Encapsulation of the septal cell wall protects Streptococcus pneumoniae from its major peptidoglycan hydrolase and host defenses.

Synthesis of the capsular polysaccharide, a major virulence factor for many pathogenic bacteria, is required for bacterial survival within the infected host. In Streptococcus pneumoniae, Wze, an autophosphorylating tyrosine kinase, and Wzd, a membrane protein required for Wze autophosphorylation, co-localize at the division septum and guarantee the presence of capsule at this subcellular location. To determine how bacteria regulate capsule synthesis, we studied pneumococcal proteins that interact with Wzd and Wze using bacterial two hybrid assays and fluorescence microscopy. We found that Wzd interacts with Wzg, the putative ligase that attaches capsule to the bacterial cell wall, and recruits it to the septal area. This interaction required residue V56 of Wzd and both the transmembrane regions and DNA-PPF domain of Wzg. When compared to the wild type, Wzd null pneumococci lack capsule at midcell, bind the peptidoglycan hydrolase LytA better and are more susceptible to LytA-induced lysis, and are less virulent in a zebrafish embryo infection model. In this manuscript, we propose that the Wzd/Wze pair guarantees full encapsulation of pneumococcal bacteria by recruiting Wzg to the division septum, ensuring that capsule attachment is coordinated with peptidoglycan synthesis. Impairing the encapsulation process, at localized subcellular sites, may facilitate elimination of bacteria by strategies that target the pneumococcal peptidoglycan.

E-cadherin variants associated with oral facial clefts trigger aberrant cell motility in a REG1A-dependent manner.

BACKGROUND: Germline mutations of E-cadherin contribute to hereditary diffuse gastric cancer (HDGC) and congenital malformations, such as oral facial clefts (OFC). However, the molecular mechanisms through which E-cadherin loss-of-function triggers distinct clinical outcomes remain unknown. We postulate that E-cadherin-mediated disorders result from abnormal interactions with the extracellular matrix and consequent aberrant intracellular signalling, affecting the coordination of cell migration. METHODS: Herein, we developed in vivo and in vitro models of E-cadherin mutants associated with either OFC or HDGC. Using a Drosophila approach, we addressed the impact of the different variants in cell morphology and migration ability. By combining gap closure migration assays and time-lapse microscopy, we further investigated the migration pattern of cells expressing OFC or HDGC variants. The adhesion profile of the variants was evaluated using high-throughput ECM arrays, whereas RNA sequencing technology was explored for identification of genes involved in aberrant cell motility. RESULTS: We have demonstrated that cells expressing OFC variants exhibit an excessive motility performance and irregular leading edges, which prevent the coordinated movement of the epithelial monolayer. Importantly, we found that OFC variants promote cell adhesion to a wider variety of extracellular matrices than HDGC variants, suggesting higher plasticity in response to different microenvironments. We unveiled a distinct transcriptomic profile in the OFC setting and pinpointed REG1A as a putative regulator of this outcome. Consistent with this, specific RNAi-mediated inhibition of REG1A shifted the migration pattern of OFC expressing cells, leading to slower wound closure with coordinated leading edges. CONCLUSIONS: We provide evidence that E-cadherin variants associated with OFC activate aberrant signalling pathways that support dynamic rearrangements of cells towards improved adaptability to the microenvironment. This proficiency results in abnormal tissue shaping and movement, possibly underlying the development of orofacial malformations.

Global declines in coral reef calcium carbonate production under ocean acidification and warming.

Ocean warming and acidification threaten the future growth of coral reefs. This is because the calcifying coral reef taxa that construct the calcium carbonate frameworks and cement the reef together are highly sensitive to ocean warming and acidification. However, the global-scale effects of ocean warming and acidification on rates of coral reef net carbonate production remain poorly constrained despite a wealth of studies assessing their effects on the calcification of individual organisms. Here, we present global estimates of projected future changes in coral reef net carbonate production under ocean warming and acidification. We apply a meta-analysis of responses of coral reef taxa calcification and bioerosion rates to predicted changes in coral cover driven by climate change to estimate the net carbonate production rates of 183 reefs worldwide by 2050 and 2100. We forecast mean global reef net carbonate production under representative concentration pathways (RCP) 2.6, 4.5, and 8.5 will decline by 76, 149, and 156%, respectively, by 2100. While 63% of reefs are projected to continue to accrete by 2100 under RCP2.6, 94% will be eroding by 2050 under RCP8.5, and no reefs will continue to accrete at rates matching projected sea level rise under RCP4.5 or 8.5 by 2100. Projected reduced coral cover due to bleaching events predominately drives these declines rather than the direct physiological impacts of ocean warming and acidification on calcification or bioerosion. Presently degraded reefs were also more sensitive in our analysis. These findings highlight the low likelihood that the world's coral reefs will maintain their functional roles without near-term stabilization of atmospheric CO2 emissions.

Human-in-the-Loop Optimization of Knee Exoskeleton Assistance for Minimizing User's Metabolic and Muscular Effort.

Lower limb exoskeletons have the potential to mitigate work-related musculoskeletal disorders; however, they often lack user-oriented control strategies. Human-in-the-loop (HITL) controls adapt an exoskeleton's assistance in real time, to optimize the user-exoskeleton interaction. This study presents a HITL control for a knee exoskeleton using a CMA-ES algorithm to minimize the users' physical effort, a parameter innovatively evaluated using the interaction torque with the exoskeleton (a muscular effort indicator) and metabolic cost. This work innovates by estimating the user's metabolic cost within the HITL control through a machine-learning model. The regression model estimated the metabolic cost, in real time, with a root mean squared error of 0.66 W/kg and mean absolute percentage error of 26% (n = 5), making faster (10 s) and less noisy estimations than a respirometer (K5, Cosmed). The HITL reduced the user's metabolic cost by 7.3% and 5.9% compared to the zero-torque and no-device conditions, respectively, and reduced the interaction torque by 32.3% compared to a zero-torque control (n = 1). The developed HITL control surpassed a non-exoskeleton and zero-torque condition regarding the user's physical effort, even for a task such as slow walking. Furthermore, the user-specific control had a lower metabolic cost than the non-user-specific assistance. This proof-of-concept demonstrated the potential of HITL controls in assisted walking.

Deep into the Apoplast: Grapevine and Plasmopara viticola Proteomes Reveal the Secret Beneath Host and Pathogen Communication at 6 h After Contact.

The apoplast is the first hub of plant-pathogen communication where pathogen effectors are recognized by plant defensive proteins and cell receptors, thus activating signal transduction pathways. As a result of this first contact, the host triggers a defense response that involves the modulation of extra- and intracellular proteins. In grapevine-pathogen interactions, little is known about the trafficking between extra- and intracellular spaces. Grapevine is an economically important crop that relies on heavy fungicide use to control several diseases, and a deeper knowledge on the activation of its immune response is crucial to define new control strategies. In this study, we focused on the first 6 h postinoculation with Plasmopara viticola to evaluate grapevine proteome modulation in the apoplast. The in planta P. viticola proteome was also assessed to enable a deeper understanding of plant-pathogen communication. Our results showed that several plant mechanisms are triggered in the tolerant grapevine cultivar Regent after inoculation, such as oomycete recognition, plant cell wall modifications, reactive oxygen species signaling, and secretion of proteins to disrupt oomycete structures. On the other hand, P. viticola proteins related to development and virulence were the most predominant. This pioneer study highlights the early dynamics of cellular communication in grapevine defense that leads to the successful establishment of an incompatible interaction.

Ketamine-dexmedetomidine combination for sedation in pediatric major surgery in a low-income country.

Anorectal malformations are one of the most frequent congenital malformations treated by pediatric surgeons. In low-income countries, the surgical and anesthetic management of children in need of these procedures can be challenging. Limited oxygen supply, lack of equipment, especially pediatric, and intensive care units make the use of regional anesthesia appealing. We present a series of four cases of anorectal malformations corrections in Guinea Bissau, in children up to 13 months of age, under regional anesthesia and sedation with ketodex, a mixture of ketamine and dexmedetomidine (in a proportion of 1 mg to 1 µg). No child developed respiratory depression requiring airway intervention or supplemental oxygen, or had hemodynamic instability.

Alteration of redox status and fatty acid profile in gills from the green crab (Carcinus aestuarii) following dimethoate exposure.

Dimethoate is a broad-spectrum organophosphate insecticide and acaricide. Through various pathways, such as runoff and drift, dimethoate can reach marine environment, and easily impact common organisms in coastal areas, close to agriculture lands, namely crustaceans. The purpose of this study was to investigate the potential effects of dimethoate exposure (50, 100, and 200 µg/l), for 1 day, on a wide range of markers of oxidative stress and neurotransmission impairment, as well as fatty acids composition and histopathological aspect in the gills of the green crab Carcinus aestuarii. A significant increase in n-3 polyunsaturated fatty acids series, namely the eicosapentaenoic acid (C20: 5n3) and its precursor alpha-linolenic acid (C 18: 3n3) in dimethoate-treated crabs was recorded. Concerning n-6 polyunsaturated fatty acids, we noted a high reduction in arachidonic acid (C20:4n-6) levels. Dimethoate exposure increased the levels of hydrogen peroxide, malondialdehyde, lipid hydroperoxides, protein carbonyl, and caused the advanced oxidation of protein products along with enzymatic and non-enzymatic antioxidant-related markers. Acetylcholinesterase activity was highly inhibited following exposure to dimethoate in a concentration-dependent manner. Finally, deleterious histopathological changes with several abnormalities were noted in exposed animals confirming our biochemical findings. The present study offered unique insights to establish a relationship between redox status and alterations in fatty acid composition, allowing a better understanding of dimethoate-triggered toxicity.

Human-Robot Joint Misalignment, Physical Interaction, and Gait Kinematic Assessment in Ankle-Foot Orthoses.

Lower limb exoskeletons and orthoses have been increasingly used to assist the user during gait rehabilitation through torque transmission and motor stability. However, the physical human-robot interface (HRi) has not been properly addressed. Current orthoses lead to spurious forces at the HRi that cause adverse effects and high abandonment rates. This study aims to assess and compare, in a holistic approach, human-robot joint misalignment and gait kinematics in three fixation designs of ankle-foot orthoses (AFOs). These are AFOs with a frontal shin guard (F-AFO), lateral shin guard (L-AFO), and the ankle modulus of the H2 exoskeleton (H2-AFO). An experimental protocol was implemented to assess misalignment, fixation displacement, pressure interactions, user-perceived comfort, and gait kinematics during walking with the three AFOs. The F-AFO showed reduced vertical misalignment (peak of 1.37 ± 0.90 cm, p-value < 0.05), interactions (median pressures of 0.39-3.12 kPa), and higher user-perceived comfort (p-value < 0.05) when compared to H2-AFO (peak misalignment of 2.95 ± 0.64 and pressures ranging from 3.19 to 19.78 kPa). F-AFO also improves the L-AFO in pressure (median pressures ranging from 8.64 to 10.83 kPa) and comfort (p-value < 0.05). All AFOs significantly modified hip joint angle regarding control gait (p-value < 0.01), while the H2-AFO also affected knee joint angle (p-value < 0.01) and gait spatiotemporal parameters (p-value < 0.05). Overall, findings indicate that an AFO with a frontal shin guard and a sports shoe is effective at reducing misalignment and pressure at the HRI, increasing comfort with slight changes in gait kinematics.

Activation of Laminin γ2 by Helicobacter pylori Promotes Invasion and Survival of Gastric Cancer Cells With E-Cadherin Defects.

BACKGROUND: Helicobacter pylori infection induces cellular phenotypes relevant for cancer progression, namely cell motility and invasion. We hypothesized that the extracellular matrix (ECM) could be involved in these deleterious effects. METHODS: Microarrays were used to uncover ECM interactors in cells infected with H. pylori. LAMC2, encoding laminin γ2, was selected as a candidate gene and its expression was assessed in vitro and in vivo. The role of LAMC2 was investigated by small interference RNA (siRNA) combined with a set of functional assays. Laminin γ2 and E-cadherin expression patterns were evaluated in gastric cancer cases. RESULTS: Laminin γ2 was found significantly overexpressed in gastric cancer cells infected with H. pylori. This finding was validated in vitro by infection with clinical isolates and in vivo by using gastric biopsies of infected and noninfected individuals. We showed that laminin γ2 overexpression is dependent on the bacterial type IV secretion system and on the CagA. Functionally, laminin γ2 promotes cell invasion and resistance to apoptosis, through modulation of Src, JNK, and AKT activity. These effects were abrogated in cells with functional E-cadherin. CONCLUSIONS: These data highlight laminin γ2 and its downstream effectors as potential therapeutic targets, and the value of H. pylori eradication to delay gastric cancer onset and progression.

Integrin ß1 orchestrates the abnormal cell-matrix attachment and invasive behaviour of E-cadherin dysfunctional cells.

BACKGROUND: Tumour progression relies on the ability of cancer cells to penetrate and invade neighbouring tissues. E-cadherin loss is associated with increased cell invasion in gastric carcinoma, and germline mutations of the E-cadherin gene are causative of hereditary diffuse gastric cancer. Although E-cadherin dysfunction impacts cell-cell adhesion, cell dissemination also requires an imbalance of adhesion to the extracellular matrix (ECM). METHODS: To identify ECM components and receptors relevant for adhesion of E-cadherin dysfunctional cells, we implemented a novel ECM microarray platform coupled with molecular interaction networks. The functional role of putative candidates was determined by combining micropattern traction microscopy, protein modulation and in vivo approaches, as well as transcriptomic data of 262 gastric carcinoma samples, retrieved from the cancer genome atlas (TCGA). RESULTS: Here, we show that E-cadherin mutations induce an abnormal interplay of cells with specific components of the ECM, which encompasses increased traction forces and Integrin ß1 activation. Integrin ß1 synergizes with E-cadherin dysfunction, promoting cell scattering and invasion. The significance of the E-cadherin-Integrin ß1 crosstalk was validated in Drosophila models and found to be consistent with evidence from human gastric carcinomas, where increased tumour grade and poor survival are associated with low E-cadherin and high Integrin ß1 levels. CONCLUSIONS: Integrin ß1 is a key mediator of invasion in carcinomas with E-cadherin impairment and should be regarded as a biomarker of poor prognosis in gastric cancer.

Synthesis and evaluation of 2,9-disubstituted-1,10-phenanthroline derivatives as G-quadruplex binders.

G-quadruplex (G4) structures are non-canonical DNA/RNA secondary structures able to form within guanine rich nucleic acids sequences. They are present in several regions of the human genome including gene promoters, untranslated sequences, and telomeres. Due to their biological relevance G4 structures are considered important drug targets, in particular for anticancer therapies, leading to the development of G4 stabilizing small molecules. Telomeric regions have received special attention in this field since they can fold into several distinct intramolecular G-quadruplexes topologies. Herein, we report the synthesis of 2,9-disubstituted-1,10-phenanthroline derivatives and their ability to stabilize different intramolecular telomeric G4 sequences. We evaluated ligand-induced stabilization, selectivity and specificity of ligands using Förster Resonance Energy Transfer (FRET) melting experiments and circular dichroism (CD). In addition, we assessed the cytotoxicity of ligands against two cancer cell lines (A549 and H1299) and one healthy cell line (NHDF).

A Review on Locomotion Mode Recognition and Prediction When Using Active Orthoses and Exoskeletons.

Understanding how to seamlessly adapt the assistance of lower-limb wearable assistive devices (active orthosis (AOs) and exoskeletons) to human locomotion modes (LMs) is challenging. Several algorithms and sensors have been explored to recognize and predict the users' LMs. Nevertheless, it is not yet clear which are the most used and effective sensor and classifier configurations in AOs/exoskeletons and how these devices' control is adapted according to the decoded LMs. To explore these aspects, we performed a systematic review by electronic search in Scopus and Web of Science databases, including published studies from 1 January 2010 to 31 August 2022. Sixteen studies were included and scored with 84.7 ± 8.7% quality. Decoding focused on level-ground walking along with ascent/descent stairs tasks performed by healthy subjects. Time-domain raw data from inertial measurement unit sensors were the most used data. Different classifiers were employed considering the LMs to decode (accuracy above 90% for all tasks). Five studies have adapted the assistance of AOs/exoskeletons attending to the decoded LM, in which only one study predicted the new LM before its occurrence. Future research is encouraged to develop decoding tools considering data from people with lower-limb impairments walking at self-selected speeds while performing daily LMs with AOs/exoskeletons.

Robotic Biofeedback for Post-Stroke Gait Rehabilitation: A Scoping Review.

This review aims to recommend directions for future research on robotic biofeedback towards prompt post-stroke gait rehabilitation by investigating the technical and clinical specifications of biofeedback systems (BSs), including the complementary use with assistive devices and/or physiotherapist-oriented cues. A literature search was conducted from January 2019 to September 2022 on Cochrane, Embase, PubMed, PEDro, Scopus, and Web of Science databases. Data regarding technical (sensors, biofeedback parameters, actuators, control strategies, assistive devices, physiotherapist-oriented cues) and clinical (participants' characteristics, protocols, outcome measures, BSs' effects) specifications of BSs were extracted from the relevant studies. A total of 31 studies were reviewed, which included 660 stroke survivors. Most studies reported visual biofeedback driven according to the comparison between real-time kinetic or spatiotemporal data from wearable sensors and a threshold. Most studies achieved statistically significant improvements on sensor-based and clinical outcomes between at least two evaluation time points. Future research should study the effectiveness of using multiple wearable sensors and actuators to provide personalized biofeedback to users with multiple sensorimotor deficits. There is space to explore BSs complementing different assistive devices and physiotherapist-oriented cues according to their needs. There is a lack of randomized-controlled studies to explore post-stroke stage, mental and sensory effects of BSs.

Provoking Artificial Slips and Trips towards Perturbation-Based Balance Training: A Narrative Review.

Humans' balance recovery responses to gait perturbations are negatively impacted with ageing. Slip and trip events, the main causes preceding falls during walking, are likely to produce severe injuries in older adults. While traditional exercise-based interventions produce inconsistent results in reducing patients' fall rates, perturbation-based balance training (PBT) emerges as a promising task-specific solution towards fall prevention. PBT improves patients' reactive stability and fall-resisting skills through the delivery of unexpected balance perturbations. The adopted perturbation conditions play an important role towards PBT's effectiveness and the acquisition of meaningful sensor data for studying human biomechanical reactions to loss of balance (LOB) events. Hence, this narrative review aims to survey the different methods employed in the scientific literature to provoke artificial slips and trips in healthy adults during treadmill and overground walking. For each type of perturbation, a comprehensive analysis was conducted to identify trends regarding the most adopted perturbation methods, gait phase perturbed, gait speed, perturbed leg, and sensor systems used for data collection. The reliable application of artificial perturbations to mimic real-life LOB events may reduce the gap between laboratory and real-life falls and potentially lead to fall-rate reduction among the elderly community.

Deep Learning-Based Energy Expenditure Estimation in Assisted and Non-Assisted Gait Using Inertial, EMG, and Heart Rate Wearable Sensors.

Energy expenditure is a key rehabilitation outcome and is starting to be used in robotics-based rehabilitation through human-in-the-loop control to tailor robot assistance towards reducing patients' energy effort. However, it is usually assessed by indirect calorimetry which entails a certain degree of invasiveness and provides delayed data, which is not suitable for controlling robotic devices. This work proposes a deep learning-based tool for steady-state energy expenditure estimation based on more ergonomic sensors than indirect calorimetry. The study innovates by estimating the energy expenditure in assisted and non-assisted conditions and in slow gait speeds similarly to impaired subjects. This work explores and benchmarks the long short-term memory (LSTM) and convolutional neural network (CNN) as deep learning regressors. As inputs, we fused inertial data, electromyography, and heart rate signals measured by on-body sensors from eight healthy volunteers walking with and without assistance from an ankle-foot exoskeleton at 0.22, 0.33, and 0.44 m/s. LSTM and CNN were compared against indirect calorimetry using a leave-one-subject-out cross-validation technique. Results showed the suitability of this tool, especially CNN, that demonstrated root-mean-squared errors of 0.36 W/kg and high correlation (ρ > 0.85) between target and estimation (R¯2 = 0.79). CNN was able to discriminate the energy expenditure between assisted and non-assisted gait, basal, and walking energy expenditure, throughout three slow gait speeds. CNN regressor driven by kinematic and physiological data was shown to be a more ergonomic technique for estimating the energy expenditure, contributing to the clinical assessment in slow and robotic-assisted gait and future research concerning human-in-the-loop control.

G-Quadruplex Aptamer-Ligand Characterization.

In this work we explore the structure of a G-rich DNA aptamer termed AT11-L2 (TGGTGGTGGTTGTTGTTGGTGGTGGTGGT; derivative of AT11) by evaluating the formation and stability of G-quadruplex (G4) conformation under different experimental conditions such as KCl concentration, temperature, and upon binding with a variety of G4 ligands (360A, BRACO-19, PDS, PhenDC3, TMPyP4). We also determined whether nucleolin (NCL) can be a target of AT11-L2 G4. Firstly, we assessed by circular dichroism, UV and NMR spectroscopies the formation of G4 by AT11-L2. We observed that, for KCl concentrations of 65 mM or less, AT11-L2 adopts hybrid or multiple topologies. In contrast, a parallel topology predominates for buffer containing 100 mM of KCl. The Tm of AT11-L2 in 100 mM of KCl is 38.9 °C, proving the weak stability of this sequence. We also found that upon titration with two molar equivalents of 360A, BRACO-19 and PhenDC3, the G4 is strongly stabilized and its topology is maintained, while the addition of 3.5 molar equivalents of TMPyP4 promotes the disruption of G4. The KD values between AT11-L2 G4, ligands and NCL were obtained by fluorescence titrations and are in the range of µM for ligand complexes and nM when adding NCL. In silico studies suggest that four ligands bind to the AT11-L2 G4 structure by stacking interactions, while the RBD1,2 domains of NCL interact preferentially with the thymines of AT11-L2 G4. Finally, AT11-L2 G4 co-localized with NCL in NCL-positive tongue squamous cell carcinoma cell line.

An apoplastic fluid extraction method for the characterization of grapevine leaves proteome and metabolome from a single sample.

The analysis of complex biological systems keeps challenging researchers. The main goal of systems biology is to decipher interactions within cells, by integrating datasets from large scale analytical approaches including transcriptomics, proteomics and metabolomics and more specialized 'OMICS' such as epigenomics and lipidomics. Studying different cellular compartments allows a broader understanding of cell dynamics. Plant apoplast, the cellular compartment external to the plasma membrane including the cell wall, is particularly demanding to analyze. Despite our knowledge on apoplast involvement on several processes from cell growth to stress responses, its dynamics is still poorly known due to the lack of efficient extraction processes adequate to each plant system. Analyzing woody plants such as grapevine raises even more challenges. Grapevine is among the most important fruit crops worldwide and a wider characterization of its apoplast is essential for a deeper understanding of its physiology and cellular mechanisms. Here, we describe, for the first time, a vacuum-infiltration-centrifugation method that allows a simultaneous extraction of grapevine apoplastic proteins and metabolites from leaves on a single sample, compatible with high-throughput mass spectrometry analyses. The extracted apoplast from two grapevine cultivars, Vitis vinifera cv 'Trincadeira' and 'Regent', was directly used for proteomics and metabolomics analysis. The proteome was analyzed by nanoLC-MS/MS and more than 700 common proteins were identified, with highly diverse biological functions. The metabolome profile through FT-ICR-MS allowed the identification of 514 unique putative compounds revealing a broad spectrum of molecular classes.

Does Gait with an Ankle Foot Orthosis Improve or Compromise Minimum Foot Clearance?

The purpose of this study was to investigate if the use of an ankle foot orthosis in passive mode (without actuation) could modify minimum foot clearance, and if there are any compensatory mechanisms to enable these changes during treadmill gait at a constant speed. Eight participants walked on an instrumented treadmill without and with an ankle foot orthosis on the dominant limb at speeds of 0.8, 1.2, and 1.6 km/h. For each gait cycle, the minimum foot clearance and some gait linear kinematic parameters were calculated by an inertial motion capture system. Additionally, maximum hip and knee flexion and maximum ankle plantar flexion were calculated. There were no significant differences in the minimum foot clearance between gait conditions and lower limbs. However, differences were found in the swing, stance and step times between gait conditions, as well as between limbs during gait with orthosis (p < 0.05). An increase in hip flexion during gait with orthosis was observed for all speeds, and different ankle ranges of motion were observed according to speed (p < 0.05). Thus, the use of an ankle foot orthosis in passive mode does not significantly hinder minimum foot clearance, but can change gait linear and angular parameters in non-pathological individuals.

Molecular Beacon Assay Development for Severe Acute Respiratory Syndrome Coronavirus 2 Detection.

The fast spread of SARS-CoV-2 has led to a global pandemic, calling for fast and accurate assays to allow infection diagnosis and prevention of transmission. We aimed to develop a molecular beacon (MB)-based detection assay for SARS-CoV-2, designed to detect the ORF1ab and S genes, proposing a two-stage COVID-19 testing strategy. The novelty of this work lies in the design and optimization of two MBs for detection of SARS-CoV-2, namely, concentration, fluorescence plateaus of hybridization, reaction temperature and real-time results. We also identify putative G-quadruplex (G4) regions in the genome of SARS-CoV-2. A total of 458 nasopharyngeal and throat swab samples (426 positive and 32 negative) were tested with the MB assay and the fluorescence levels compared with the cycle threshold (Ct) values obtained from a commercial RT-PCR test in terms of test duration, sensitivity, and specificity. Our results show that the samples with higher fluorescence levels correspond to those with low Ct values, suggesting a correlation between viral load and increased MB fluorescence. The proposed assay represents a fast (total duration of 2 h 20 min including amplification and fluorescence reading stages) and simple way of detecting SARS-CoV-2 in clinical samples from the upper respiratory tract.

Ligands as Stabilizers of G-Quadruplexes in Non-Coding RNAs.

The non-coding RNAs (ncRNA) are RNA transcripts with different sizes, structures and biological functions that do not encode functional proteins. RNA G-quadruplexes (rG4s) have been found in small and long ncRNAs. The existence of an equilibrium between rG4 and stem-loop structures in ncRNAs and its effect on biological processes remains unexplored. For example, deviation from the stem-loop leads to deregulated mature miRNA levels, demonstrating that miRNA biogenesis can be modulated by ions or small molecules. In light of this, we report several examples of rG4s in certain types of ncRNAs, and the implications of G4 stabilization using small molecules, also known as G4 ligands, in the regulation of gene expression, miRNA biogenesis, and miRNA-mRNA interactions. Until now, different G4 ligands scaffolds were synthesized for these targets. The regulatory role of the above-mentioned rG4s in ncRNAs can be used as novel therapeutic approaches for adjusting miRNA levels.