Opportunities and challenges with G-quadruplexes as promising targets for drug design.

INTRODUCTION: G-quadruplexes (G4s) are secondary structures formed in guanine-rich regions of nucleic acids (both DNA and RNA). G4s are significantly enriched at regulatory genomic regions and are associated with important biological processes ranging from telomere homeostasis and genome instability to transcription and translation. Importantly, G4s are related to health and diseases such as cancer, neurological diseases, as well as infections with viruses and microbial pathogens. Increasing evidence suggests the potential of G4s for designing new diagnostic and therapeutic strategies although in vivo studies are still at early stages. AREAS COVERED: This review provides an updated summary of the literature describing the impact of G4s in human diseases and different approaches based on G4 targeting in therapy. EXPERT OPINION: Within the G4 field, most of the studies have been performed in vitro and in a descriptive manner. Therefore, detailed mechanistic understanding of G4s in the biological context remains to be deciphered. In clinics, the use of G4s as therapeutic targets has been hindered due to the low selectivity profile and poor drug-like properties of G4 ligands. Future research on G4s may overcome current methodological and interventional limitations and shed light on these unique structural elements in the pathogenesis and treatment of diseases.

Increased blood draws for ultrasensitive ctDNA and CTCs detection in early breast cancer patients.

Early breast cancer patients often experience relapse due to residual disease after treatment. Liquid biopsy is a methodology capable of detecting tumor components in blood, but low concentrations at early stages pose challenges. To detect them, next-generation sequencing has promise but entails complex processes. Exploring larger blood volumes could overcome detection limitations. Herein, a total of 282 high-volume plasma and blood-cell samples were collected for dual ctDNA/CTCs detection using a single droplet-digital PCR assay per patient. ctDNA and/or CTCs were detected in 100% of pre-treatment samples. On the other hand, post-treatment positive samples exhibited a minimum variant allele frequency of 0.003% for ctDNA and minimum cell number of 0.069 CTCs/mL of blood, surpassing previous investigations. Accurate prediction of residual disease before surgery was achieved in patients without a complete pathological response. A model utilizing ctDNA dynamics achieved an area under the ROC curve of 0.92 for predicting response. We detected disease recurrence in blood in the three patients who experienced a relapse, anticipating clinical relapse by 34.61, 9.10, and 7.59 months. This methodology provides an easily implemented alternative for ultrasensitive residual disease detection in early breast cancer patients.

Comparative study of droplet-digital PCR and absolute Q digital PCR for ctDNA detection in early-stage breast cancer patients.

BACKGROUND: Analysis of circulating tumor DNA (ctDNA) is increasingly used for clinical decision-making in oncology. However, ctDNA could represent ≤ 0.1 % of cell-free DNA in early-stage tumors and its detection requires high-sensitive techniques such as digital PCR (dPCR). METHODS: In 46 samples from patients with early-stage breast cancer, we compared two leading dPCR assays for ctDNA analysis: QX200 droplet digital PCR (ddPCR) system from Bio-Rad which is the gold-standard in the field, and Absolute Q plate-based digital PCR (pdPCR) system from Thermo Fisher Scientific which has not been reported before. We analyzed 5 mL of baseline plasma samples prior to any treatment. RESULTS: Both systems displayed a comparable sensitivity with no significant differences observed in mutant allele frequency. In fact, ddPCR and pdPCR possessed a concordance > 90 % in ctDNA positivity. Nevertheless, ddPCR exhibited higher variability and a longer workflow. Finally, we explored the association between ctDNA levels and clinicopathological features. Significantly higher ctDNA levels were present in patients with a Ki67 score > 20 % or with estrogen receptor-negative or triple-negative breast cancer subtypes. CONCLUSION: Both ddPCR and pdPCR may constitute sensitive and reliable tools for ctDNA analysis with an adequate agreement in early-stage breast cancer patients.

Fermented Goat's Milk Contributes to the Recovery of Iron Deficiency Anemia via Modulation of the Gut Microbiome.

Iron deficiency anemia (IDA) is a global public health concern affecting 1.6 billion people worldwide. The administration of iron supplements during the treatment of IDA adversely affects the intestinal barrier function and the composition and functionality of the intestinal microbiome, both of which are already altered during IDA. For this reason, it is of great interest to develop nutritional strategies aimed at alleviating these gut alterations associated with IDA and its treatment. In this sense, fermented goat's milk (FGM) was studied due to its nutritional quality. Our findings showed that in anemic animals the consumption of a FGM-based diet, compared to a standard diet, had positive modulatory effects on the intestinal microbiome. FGM-based diet restored intestinal dysbiosis, the intestinal barrier functionality, and bacterial translocation, contributing to a more efficient recovery of IDA. Therefore, FGM is a useful nutritional tool to ease intestinal alterations occurring during IDA and during its treatment.

Benefits for cardiovascular system, bone density, and quality of life of a long-term hormone therapy in hysterectomized women: a 20-year follow-up study.

OBJECTIVE: The safety, consequences, and dosage of long-term hormone therapy (HT) for postmenopausal women remain unclear. Our aim was to analyze the effects of HT after 20 years of therapy in women after hysterectomy, focusing on the symptoms of menopause, blood pressure, lipid profiles, and bone density. METHODS: A prospective observational longitudinal study was designed. The initial transdermal estradiol dose was reduced in half (0.025 mg/d) at 60 years of age. Different parameters including demographic, cardiovascular, bone density, and metabolic variables, as well as quality of life characteristics, were analyzed using bivariate analyses. Multivariate generalized estimating equations for longitudinal data were fitted for differences over time and between doses (<60 vs ≥60 y) using the R package geepack. RESULTS: After 20 years of HT, the mean age of 56 studied hysterectomized women was 67.1 years. The mean Kupperman index score decreased from 26.7 to 12.0 ( P < 0.001). A trend with total and low-density lipoprotein cholesterol reduction and high-density lipoprotein cholesterol increase was observed over time. A decrease in very-low-density lipoprotein cholesterol ( P = 0.05) and an increase in T score vertebral densitometry ( P = 0.014) were detected after HT. No changes in health outcome were detected in women older than 60 years with the reduced dose of HT. Breast cancer was the reason for dropouts in 0.02% women. CONCLUSIONS: HT for up to 20 years after hysterectomy may be beneficial for bone and cardiovascular health and for the overall quality of life. Our data suggest the importance of evaluating the dose and the timing of HT.

Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes.

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.

Curvicollide D Isolated from the Fungus Amesia sp. Kills African Trypanosomes by Inhibiting Transcription.

Sleeping sickness or African trypanosomiasis is a serious health concern with an added socio-economic impact in sub-Saharan Africa due to direct infection in both humans and their domestic livestock. There is no vaccine available against African trypanosomes and its treatment relies only on chemotherapy. Although the current drugs are effective, most of them are far from the modern concept of a drug in terms of toxicity, specificity and therapeutic regime. In a search for new molecules with trypanocidal activity, a high throughput screening of 2000 microbial extracts was performed. Fractionation of one of these extracts, belonging to a culture of the fungus Amesia sp., yielded a new member of the curvicollide family that has been designated as curvicollide D. The new compound showed an inhibitory concentration 50 (IC50) 16-fold lower in Trypanosoma brucei than in human cells. Moreover, it induced cell cycle arrest and disruption of the nucleolar structure. Finally, we showed that curvicollide D binds to DNA and inhibits transcription in African trypanosomes, resulting in cell death. These results constitute the first report on the activity and mode of action of a member of the curvicollide family in T. brucei.

Gut microbiome-short-chain fatty acids interplay in the context of iron deficiency anaemia.

PURPOSE: Anaemia is a global health concern, with iron deficiency anaemia (IDA) causing approximately 50% of cases. Affecting mostly the elderly, pregnant and adult women and children, physiopathology of IDA in relation to the gut microbiome is poorly understood. Therefore, the objective of this study is to analyse, in an animal model, the effect of IDA on the gut microbiome along the gastrointestinal tract, as well as to relate intestinal dysbiosis to changes in microbial metabolites such as short chain fatty acids (SCFA). METHODS: IDA was experimentally induced through an iron deficient diet for a period of 40 days, with twenty weaned male Wistar rats being randomly divided into control or anaemic groups. Blood samples were collected to control haematological parameters, and so were faecal and intestinal content samples to study gut microbial communities and SCFA, using 16S rRNA sequencing and HPLC-UV respectively. RESULTS: An intestinal dysbiosis was observed as a consequence of IDA, especially towards the distal segments of the gastrointestinal tract and the colon. An increase in SCFA was also noticed during IDA, with the major difference appearing in the colon and correlating with changes in the composition of the gut microbiome. Clostridium_sensu_stricto_1 and Clostridium_sensu_stricto_4 showed the greatest correlation with variations in butyric and propionic concentrations in the colon of anaemic animals. CONCLUSIONS: Composition of intestinal microbial communities was affected by the generation of IDA. An enrichment in certain SCFA-producing genera and SCFA concentrations was found in the colon of anaemic animals, suggesting a trade-off mechanism against disease.

A rare cause of bacteremia due to Porphyromonas asaccharolytica in a patient with necrotizing fasciitis.

Porphyromonas species are Gram-negative anaerobic bacilli mainly involved in human periodontal diseases. We report an uncommon case of bacteremia due to P. asaccharolytica in a patient with necrotizing fasciitis. A 52-year-old woman with a history of diabetes mellitus was admitted for an extensive necrotizing lesion on the left lower limb. After she developed septic shock, two sets of blood cultures were taken. Anaerobic bottles yielded a pure culture of a microorganism initially identified as P. uenonis by MALDI-TOF MS but with a low log score, and a gene sequencing technique was therefore applied, identifying the isolate as P. asaccharolytica. Only resistance to penicillin and clindamycin was documented. Treatment with meropenem was administered, and the patient was discharged following her recovery.

Quadruplex Ligands in Cancer Therapy.

Nucleic acids can adopt alternative secondary conformations including four-stranded structures known as quadruplexes. To date, quadruplexes have been demonstrated to exist both in human chromatin DNA and RNA. In particular, quadruplexes are found in guanine-rich sequences constituting G-quadruplexes, and in cytosine-rich sequences forming i-Motifs as a counterpart. Quadruplexes are associated with key biological processes ranging from transcription and translation of several oncogenes and tumor suppressors to telomeres maintenance and genome instability. In this context, quadruplexes have prompted investigations on their possible role in cancer biology and the evaluation of small-molecule ligands as potential therapeutic agents. This review aims to provide an updated close-up view of the literature on quadruplex ligands in cancer therapy, by grouping together ligands for DNA and RNA G-quadruplexes and DNA i-Motifs.

Targeting ribosomal G-quadruplexes with naphthalene-diimides as RNA polymerase I inhibitors for colorectal cancer treatment.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures commonly found in regulatory genomic regions. G4 targeting has emerged as a therapeutic approach in cancer. We have screened naphthalene-diimides (NDIs), a class of G4 ligands, in a cellular model of colorectal cancer (CRC). Here, we identify the leading compound T5 with a potent and selective inhibition of cell growth by high-affinity binding to G4s in ribosomal DNA, impairing RNA polymerase I (Pol I) elongation. Consequently, T5 induces a rapid inhibition of Pol I transcription, nucleolus disruption, proteasome-dependent Pol I catalytic subunit A degradation and autophagy. Moreover, we attribute the higher selectivity of carbohydrate-conjugated T5 for tumoral cells to its preferential uptake through the overexpressed glucose transporter 1. Finally, we succinctly demonstrate that T5 could be explored as a therapeutic agent in a patient cohort with CRC. Therefore, we report a mode of action for these NDIs involving ribosomal G4 targeting.

Bacteremia caused by Anaerococcus SPP: Is this an underdiagnosed infection?

The objectives of this study were to report 10 episodes of clinically significant bacteremia caused by species of the genus Anaerococcus isolated between July 2018 and February 2021 from the microbiology laboratory of a tertiary hospital in Granada (Spain). None of the isolates were identified by MALDI-TOF MS, and the definitive species identification was performed by 16 S rRNA gene sequencing. No reference spectra of the Anaerococcus species were present in the MALDI-TOF MS database. Eight isolates were finally identified as A. octavius, one isolate as A. tetradius and the other as A. urinomassiliensis. The majority of these infections were seen in patients aged >70 years. Risk factors for anaerobic infection were observed in eight patients, especially diabetes mellitus, surgery, and the presence of cancer. Fever was present in all patients. Three patients died, but only one death was attributed to the infection. Mean detection time of positive blood cultures was 47.5 h (range 24-92 h). Antimicrobial susceptibility to penicillin, amoxicillin-clavulanate, imipenem, moxifloxacin, clindamycin, metronidazole, and piperacillin-tazobactam was tested using the gradient diffusion technique and EUCAST breakpoints (except for moxifloxacin). No resistance to amoxicillin-clavulanate, metronidazole, imipenem, or piperacillin-tazobactam was detected; however, the majority of isolates were resistant to clindamycin. When MALDI-TOF MS does not provide a correct identification at genus or species level, as in some isolates of Gram-positive anaerobic cocci, microbiologists should perform an additional confirmatory technique, such as gene sequencing analysis, to obtain a definitive diagnosis.

An Updated Focus on Quadruplex Structures as Potential Therapeutic Targets in Cancer.

Non-canonical, four-stranded nucleic acids secondary structures are present within regulatory regions in the human genome and transcriptome. To date, these quadruplex structures include both DNA and RNA G-quadruplexes, formed in guanine-rich sequences, and i-Motifs, found in cytosine-rich sequences, as their counterparts. Quadruplexes have been extensively associated with cancer, playing an important role in telomere maintenance and control of genetic expression of several oncogenes and tumor suppressors. Therefore, quadruplex structures are considered attractive molecular targets for cancer therapeutics with novel mechanisms of action. In this review, we provide a general overview about recent research on the implications of quadruplex structures in cancer, firstly gathering together DNA G-quadruplexes, RNA G-quadruplexes as well as DNA i-Motifs.

Influence of 16S rRNA target region on the outcome of microbiome studies in soil and saliva samples.

Next generation sequencing methods are widely used in evaluating the structure and functioning of microbial communities, especially those centered on 16S rRNA subunit. Since Illumina Miseq, the most used sequencing platform, does not allow the full sequencing of 16S rRNA gene, this study aims to evaluate whether the choice of different target regions might affect the outcome of microbiome studies regarding soil and saliva samples. V1V3, V3V4, V4V5 and V6V8 domains were studied, finding that while some regions showed differences in the detection of certain bacterial taxa and in the calculation of alpha diversity, especially in soil samples, the overall effect did not compromise the differentiation of any sample type in terms of taxonomic analysis at the genus level. 16S rRNA target regions did affect the detection of specific bacteria related to soil quality and development, and microbial genera used as health biomarkers in saliva. V1V3 region showed the closest similarity to internal sequencing control mock community B, suggesting it might be the most preferable choice regarding data reliability.

Correction: Preoperative chemoradiotherapy for rectal cancer: the sensitizer role of the association between miR-375 and c-Myc.

[This corrects the article DOI: 10.18632/oncotarget.19393.].

Preoperative chemoradiotherapy for rectal cancer: the sensitizer role of the association between miR-375 and c-Myc.

Administration of chemoradiation before tumor resection has revolutionized the management of locally advanced rectal cancer, but many patients have proven resistant to this preoperative therapy. Our group recently reported a negative correlation between c-Myc gene expression and this resistance. In the present study, integrated analysis of miRNA and mRNA expression profiles was conducted in 45 pre-treatment rectal tumors in order to analyze the expressions of miRNAs and c-Myc and their relationship with clinicopathological factors and patient survival. Twelve miRNAs were found to be differentially expressed by responders and non-responders to the chemoradiation. Functional classification revealed an association between the differentially expressed miRNAs and c-Myc. Quantitative real-time PCR results showed that miRNA-148 and miRNA-375 levels were both significantly lower in responders than in non-responders. Notably, a significant negative correlation was found between miRNA-375 expression and c-Myc expression. According to these findings, miRNA-375 and its targeted c-Myc may be useful as a predictive biomarker of the response to neoadjuvant treatment in patients with locally advanced rectal cancer.