Splice-Switching Antisense Oligonucleotides Correct Phenylalanine Hydroxylase Exon 11 Skipping Defects and Rescue Enzyme Activity in Phenylketonuria.

The PAH gene encodes the hepatic enzyme phenylalanine hydroxylase (PAH), and its deficiency, known as phenylketonuria (PKU), leads to neurotoxic high levels of phenylalanine. PAH exon 11 is weakly defined, and several missense and intronic variants identified in patients affect the splicing process. Recently, we identified a novel intron 11 splicing regulatory element where U1snRNP binds, participating in exon 11 definition. In this work, we describe the implementation of an antisense strategy targeting intron 11 sequences to correct the effect of PAH mis-splicing variants. We used an in vitro assay with minigenes and identified splice-switching antisense oligonucleotides (SSOs) that correct the exon skipping defect of PAH variants c.1199+17G>A, c.1199+20G>C, c.1144T>C, and c.1066-3C>T. To examine the functional rescue induced by the SSOs, we generated a hepatoma cell model with variant c.1199+17G>A using CRISPR/Cas9. The edited cell line reproduces the exon 11 skipping pattern observed from minigenes, leading to reduced PAH protein levels and activity. SSO transfection results in an increase in exon 11 inclusion and corrects PAH deficiency. Our results provide proof of concept of the potential therapeutic use of a single SSO for different exonic and intronic splicing variants causing PAH exon 11 skipping in PKU.

A 3D bioprinted hydrogel gut-on-chip with integrated electrodes for transepithelial electrical resistance (TEER) measurements.

Conventional gut-on-chip (GOC) models typically represent the epithelial layer of the gut tissue, neglecting other important components such as the stromal compartment and the extracellular matrix (ECM) that play crucial roles in maintaining intestinal barrier integrity and function. These models often employ hard, flat porous membranes for cell culture, thus failing to recapitulate the soft environment and complex 3D architecture of the intestinal mucosa. Alternatively, hydrogels have been recently introduced in GOCs as ECM analogs to support the co-culture of intestinal cells inin vivo-like configurations, and thus opening new opportunities in the organ-on-chip field. In this work, we present an innovative GOC device that includes a 3D bioprinted hydrogel channel replicating the intestinal villi architecture containing both the epithelial and stromal compartments of the gut mucosa. The bioprinted hydrogels successfully support both the encapsulation of fibroblasts and their co-culture with intestinal epithelial cells under physiological flow conditions. Moreover, we successfully integrated electrodes into the microfluidic system to monitor the barrier formation in real time via transepithelial electrical resistance measurements.

PAH deficient pathology in humanized c.1066-11G>A phenylketonuria mice.

We have generated using CRISPR/Cas9 technology a partially humanized mouse model of the neurometabolic disease phenylketonuria (PKU), carrying the highly prevalent PAH variant c.1066-11G>A. This variant creates an alternative 3' splice site, leading to the inclusion of 9 nucleotides coding for 3 extra amino acids between Q355 and Y356 of the protein. Homozygous Pah c.1066-11A mice, with a partially humanized intron 10 sequence with the variant, accurately recapitulate the splicing defect and present almost undetectable hepatic PAH activity. They exhibit fur hypopigmentation, lower brain and body weight and reduced survival. Blood and brain phenylalanine levels are elevated, along with decreased tyrosine, tryptophan and monoamine neurotransmitter levels. They present behavioral deficits, mainly hypoactivity and diminished social interaction, locomotor deficiencies and an abnormal hind-limb clasping reflex. Changes in the morphology of glial cells, increased GFAP and Iba1 staining signals and decreased myelinization are observed. Hepatic tissue exhibits nearly absent PAH protein, reduced levels of chaperones DNAJC12 and HSP70 and increased autophagy markers LAMP1 and LC3BII, suggesting possible coaggregation of mutant PAH with chaperones and subsequent autophagy processing. This PKU mouse model with a prevalent human variant represents a useful tool for pathophysiology research and for novel therapies development.

Características sociales de la población con infección COVID-19 de un centro de salud en cuatro olas consecutivas de la pandemia / Social characteristics of the population with COVID-19 infection in a primary care center in four consecutive pandemic waves

El objetivo de este estudio es describir algunas características sociales de la población con enfermedad por coronavirus (COVID-19) de un centro de salud de Fuenlabrada. Se incluyeron todos los pacientes valorados en consulta de forma presencial o telefónica, que fueron atendidos, tras el diagnóstico por prueba de detección de infección activa (PDIA), por las gestoras COVID-19 del centro. Los resultados principales describen una mayoría de hogares compuestos por tres habitaciones, siendo esto acorde al número de convivientes; con un baño de media y con terraza en la mayoría de ellos. Hay una gran incidencia de hogares sin personas activas laboralmente y con escasa ayuda social. Se observa ligeramente una mayor incidencia de mujeres diagnosticadas. Los determinantes sociales de la salud como las condiciones de vivienda, el estado laboral o las ayudas sociales influyen en la distribución de los recursos sanitarios. Este estudio refuerza la importancia de la Atención Primaria y sus recursos en situaciones de emergencia. (AU)

This study aims to describe some social characteristics of the population with Coronavirus Disease (COVID) infection in a Primary Care Centre in Fuenlabrada. All patients assessed in the consultation in person or by telephone, who were attended after the diagnosis by active infection diagnostic test (AIDT), by the centre's COVID tracing team, were included. The main results describe a majority of households comprising three rooms, this being in line with the number of cohabitants; with one bathroom on average and a terrace in the majority. There is a high incidence of households with unemployed people and with little social assistance. There is a slightly higher incidence of diagnosed women. The social determinants of health such as housing conditions, employment status, or social assistance influence the distribution of health resources. This study reinforces the importance of Primary Care and its emergency resources. (AU)

High performance of the automated ADVIA Centaur Systems SARS-CoV-2 Antigen Assay in nasopharyngeal samples with high viral load / Alto rendimiento del ensayo de antígeno ADVIA Centaur Systems SARS-CoV-2 automatizado en muestras nasofaríngeas con alta carga viral

ADVIA Centaur SARS-CoV-2 Antigen (COV2Ag) Assay (Siemens Healthineers) was evaluated for SARS-CoV-2 detection. A total of 141 nasopharyngeal samples were analyzed by this technique and results were compared with those obtained by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The overall sensitivity and specificity of the test were 68.70% and 70%, respectively. Regarding cycle threshold (Ct) values, the COV2Ag test showed a sensitivity of 93.75% and 100% for nasopharyngeal samples with Ct < 25 and < 20, respectively. ADVIA Centaur COV2Ag Assay is a useful, automated, and rapid technique for early SARS-CoV-2 diagnosis and isolation of the infected individuals, avoiding its transmission.(AU)

Experiential COVID-19 factors predicting resilience among Spanish adults.

BACKGROUND: The pandemic caused by COVID-19 has meant for spanish citizens a constant adaptation to health measures in order to try to stop transmission of the virus. During this adaptation process, different psychosocial aspects have caused consequences for people?s mental health to a greater or lesser extent. Makes sense of an emotional torrent who has gone through fear, anxiety, loneliness and anger. The interaction between perception and reality has given rise to situations where loneliness and social isolation have been imposed and lived with a load of emotional discomfort. In others, social isolation and measures to stop the pandemic have been accepted as a protection system and has been experienced since serenity and the feeling of self-protection fostering individual resilience. Studying the predictors of resilience is going to be key since it is the ideal antidote to stop the appearance of mental disorders associated with the pandemic (such as depression, anxiety, post-traumatic stress, social phobia, cleaning obsessions, and generalized anxiety disorder). The objective of this research is to analyze the relationship between resilience and experiential COVID-19 factors. METHODS: Sample was comprised of Spanish adults (n = 1000; age 18-79 [mean =40.43],793 female, 201 male, and 2 non binary sex). These people participating in an online study focused on the impact of COVID-19 experiences. The research has been cross-sectional, descriptive and correlational design. The instrument created for this research was a specific online questionnaire, including the "Scale of resilience" (RS, Wagnild & Young, 1993, Spanish version, Sánchez-Teruel, et al., 2015). That questionnaire has been administered during the months of April 2022 to July 2022. RESULTS: The results obtained show how people who have been able to face the pandemic in a responsive and adaptive way have high resilience. Specifically, those participants that accepting the use of masks, vaccinations and confinement obtained high resilience. CONCLUSIONS: Using public funding and allocating research to the development of programs to promote resilience, adaptative beliefs and prosocial behaviors becomes basic to live in a world in constant change.

Correction: Organ-on-a-chip with integrated semitransparent organic electrodes for barrier function monitoring.

Correction for 'Organ-on-a-chip with integrated semitransparent organic electrodes for barrier function monitoring' by Denise Marrero et al., Lab Chip, 2023, https://doi.org/10.1039/d2lc01097f.

Organ-on-a-chip with integrated semitransparent organic electrodes for barrier function monitoring.

Organs-on-a-chip (OoC) are cell culture platforms that replicate key functional units of tissues in vitro. Barrier integrity and permeability evaluation are of utmost importance when studying barrier-forming tissues. Impedance spectroscopy is a powerful tool and is widely used to monitor barrier permeability and integrity in real-time. However, data comparison across devices is misleading due to the generation of a non-homogenous field across the tissue barrier, making impedance data normalization very challenging. In this work, we address this issue by integrating PEDOT:PSS electrodes for barrier function monitoring with impedance spectroscopy. The semitransparent PEDOT:PSS electrodes cover the entire cell culture membrane providing a homogenous electric field across the entire membrane making the cell culture area equally accountable to the measured impedance. To the best of our knowledge, PEDOT:PSS has never been used solely to monitor the impedance of cellular barriers while enabling optical inspection in the OoC. The performance of the device is demonstrated by lining the device with intestinal cells where we monitored barrier formation under flow conditions, as well as barrier disruption and recovery under exposure to a permeability enhancer. The barrier tightness and integrity, and the intercellular cleft have been evaluated by analyzing the full impedance spectrum. Furthermore, the device is autoclavable paving the way toward more sustainable OoC options.

Dysregulated Cell Homeostasis and miRNAs in Human iPSC-Derived Cardiomyocytes from a Propionic Acidemia Patient with Cardiomyopathy.

Propionic acidemia (PA) disorder shows major involvement of the heart, among other alterations. A significant number of PA patients develop cardiac complications, and available evidence suggests that this cardiac dysfunction is driven mainly by the accumulation of toxic metabolites. To contribute to the elucidation of the mechanistic basis underlying this dysfunction, we have successfully generated cardiomyocytes through the differentiation of induced pluripotent stem cells (iPSCs) from a PCCB patient and its isogenic control. In this human cellular model, we aimed to examine microRNAs (miRNAs) profiles and analyze several cellular pathways to determine miRNAs activity patterns associated with PA cardiac phenotypes. We have identified a series of upregulated cardiac-enriched miRNAs and alterations in some of their regulated signaling pathways, including an increase in the expression of cardiac damage markers and cardiac channels, an increase in oxidative stress, a decrease in mitochondrial respiration and autophagy; and lipid accumulation. Our findings indicate that miRNA activity patterns from PA iPSC-derived cardiomyocytes are biologically informative and advance the understanding of the molecular mechanisms of this rare disease, providing a basis for identifying new therapeutic targets for intervention strategies.

Pathogenic variants of the coenzyme A biosynthesis-associated enzyme phosphopantothenoylcysteine decarboxylase cause autosomal-recessive dilated cardiomyopathy.

Coenzyme A (CoA) is an essential cofactor involved in a range of metabolic pathways including the activation of long-chain fatty acids for catabolism. Cells synthesize CoA de novo from vitamin B5 (pantothenate) via a pathway strongly conserved across prokaryotes and eukaryotes. In humans, it involves five enzymatic steps catalyzed by four enzymes: pantothenate kinase (PANK [isoforms 1-4]), 4'-phosphopantothenoylcysteine synthetase (PPCS), phosphopantothenoylcysteine decarboxylase (PPCDC), and CoA synthase (COASY). To date, inborn errors of metabolism associated with all of these genes, except PPCDC, have been described, two related to neurodegeneration with brain iron accumulation (NBIA), and one associated with a cardiac phenotype. This paper reports another defect in this pathway (detected in two sisters), associated with a fatal cardiac phenotype, caused by biallelic variants (p.Thr53Pro and p.Ala95Val) of PPCDC. PPCDC enzyme (EC 4.1.1.36) catalyzes the decarboxylation of 4'-phosphopantothenoylcysteine to 4'-phosphopantetheine in CoA biosynthesis. The variants p.Thr53Pro and p.Ala95Val affect residues highly conserved across different species; p.Thr53Pro is involved in the binding of flavin mononucleotide, and p.Ala95Val is likely a destabilizing mutation. Patient-derived fibroblasts showed an absence of PPCDC protein, and nearly 50% reductions in CoA levels. The cells showed clear energy deficiency problems, with defects in mitochondrial respiration, and mostly glycolytic ATP synthesis. Functional studies performed in yeast suggest these mutations to be functionally relevant. In summary, this work describes a new, ultra-rare, severe inborn error of metabolism due to pathogenic variants of PPCDC.

High performance of the automated ADVIA Centaur Systems SARS-CoV-2 Antigen Assay in nasopharyngeal samples with high viral load.

ADVIA Centaur SARS-CoV-2 Antigen (COV2Ag) Assay (Siemens Healthineers) was evaluated for SARS-CoV-2 detection. A total of 141 nasopharyngeal samples were analyzed by this technique and results were compared with those obtained by quantitative reverse-transcription polymerase chain reaction (RT-PCR). The overall sensitivity and specificity of the test were 68.70% and 70%, respectively. Regarding cycle threshold (Ct) values, the COV2Ag test showed a sensitivity of 93.75% and 100% for nasopharyngeal samples with Ct < 25 and < 20, respectively. ADVIA Centaur COV2Ag Assay is a useful, automated, and rapid technique for early SARS-CoV-2 diagnosis and isolation of the infected individuals, avoiding its transmission.

Direct Color Observation of Light-Driven Molecular Conformation-Induced Stress.

Although usually complex to handle, nanomechanical sensors are exceptional, label-free tools for monitoring molecular conformational changes, which makes them of paramount importance in understanding biomolecular interactions. Herein, a simple and inexpensive mechanical imaging approach based on low-stiffness cantilevers with structural coloration (mechanochromic cantilevers (MMC)) is demonstrated, able to monitor and quantify molecular conformational changes with similar sensitivity to the classical optical beam detection method of cantilever-based sensors (≈4.6 × 10-3  N m-1 ). This high sensitivity is achieved by using a white light and an RGB camera working in the reflection configuration. The sensor performance is demonstrated by monitoring the UV-light induced reversible conformational changes of azobenzene molecules coating. The trans-cis isomerization of the azobenzene molecules induces a deflection of the cantilevers modifying their diffracted color, which returns to the initial state by cis-trans relaxation. Interestingly, the mechanical imaging enables a simultaneous 2D mapping of the response thus enhancing the spatial resolution of the measurements. A tight correlation is found between the color output and the cantilever's deflection and curvature angle (sensitivities of 5 × 10-3  Hue µm-1 and 1.5 × 10-1  Hue (°)-1 ). These findings highlight the suitability of low-stiffness MMC as an enabling technology for monitoring molecular changes with unprecedented simplicity, high-throughput capability, and functionalities.

Elastic Plasmonic-Enhanced Fabry-Pérot Cavities with Ultrasensitive Stretching Tunability.

The emerging stretchable photonics field faces challenges, like the robust integration of optical elements into elastic matrices or the generation of large optomechanical effects. Here, the first stretchable plasmonic-enhanced and wrinkled Fabry-Pérot (FP) cavities are demonstrated, which are composed of self-embedded arrays of Au nanostructures at controlled depths into elastomer films. The novel self-embedding process is triggered by the Au nanostructures' catalytic activity, which locally increases the polymer curing rate, thereby inducing a mechanical stress that simultaneously pulls the Au nanostructures into the polymer and forms a wrinkled skin layer. This geometry yields unprecedented optomechanical effects produced by the coupling of the broad plasmonic modes of the Au nanostructures and the FP modes, which are modulated by the wrinkled optical cavity. As a result, film stretching induces drastic changes in both the spectral position and intensity of the plasmonic-enhanced FP resonances due to the simultaneous cavity thickness reduction and cavity wrinkle flattening, thus increasing the cavity finesse. These optomechanical effects are exploited to demonstrate new strain-sensing approaches, achieving a strain detection limit of 0.006%, i.e., 16-fold lower than current optical strain-detection schemes.

Mechanochromic Detection for Soft Opto-Magnetic Actuators.

New multi-stimuli responsive materials are required in smart systems applications to overcome current limitations in remote actuation and to achieve versatile operation in inaccessible environments. The incorporation of detection mechanisms to quantify in real time the response to external stimuli is crucial for the development of automated systems. Here, we present the first wireless opto-magnetic actuator with mechanochromic response. The device, based on a nanostructured-iron (Fe) layer transferred onto suspended elastomer structures with a periodically corrugated backside, can be actuated both optically (in a broadband spectral range) and magnetically. The combined opto-magnetic stimulus can accurately modulate the mechanical response (strength and direction) of the device. The structural coloration generated at the corrugated back surface enables to easily map and quantify, in 2D, the mechanical deflections by analyzing in real time the hue changes of images taken using a conventional RGB smartphone camera, with a precision of 0.05°. We demonstrate the independent and synergetic optical and magnetic actuation and detection with a detection limit of 1.8 mW·cm-2 and 0.34 mT, respectively. The simple operation, versatility, and cost-effectiveness of the wireless multiactuated device with highly sensitive mechanochromic mapping paves the way to a new generation of wirelessly controlled smart systems.

Discovery, optimization, and target identification of novel coumarin derivatives as HIV-1 reverse transcriptase-associated ribonuclease H inhibitors.

Despite significant advances in antiretroviral therapy, acquired immunodeficiency syndrome remains as one of the leading causes of death worldwide. New antiretroviral drugs combined with updated treatment strategies are needed to improve convenience, tolerability, safety, and antiviral efficacy of available therapies. In this work, a focused library of coumarin derivatives was exploited by cell phenotypic screening to discover novel inhibitors of HIV-1 replication. Five compounds (DW-3, DW-4, DW-11, DW-25 and DW-31) showed moderate activity against wild-type and drug-resistant strains of HIV-1 (IIIB and RES056). Four of those molecules were identified as inhibitors of the viral RT-associated RNase H. Structural modification of the most potent DW-3 and DW-4 led to the discovery of compound 8a. This molecule showed increased potency against wild-type HIV-1 strain (EC50 = 3.94 ± 0.22 µM) and retained activity against a panel of mutant strains, showing EC50 values ranging from 5.62 µM to 202 µM. In enzymatic assays, 8a was found to inhibit the viral RNase H with an IC50 of 12.3 µM. Molecular docking studies revealed that 8a could adopt a binding mode similar to that previously reported for other active site HIV-1 RNase H inhibitors.

[Improvement of flu vaccination coverage in people over 65 years old. Strategy developed in a health area of Asturias.] / Mejora de la cobertura de vacunación antigripal en persona mayores de 65 años. Estrategia desarrollada en un área de salud de Asturias.

OBJECTIVE: Influenza vaccination rates among adults over 65 years have not reached the minimum levels recommended by the health authorities in Asturias during previous vaccination campaigns. The objective of this study is to describe the characteristics of an influenza vaccination strategy and its effectiveness. METHODS: Strategy developed during the 2019-2020 influenza vaccination campaign in the Área Sanitaria VII in the Servicio de Salud of the Principado de Asturias. The target population were people over 65 years. The strategy consisted of training sessions for professionals and the general population, information dissemination through the media, overt recommendations for the vaccination of hospital personnel and social recognition of the professionals involved. The vaccination rates were described and a two-tailed hypothesis test was used to determine the differences in the vaccination percentages. RESULTS: The percentage of vaccination in the Área Sanitaria VII went from 56.29% in the 2018-2019 campaign to 65.82% in the 2019-2020 campaign (+9.53; p<0.001). During the same campaign, the percentage of vaccination in Asturias was 57,38%, that is, 8,44% less than in the intervened Área Sanitaria. CONCLUSIONS: The strategy proved effective in increasing the rates of influenza vaccination among adults over 65 years in the Área Sanitaria VII of the Principado de Asturias.

OBJETIVO: La vacunación de la gripe en población mayor de 65 años en Asturias no ha alcanzado el mínimo recomendado por las autoridades sanitarias españolas en las últimas campañas de vacunación. El objetivo de este trabajo consistió en presentar las características de una estrategia de vacunación frente a la gripe y su efectividad. METODOS: Estrategia desarrollada en la campaña de vacunación 2019-2020, en el Área Sanitaria VII del Servicio de Salud del Principado de Asturias. Su población objeto fueron las personas mayores de 65 años. Incluyó sesiones formativas a profesionales y población, información a la población a través de medios de comunicación, recomendación activa de la vacunación en el ámbito hospitalario y reconocimiento social a los profesionales implicados. Se realizó la descripción de las tasas de vacunación y, para determinar las diferencias en los porcentajes de vacunación, se utilizó un contraste de hipótesis para proporciones de dos colas. RESULTADOS: El porcentaje de vacunación en el Área Sanitaria VII pasó de un 56,29% en la campaña 2018-2019 a un 65,82% en la 2019-2020 (+9,53; p<0,001). Esa misma campaña el porcentaje de vacunación en Asturias fue 57,38%, es decir un 8,44% menor que en Área Sanitaria intervenida. CONCLUSIONES: La estrategia ha mostrado ser efectiva para aumentar el porcentaje de vacunación frente a la gripe en población mayor de 65 años en el Área Sanitaria VII del Principado de Asturias.

Engineering Tissue Barrier Models on Hydrogel Microfluidic Platforms.

Tissue barriers play a crucial role in human physiology by establishing tissue compartmentalization and regulating organ homeostasis. At the interface between the extracellular matrix (ECM) and flowing fluids, epithelial and endothelial barriers are responsible for solute and gas exchange. In the past decade, microfluidic technologies and organ-on-chip devices became popular as in vitro models able to recapitulate these biological barriers. However, in conventional microfluidic devices, cell barriers are primarily grown on hard polymeric membranes within polydimethylsiloxane (PDMS) channels that do not mimic the cell-ECM interactions nor allow the incorporation of other cellular compartments such as stromal tissue or vascular structures. To develop models that accurately account for the different cellular and acellular compartments of tissue barriers, researchers have integrated hydrogels into microfluidic setups for tissue barrier-on-chips, either as cell substrates inside the chip, or as self-contained devices. These biomaterials provide the soft mechanical properties of tissue barriers and allow the embedding of stromal cells. Combining hydrogels with microfluidics technology provides unique opportunities to better recreate in vitro the tissue barrier models including the cellular components and the functionality of the in vivo tissues. Such platforms have the potential of greatly improving the predictive capacities of the in vitro systems in applications such as drug development, or disease modeling. Nevertheless, their development is not without challenges in their microfabrication. In this review, we will discuss the recent advances driving the fabrication of hydrogel microfluidic platforms and their applications in multiple tissue barrier models.

Gut-on-a-chip: Mimicking and monitoring the human intestine.

Over the last few years, the intestine has been extensively studied using in vitro microfluidic systems, commonly known as gut-on-a-chip (GOC) devices. This interest has been due not only to the importance of the intestine's proper functions but also to the relationship that this organ and the microbiota that inhabits it has with the rest of the body's organs. The increased complexity of these in vitro systems, together with the need to improve our understanding of intestinal physiology interdependencies, has led to greater focus on the integration of biosensors within these devices. However, the current number of GOC devices with integrated sensors for monitoring relevant physiological parameters are very limited and demand the use of external analytical techniques that delay the analysis and prevent real-time decision-making. This paper reviews the various materials, technologies, and structures that have been used both for mimicking the physiology of the intestine and monitoring relevant physiological parameters, such as permeability of the gut barrier, dissolved oxygen concentration, cytokines profile and the production of microbial short-chain fatty acids. We also propose alternative biosensing techniques demonstrated in other in vitro and lab-on-a-chip devices that could be translated to GOC models. A critical analysis of the requirements, limitations, and current challenges on the microenvironment replication and monitorization of GOC models is included, with a particular focus on the physiological parameters and biomarkers that should be detected simultaneously in real-time to get a proper framework of the gut function that until now, have not received the necessary attention.

Analysis and Molecular Determinants of HIV RNase H Cleavage Specificity at the PPT/U3 Junction.

HIV reverse transcriptases (RTs) convert viral genomic RNA into double-stranded DNA. During reverse transcription, polypurine tracts (PPTs) resilient to RNase H cleavage are used as primers for plus-strand DNA synthesis. Nonnucleoside RT inhibitors (NNRTIs) can interfere with the initiation of plus-strand DNA synthesis by enhancing PPT removal, while HIV RT connection subdomain mutations N348I and N348I/T369I mitigate this effect by altering RNase H cleavage specificity. Now, we demonstrate that among approved nonnucleoside RT inhibitors (NNRTIs), nevirapine and doravirine show the largest effects. The combination N348I/T369I in HIV-1BH10 RT has a dominant effect on the RNase H cleavage specificity at the PPT/U3 site. Biochemical studies showed that wild-type HIV-1 and HIV-2 RTs were able to process efficiently and accurately all tested HIV PPT sequences. However, the cleavage accuracy at the PPT/U3 junction shown by the HIV-2EHO RT was further improved after substituting the sequence YQEPFKNLKT of HIV-1BH10 RT (positions 342-351) for the equivalent residues of the HIV-2 enzyme (HQGDKILKV). Our results highlight the role of ß-sheets 17 and 18 and their connecting loop (residues 342-350) in the connection subdomain of the large subunit, in determining the RNase H cleavage window of HIV RTs.