findings demonstrate the effectiveness of molecules disrupting DNA replication processes in targeting hyperproliferating cells, highlighting their potential as anti-cancer molecules.

"Bridging the DNA divide": Understanding the interplay between replication- gaps and homologous recombination proteins RAD51 and BRCA1/2.

A key but often neglected component of genomic instability is the emergence of single-stranded DNA (ssDNA) gaps during DNA replication in the absence of functional homologous recombination (HR) proteins, such as RAD51 and BRCA1/2. Research in prokaryotes has shed light on the dual role of RAD51's bacterial ortholog, RecA, in HR and the protection of replication forks, emphasizing its essential role in preventing the formation of ssDNA gaps, which is vital for cellular viability. This phenomenon was corroborated in eukaryotic cells deficient in HR, where the formation of ssDNA gaps within newly synthesized DNA and their subsequent processing by the MRE11 nuclease were observed. Without functional HR proteins, cells employ alternative ssDNA gap-filling mechanisms to ensure survival, though this compensatory response can compromise genomic stability. A notable example is the involvement of the translesion synthesis (TLS) polymerase POLζ, along with the repair protein POLθ, in the suppression of replicative ssDNA gaps. Persistent ssDNA gaps may result in replication fork collapse, chromosomal anomalies, and cell death, which contribute to cancer progression and resistance to therapy. Elucidating the processes that avert ssDNA gaps and safeguard replication forks is critical for enhancing cancer treatment approaches by exploiting the vulnerabilities of cancer cells in these pathways.

Who Is the Intermediate Host of RNA Viruses? A Study Focusing on SARS-CoV-2 and Poliovirus.

The COVID-19 pandemic has sparked a surge in research on microbiology and virology, shedding light on overlooked aspects such as the infection of bacteria by RNA virions in the animal microbiome. Studies reveal a decrease in beneficial gut bacteria during COVID-19, indicating a significant interaction between SARS-CoV-2 and the human microbiome. However, determining the origins of the virus remains complex, with observed phenomena such as species jumps adding layers to the narrative. Prokaryotic cells play a crucial role in the disease's pathogenesis and transmission. Analyzing previous studies highlights intricate interactions from clinical manifestations to the use of the nitrogen isotope test. Drawing parallels with the history of the Poliovirus underscores the need to prioritize investigations into prokaryotic cells hosting RNA viruses.

A Comprehensive Review of Cancer Drug-Induced Cardiotoxicity in Blood Cancer Patients: Current Perspectives and Therapeutic Strategies.

OPINION STATEMENT: Cardiotoxicity has emerged as a serious outcome catalyzed by various therapeutic targets in the field of cancer treatment, which includes chemotherapy, radiation, and targeted therapies. The growing significance of cancer drug-induced cardiotoxicity (CDIC) and radiation-induced cardiotoxicity (CRIC) necessitates immediate attention. This article intricately unveils how cancer treatments cause cardiotoxicity, which is exacerbated by patient-specific risks. In particular, drugs like anthracyclines, alkylating agents, and tyrosine kinase inhibitors pose a risk, along with factors such as hypertension and diabetes. Mechanistic insights into oxidative stress and topoisomerase-II-B inhibition are crucial, while cardiac biomarkers show early damage. Timely intervention and prompt treatment, especially with specific agents like dexrazoxane and beta-blockers, are pivotal in the proactive management of CDIC.

Cell stretching activates an ATM mechano-transduction pathway that remodels cytoskeleton and chromatin.

Ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3-related (ATR) DNA damage response (DDR) kinases contain elastic domains. ATM also responds to reactive oxygen species (ROS) and ATR to nuclear mechanical stress. Mre11 mediates ATM activation following DNA damage; ATM mutations cause ataxia telangiectasia (A-T). Here, using in vivo imaging, electron microscopy, proteomic, and mechano-biology approaches, we study how ATM responds to mechanical stress. We report that cytoskeleton and ROS, but not Mre11, mediate ATM activation following cell deformation. ATM deficiency causes hyper-stiffness, stress fiber accumulation, Yes-associated protein (YAP) nuclear enrichment, plasma and nuclear membrane alterations during interstitial migration, and H3 hyper-methylation. ATM locates to the actin cytoskeleton and, following cytoskeleton stress, promotes phosphorylation of key cytoskeleton and chromatin regulators. Our data contribute to explain some clinical features of patients with A-T and pinpoint the existence of an integrated mechano-response in which ATM and ATR have distinct roles unrelated to their canonical DDR functions.

The SGLT2 inhibitor dapagliflozin improves kidney function in glycogen storage disease XI.

Glycogen storage disease XI, also known as Fanconi-Bickel syndrome (FBS), is a rare autosomal recessive disorder caused by mutations in the SLC2A2 gene that encodes the glucose-facilitated transporter type 2 (GLUT2). Patients develop a life-threatening renal proximal tubule dysfunction for which no treatment is available apart from electrolyte replacement. To investigate the renal pathogenesis of FBS, SLC2A2 expression was ablated in mouse kidney and HK-2 proximal tubule cells. GLUT2Pax8Cre+ mice developed time-dependent glycogen accumulation in proximal tubule cells and recapitulated the renal Fanconi phenotype seen in patients. In vitro suppression of GLUT2 impaired lysosomal autophagy as shown by transcriptomic and biochemical analysis. However, this effect was reversed by exposure to a low glucose concentration, suggesting that GLUT2 facilitates the homeostasis of key cellular pathways in proximal tubule cells by preventing glucose toxicity. To investigate whether targeting proximal tubule glucose influx can limit glycogen accumulation and correct symptoms in vivo, we treated mice with the selective SGLT2 inhibitor dapagliflozin. Dapagliflozin reduced glycogen accumulation and improved metabolic acidosis and phosphaturia in the animals by normalizing the expression of Napi2a and NHE3 transporters. In addition, in a patient with FBS, dapagliflozin was safe, improved serum potassium and phosphate concentrations, and reduced glycogen content in urinary shed cells. Overall, this study provides proof of concept for dapagliflozin as a potentially suitable therapy for FBS.

Distinct Mitotic Functions of Nucleolar and Spindle-Associated Protein 1 (NuSAP1) Are Controlled by Two Consensus SUMOylation Sites.

Nucleolar and Spindle-Associated Protein 1 (NuSAP1) is an important mitotic regulator, implicated in control of mitotic microtubule stability and chromosome segregation. NuSAP1 regulates these processes by interacting with several protein partners. Its abundance, activity and interactions are therefore tightly regulated during mitosis. Protein conjugation with SUMO (Small Ubiquitin-like MOdifier peptide) is a reversible post-translational modification that modulates rapid changes in the structure, interaction(s) and localization of proteins. NuSAP1 was previously found to interact with RANBP2, a nucleoporin with SUMO ligase and SUMO-stabilizing activity, but how this interaction affects NuSAP1 activity has remained elusive. Here, we show that NuSAP1 interacts with RANBP2 and forms proximity ligation products with SUMO2/3 peptides in a RANBP2-dependent manner at key mitotic sites. A bioinformatic search identified two putative SUMO consensus sites in NuSAP1, within the DNA-binding and the microtubule-binding domains, respectively. Site-specific mutagenesis, and mitotic phenotyping in cell lines expressing each NuSAP1 mutant version, revealed selective roles of each individual site in control of NuSAP1 localization and in generation of specific mitotic defects and distinct fates in daughter cells. These results identify therefore two new regulatory sites for NuSAP1 functions and implicate RANBP2 in control of NuSAP1 activity.

RFWD3 promotes ZRANB3 recruitment to regulate the remodeling of stalled replication forks.

Replication fork reversal is an important mechanism to protect the stability of stalled forks and thereby preserve genomic integrity. While multiple enzymes have been identified that can remodel forks, their regulation remains poorly understood. Here, we demonstrate that the ubiquitin ligase RFWD3, whose mutation causes Fanconi Anemia, promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. Using electron microscopy, we show that RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner. Fork reversal is known to promote nascent DNA degradation in BRCA2-deficient cells. Consistent with a role for RFWD3 in fork reversal, inactivation of RFWD3 in these cells rescues fork degradation and collapse, analogous to ZRANB3 inactivation. RFWD3 loss impairs ZRANB3 localization to spontaneous nuclear foci induced by inhibition of the PCNA deubiquitinase USP1. We demonstrate that RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing a mechanism for RFWD3-dependent recruitment of ZRANB3. Together, these results uncover a new role for RFWD3 in regulating ZRANB3-dependent fork remodeling.

GEMC1 and MCIDAS interactions with SWI/SNF complexes regulate the multiciliated cell-specific transcriptional program.

Multiciliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex while MCIDAS interacts primarily with BRD9 containing ncBAF complexes. Treatment with a BRD9 inhibitor impaired MCIDAS-mediated activation of several target genes and compromised the MCC differentiation program in multiple cell based models. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation and mediated by their distinct C-terminal domains.

The Potential Role of Vaccines in Preventing Antimicrobial Resistance (AMR): An Update and Future Perspectives.

In the modern era, the consumption of antibiotics represents a revolutionary weapon against several infectious diseases, contributing to the saving of millions of lives worldwide. However, the misuse of antibiotics for human and animal purposes has fueled the process of antimicrobial resistance (AMR), considered now a global emergency by the World Health Organization (WHO), which significantly increases the mortality risk and related medical costs linked to the management of bacterial diseases. The current research aiming at developing novel efficient antibiotics is very challenging, and just a few candidates have been identified so far due to the difficulties connected with AMR. Therefore, novel therapeutic or prophylactic strategies to fight AMR are urgently needed. In this scenario, vaccines constitute a promising approach that proves to be crucial in preventing pathogen spreading in primary infections and in minimizing the usage of antibiotics following secondary bacterial infections. Unfortunately, most of the vaccines developed against the main resistant pathogens are still under preclinical and clinical evaluation due to the complexity of pathogens and technical difficulties. In this review, we describe not only the main causes of AMR and the role of vaccines in reducing the burden of infectious diseases, but we also report on specific prophylactic advancements against some of the main pathogens, focusing on new strategies that aim at improving vaccine efficiency.

Analysis of Bacteriophage Behavior of a Human RNA Virus, SARS-CoV-2, through the Integrated Approach of Immunofluorescence Microscopy, Proteomics and D-Amino Acid Quantification.

SARS-CoV-2, one of the human RNA viruses, is widely studied around the world. Significant efforts have been made to understand its molecular mechanisms of action and how it interacts with epithelial cells and the human microbiome since it has also been observed in gut microbiome bacteria. Many studies emphasize the importance of surface immunity and also that the mucosal system is critical in the interaction of the pathogen with the cells of the oral, nasal, pharyngeal, and intestinal epithelium. Recent studies have shown how bacteria in the human gut microbiome produce toxins capable of altering the classical mechanisms of interaction of viruses with surface cells. This paper presents a simple approach to highlight the initial behavior of a novel pathogen, SARS-CoV-2, on the human microbiome. The immunofluorescence microscopy technique can be combined with spectral counting performed at mass spectrometry of viral peptides in bacterial cultures, along with identification of the presence of D-amino acids within viral peptides in bacterial cultures and in patients' blood. This approach makes it possible to establish the possible expression or increase of viral RNA viruses in general and SARS-CoV-2, as discussed in this study, and to determine whether or not the microbiome is involved in the pathogenetic mechanisms of the viruses. This novel combined approach can provide information more rapidly, avoiding the biases of virological diagnosis and identifying whether a virus can interact with, bind to, and infect bacteria and epithelial cells. Understanding whether some viruses have bacteriophagic behavior allows vaccine therapies to be focused either toward certain toxins produced by bacteria in the microbiome or toward finding inert or symbiotic viral mutations with the human microbiome. This new knowledge opens a scenario on a possible future vaccine: the probiotics vaccine, engineered with the right resistance to viruses that attach to both the epithelium human surface and gut microbiome bacteria.

POLθ prevents MRE11-NBS1-CtIP-dependent fork breakage in the absence of BRCA2/RAD51 by filling lagging-strand gaps.

POLθ promotes repair of DNA double-strand breaks (DSBs) resulting from collapsed forks in homologous recombination (HR) defective tumors. Inactivation of POLθ results in synthetic lethality with the loss of HR genes BRCA1/2, which induces under-replicated DNA accumulation. However, it is unclear whether POLθ-dependent DNA replication prevents HR-deficiency-associated lethality. Here, we isolated Xenopus laevis POLθ and showed that it processes stalled Okazaki fragments, directly visualized by electron microscopy, thereby suppressing ssDNA gaps accumulating on lagging strands in the absence of RAD51 and preventing fork reversal. Inhibition of POLθ DNA polymerase activity leaves fork gaps unprotected, enabling their cleavage by the MRE11-NBS1-CtIP endonuclease, which produces broken forks with asymmetric single-ended DSBs, hampering BRCA2-defective cell survival. These results reveal a POLθ-dependent genome protection function preventing stalled forks rupture and highlight possible resistance mechanisms to POLθ inhibitors.

POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells.

Polymerase theta (POLθ) is an error-prone DNA polymerase whose loss is synthetically lethal in cancer cells bearing breast cancer susceptibility proteins 1 and 2 (BRCA1/2) mutations. To investigate the basis of this genetic interaction, we utilized a small-molecule inhibitor targeting the POLθ polymerase domain. We found that POLθ processes single-stranded DNA (ssDNA) gaps that emerge in the absence of BRCA1, thus promoting unperturbed replication fork progression and survival of BRCA1 mutant cells. A genome-scale CRISPR-Cas9 knockout screen uncovered suppressors of the functional interaction between POLθ and BRCA1, including NBN, a component of the MRN complex, and cell-cycle regulators such as CDK6. While the MRN complex nucleolytically processes ssDNA gaps, CDK6 promotes cell-cycle progression, thereby exacerbating replication stress, a feature of BRCA1-deficient cells that lack POLθ activity. Thus, ssDNA gap formation, modulated by cell-cycle regulators and MRN complex activity, underlies the synthetic lethality between POLθ and BRCA1, an important insight for clinical trials with POLθ inhibitors.

Dapagliflozin Prevents Kidney Glycogen Accumulation and Improves Renal Proximal Tubule Cell Functions in a Mouse Model of Glycogen Storage Disease Type 1b.

BACKGROUND: Mutations in SLC37A4, which encodes the intracellular glucose transporter G6PT, cause the rare glycogen storage disease type 1b (GSD1b). A long-term consequence of GSD1b is kidney failure, which requires KRT. The main protein markers of proximal tubule function, including NaPi2A, NHE3, SGLT2, GLUT2, and AQP1, are downregulated as part of the disease phenotype. METHODS: We utilized an inducible mouse model of GSD1b, TM-G6PT-/-, to show that glycogen accumulation plays a crucial role in altering proximal tubule morphology and function. To limit glucose entry into proximal tubule cells and thus to prevent glycogen accumulation, we administered an SGLT2-inhibitor, dapagliflozin, to TM-G6PT-/- mice. RESULTS: In proximal tubule cells, G6PT suppression stimulates the upregulation and activity of hexokinase-I, which increases availability of the reabsorbed glucose for intracellular metabolism. Dapagliflozin prevented glycogen accumulation and improved kidney morphology by promoting a metabolic switch from glycogen synthesis toward lysis and by restoring expression levels of the main proximal tubule functional markers. CONCLUSION: We provide proof of concept for the efficacy of dapagliflozin in preserving kidney function in GSD1b mice. Our findings could represent the basis for repurposing this drug to treat patients with GSD1b.

Single nephron glomerular filtration rate measured by linescan multiphoton microscopy compared to conventional micropuncture.

Renal micropuncture, which requires the direct access to the renal tubules, has for long time been the technique of choice to measure the single nephron glomerular filtration rate (SNGFR) in animal models. This approach is challenging by virtue of complex animal preparation and numerous technically difficult steps. The introduction of intravital multiphoton microscopy (MPM) offers another approach to the measure of the SNGFR by mean of the high laser-tissue penetration and the optical sectioning capacity. Previous MPM studies measuring SNGFR in vivo relied on fast full-frame acquisition during the filtration process obtainable with high performance resonant scanners. In this study, we describe an innovative linescan-based MPM method. The new method can discriminate SNGFR variations both in conditions of low and high glomerular filtration, and shows results comparable to conventional micropuncture both for rats and mice. Moreover, this novel approach has improved spatial and time resolution and is faster than previous methods, thus enabling the investigation of SNGFR from more tubules and improving options for data-analysis.

The Probe for Renal Organic Cation Secretion (4-Dimethylaminostyryl)-N-Methylpyridinium (ASP+)) Shows Amplified Fluorescence by Binding to Albumin and Is Accumulated In Vivo.

Accumulation of uremic toxins may lead to the life-threatening condition "uremic syndrome" in patients with advanced chronic kidney disease (CKD) requiring renal replacement therapy. Clinical evaluation of proximal tubular secretion of organic cations (OC), of which some are uremic toxins, is desired, but difficult. The biomedical knowledge on OC secretion and cellular transport partly relies on studies using the fluorescent tracer 4-dimethylaminostyryl)-N-methylpyridinium (ASP+), which has been used in many studies of renal excretion mechanisms of organic ions and which could be a candidate as a PET tracer. This study is aimed at expanding the knowledge of the tracer characteristics of ASP+ by recording the distribution and intensity of ASP+ signals in vivo both by fluorescence and by positron emission tomography (PET) imaging and at investigating if the fluorescence signal of ASP+ is influenced by the presence of albumin. Two-photon in vivo microscopy of male Münich Wistar Frömter rats showed that a bolus injection of ASP+ conferred a fluorescence signal to the blood plasma lasting for about 30 minutes. In the renal proximal tubule, the bolus resulted in a complex pattern of fluorescence including a rapid and strong transient signal at the brush border, a very low signal in the luminal fluid, and a slow transient intracellular signal. PET imaging using 11C-labelled ASP+ showed accumulation in the liver, heart, and kidney. Fluorescence emission spectra recorded in vitro of ASP+ alone and in the presence of albumin using both 1-photon excitation and two-photon excitation showed that albumin strongly enhance the emission from ASP+ and induce a shift of the emission maximum from 600 to 570 nm. Conclusion. The renal pattern of fluorescence observed from ASP+ in vivo is likely affected by the local concentration of albumin, and quantification of ASP+ fluorescent signals in vivo cannot be directly translated to ASP+ concentrations.

Physical Activity and Nutritional Influence on Immune Function: An Important Strategy to Improve Immunity and Health Status.

Physical activity (PA) and nutrition are the essential components of a healthy lifestyle, as they can influence energy balance, promote functional ability of various systems and improve immunity. Infections and their associated symptoms are the common and frequent challenges to human health that are causing severe economic and social consequences around the world. During aging, human immune system undergoes dramatic aging-related changes/dysfunctions known as immunosenescence. Clinically, immunosenescence refers to the gradual deterioration of immune system that increases exposure to infections, and reduces vaccine efficacy. Such phenomenon is linked to impaired immune responses that lead to dysfunction of multiple organs, while lack of physical activity, progressive loss of muscle mass, and concomitant decline in muscle strength facilitate immunosenescence and inflammation. In the present review, we have discussed the role of nutrition and PA, which can boost the immune system alone and synergistically. Evidence suggests that long-term PA is beneficial in improving immune system and preventing various infections. We have further discussed several nutritional strategies for improving the immune system. Unfortunately, the available evidence shows conflicting results. In terms of interaction with food intake, PA does not tend to increase energy intake during a short time course. However, overcoming nutritional deficiencies appears to be the most practical recommendation. Through the balanced nutritious diet intake one can fulfill the bodily requirement of optimal nutrition that significantly impacts the immune system. Supplementation of a single nutrient as food is generally not advisable. Rather incorporating various fruits and vegetables, whole grains, proteins and probiotics may ensure adequate nutrient intake. Therefore, multi-nutrient supplements may benefit people having deficiency in spite of sufficient diet. Along with PA, supplementation of probiotics, bovine colostrum, plant-derived products and functional foods may provide additional benefits in improving the immune system.