Phylogenetic relationships of the Mangrove Hummingbird, "Amazilia" boucardi (Apodiformes: Trochilidae) of Costa Rica / Relaciones filogenéticas del Colibrí manglero "Amazilia" boucardi (Apodiformes: Trochilidae) de Costa Rica

Abstract Introduction: A recent revision of the generic classification of the Trochilidae based on DNA sequences revealed many inconsistencies with the current generic classification, largely based on plumage characters subject to homoplasy, especially in the Trochilini, the largest tribe. A thorough generic reorganization brought the classification into accord with the phylogeny, but due to lack of genetic data, two species remained unclassified. One of these was the Mangrove Hummingbird, "Amazilia" boucardi, endemic to Costa Rica and included in the IUCN red list of threatened species. Objective: To obtain molecular evidence to clarify the generic relationships of "A." boucardi. Methods: We isolated DNA from tissues of this species and amplified 4 nuclear and 4 mitochondrial fragments and compared these with homologous fragments from 56 species in the Trochilini, constructing phylogenetic trees with maximum likelihood and Bayesian methods. Results: Our phylogenetic analyses confirmed the placement of boucardi in the Trochilini and definitely excluded it from Amazilia but placed it with high confidence in the genus Chrysuronia Bonaparte, 1850, within which its closest relative is C. coeruleogularis, which also inhabits mangroves. Conclusions: Our genetic data based on nuclear and mitochondrial regions clearly indicate the relationship of A. boucardi and L. coeruleogularis. Moreover, it is also supported by their habitat distribution in the mangroves of the Pacific coast of Costa Rica and Western Panama. Therefore, we suggested to exclude A. boucardi as "incertae sedis".

Resumen Introducción: Una revisión reciente de la clasificación de la familia Trochilidae con base en secuencias de ADN demostró muchas incongruencias con la clasificación genérica previa, que había sido hecho con base en caracteres del plumaje muy sujetos a homoplasia, especialmente en la tribu más grande, Trochillini. Una reorganización de los géneros logró llevar su clasificación genérica a la concordancia con la filogenia, pero debido a la ausencia de datos genéticos, dos especies permanecieron sin clasificar. Una de estas fue el colibrí de manglar Amazilia boucardi, una especie endémica de Costa Rica, considerada como amenazada en la lista roja de la UICN. Objetivo: Obtener evidencia molecular para esclarecer las relaciones genéricas de A. boucardi. Métodos: Se aisló ADN de tejidos de esta especie y se amplificaron 4 fragmentos de ADN del núcleo y 5 de la mitocondria, y se compararon con fragmentos homólogos de 56 especies en la tribu Trochillini, generando árboles filogenéticos con métodos de máxima verosimilitud y bayesiano. Resultados: Los análisis filogénticos obtenidos confirmaron la ubicación de boucardi en Trochilini y definitivamente la excluyó del género Amazilia, pero la ubicó con un alto grado de confianza en el género Chrysuronia Bonaparte, 1850, dentro los cuales su pariente más cercano es C. coeruleogularis, que también habita manglares. Conclusiones: Nuestros datos genéticos basados en regiones nucleares y mitocondriales indican claramente la relación entre A. boucardi and L. coeruleogularis. Es más, lo anterior se sustenta por su distribución en los manglares de la costa Pacífica de Costa Rica y oeste de Panamá. Por lo tanto, sugerimos excluir a A. boucardi como "incertae sedis".

Evaluating the reliability of DNA Barcoding for Central American Pacific shallow water echinoderms identification: a molecular taxonomy and database accuracy analysis / Evaluando la fiabilidad del Código de Barras de ADN para la identificación de equinodermos en aguas poco profundas del Pacífico de América Central: un análisis de taxonomía molecular y precisión de bases de datos

Abstract Introduction: Molecular divergence thresholds have been proposed to distinguish recently separated evolutive units, often displaying more accurate putative species assignments in taxonomic research compared to traditional morphological approaches. This makes DNA barcoding an attractive identification tool for a variety of marine invertebrates, especially for cryptic species complexes. Although GenBank and the Barcode of Life Data System (BOLD) are the major sequence repositories worldwide, very few have tested their performance in the identification of echinoderm sequences. Objective: We use COI echinoderm sequences from local samples and the molecular identification platforms from GenBank and BOLD, in order to test their accuracy and reliability in the DNA barcoding identification for Central American shallow water echinoderms, at genus and species level. Methods: We conducted sampling, tissue extraction, COI amplification, sequencing, and taxonomic identification for 475 specimens. The 348 obtained sequences were individually enquired with BLAST in GenBank as well as using the Identification System (IDS) in BOLD. Query sequences were classified depending on the best match result. McNemar's chi-squared, Kruskal-Wallis's and Mann-Whitney's U tests were performed to prove differences between the results from both databases. Additionally, we recorded an updated list of species reported for the shallow waters of the Central American Pacific. Results: We found 324 echinoderm species reported for Central American Pacific shallow waters. Only 118 and 110 were present in GenBank and BOLD databases respectively. We proposed 325 solved morphology-based identities and 21 provisional identifications in 50 putative taxa. GenBank retrieved 348 molecular-based identifications in 58 species, including twelve provisional identifications in tree taxa. BOLD recovered 170 COI identifications in 23 species with one provisional identification. Nevertheless, 178 sequences retrieved unmatched terms (in 34 morphology-based taxa). Only 86 sequences (25 %) were retrieved as correct identifications and 128 (37 %) as identification errors in both platforms. We include 84 sequences for eleven species not represented in GenBank and 65 sequences for ten species in BOLD Echinoderm COI databases. The identification accuracy using BLAST (175 correct and 152 incorrect identifications) was greater than with IDS engine (110 correct and 218 identification errors), therefore GenBank outperforms BOLD (Kruskal-Wallis = 41.625, df = 1, p < 0.001). Conclusions: Additional echinoderm sample references are needed to improve the utility of the evaluated DNA barcoding identification tools. Identification discordances in both databases may obey specific parameters used in each search algorithm engine and the available sequences. We recommend the use of barcoding as a complementary identification source for Central American Pacific shallow water echinoderm species.

Resumen Introducción: Se han propuesto los umbrales de divergencia molecular para distinguir unidades evolutivas recientemente separadas, que a menudo muestran asignaciones de especies putativas más precisas en la investigación taxonómica en comparación con los enfoques morfológicos tradicionales. Esto hace que los Códigos de Barras de ADN sean una herramienta de identificación atractiva para una variedad de invertebrados marinos, especialmente para complejos de especies crípticas. Aunque GenBank y Barcode of Life Data System (BOLD) son los principales repositorios de secuencias en todo el mundo, muy pocos han probado su desempeño en la identificación de secuencias de equinodermos. Objetivo: Utilizamos secuencias de equinodermos COI de muestras locales y las plataformas de identificación molecular de GenBank y BOLD, para probar su precisión y confiabilidad en la implementación de códigos de barras de ADN para equinodermos de aguas someras de Centroamérica, a nivel de género y especie. Métodos: Realizamos muestreo, extracción de tejido, amplificación de COI, secuenciación e identificación taxonómica de 475 especímenes. Las 348 secuencias obtenidas fueron consultadas individualmente con BLAST en GenBank así como utilizando el Sistema de Identificación (IDS) en BOLD. Las secuencias consultadas se clasificaron según el mejor resultado de coincidencia. Se realizaron las pruebas chi-cuadrado de McNemar, Kruskal-Wallis y U de Mann-Whitney para comprobar diferencias entre los resultados de ambas bases de datos. Además, registramos una lista actualizada de especies reportadas para las aguas someras del Pacífico Centroamericano. Resultados: Encontramos 324 especies de equinodermos reportadas para aguas someras (< 200 m) del Pacífico centroamericano. Sólo 118 y 110 estaban presentes en las bases de datos GenBank y BOLD respectivamente. Propusimos 325 identidades resueltas basadas en morfología y 21 identificaciones provisionales en 50 taxones putativos. GenBank recuperó 348 identificaciones de base molecular en 58 especies, incluidas doce identificaciones provisionales en tres taxones. BOLD recuperó 170 identificaciones de COI en 23 especies con una identificación provisional. Sin embargo, 178 secuencias recuperaron términos no coincidentes (en 34 taxones basados en morfología). Sólo 86 secuencias (25 %) se recuperaron como identificaciones correctas y 128 (37 %) como errores de identificación en ambas plataformas. Incluimos 84 secuencias para once especies no representadas en GenBank y 65 secuencias para diez especies ausentes en las bases de datos BOLD Echinoderm COI. La precisión de la identificación usando BLAST (175 identificaciones correctas y 152 incorrectas) fue mayor que con el motor IDS (110 correctas y 218 errores de identificación), por lo tanto, GenBank supera a BOLD (Kruskal-Wallis = 41.625, df = 1, p < 0.001). Conclusiones: Se necesitan muestras adicionales de equinodermos de referencia para mejorar la utilidad de las herramientas de identificación de códigos de barras de ADN evaluadas. Las discordancias de identificación en ambas bases de datos pueden obedecer a parámetros específicos utilizados en cada algoritmo de búsqueda y a las secuencias disponibles. Recomendamos el uso de códigos de barras como fuente de identificación complementaria para las especies de equinodermos de aguas someras del Pacífico centroamericano.

Recent molecular techniques to strengthen ecological studies in echinoderms / Técnicas moleculares recientes que fortalecen los estudios ecológicos de equinodermos

Abstract Introduction: Echinoderms, an integral component of marine ecosystems worldwide, have captivated scientific interest for centuries. Despite this longstanding attention, comprehending key facets such as trophic relationships, diet composition, and host-microbiota relationships still represents a challenge using traditional techniques. Recent years, however, have witnessed a transformative shift, thanks to the emergence of advanced molecular techniques, offering new approaches to strengthen ecological studies in echinoderms. Objective: Explore how recent advancements in molecular tools have impacted ecological research on echinoderms. Specifically, we aim to investigate the potential of these tools to shed light on trophic interactions, diet composition, and the characterization of gut microbial communities in these organisms. Methods: Available literature was used to clarify how novel molecular techniques can improve ecological studies. The focus is diet, trophic relationships, and gut microbiota. Results: Traditionally, studies of stomach contents using compound microscopy have provided an idea of ingested material; nevertheless, sometimes a simple magnified visualization of dietary content does not allow exhaustive identification of the entire food spectrum, as it is limited due to the rapid digestion and maceration of food items within the echinoderm's digestive tract. The use of DNA-metabarcoding, targeting specific DNA regions, such as the mitochondrial COI gene, has allowed us to enhance the accuracy and precision of diet characterization by enabling the identification of prey items down to the species or even genetic variant level, providing valuable insights into specific dietary preferences. Another approach is the use of stable isotopes, particularly carbon and nitrogen, which provide a powerful tool to trace the origin and flow of nutrients through food webs. By analyzing the isotopic signatures in muscular tissues and food items, we can discern the sources of their primary food items and gain insights into their trophic position within the ecosystem. Lastly, a third new technique used to elucidate the characterization of the prokaryotic community is 16S rRNA sequencing. This method allows us to explore the composition and dynamics of the digestive tract microbial communities. Conclusions: This is a promising era for ecological research on echinoderms, where advances of molecular tools have enabled an unprecedented level of detail, resolving longstanding challenges in comprehending their trophic interactions, diet composition, and host-microbiota relationships, and opening new avenues of investigation in ecological studies.

Resumen Introducción: Los equinodermos, un componente integral de los ecosistemas marinos en todo el mundo, han captado el interés científico durante siglos. A pesar de esta prolongada atención, el comprender facetas clave como las relaciones tróficas, la composición de la dieta y las relaciones huésped-microbiota todavía representa un desafío utilizando técnicas tradicionales. Sin embargo, los últimos años han sido testigos de un cambio transformador, gracias a la aparición de técnicas moleculares avanzadas, que ofrecen nuevos enfoques para fortalecer los estudios ecológicos en equinodermos. Objetivo: Explorar cómo los avances recientes en herramientas moleculares han impactado la investigación ecológica sobre equinodermos. Específicamente, nuestro objetivo es investigar el potencial de estas herramientas para arrojar luz sobre las interacciones tróficas, la composición de la dieta y la caracterización de las comunidades microbianas intestinales en estos organismos. Métodos: Se utilizó la literatura disponible para aclarar cómo las nuevas técnicas moleculares pueden mejorar los estudios ecológicos. La atención se centra en la dieta, las relaciones tróficas y la microbiota intestinal. Resultados: Tradicionalmente, los estudios del contenido estomacal mediante microscopía compuesta han proporcionado una idea del material ingerido; Sin embargo, a veces una simple visualización ampliada del contenido dietético no permite una identificación exhaustiva de todo el espectro alimentario, ya que está limitado debido a la rápida digestión y maceración de los alimentos dentro del tracto digestivo del equinodermo. El uso de metabarcoding de ADN, dirigidos a regiones específicas del ADN, como el gen COI mitocondrial, nos ha permitido mejorar la exactitud y precisión de la caracterización de la dieta al permitir la identificación de presas hasta el nivel de especie o incluso de variante genética, lo que proporciona valiosos resultados sobre preferencias dietéticas específicas. Otro enfoque es el uso de isótopos estables, en particular carbono y nitrógeno, que proporcionan una poderosa herramienta para rastrear el origen y el flujo de nutrientes a través de las redes alimentarias. Al analizar las firmas isotópicas en los tejidos musculares y los alimentos, podemos discernir las fuentes de sus alimentos primarios y obtener información sobre su posición trófica dentro del ecosistema. Por último, una tercera técnica nueva utilizada para dilucidar la caracterización de la comunidad procariótica es la secuenciación del ARNr 16S. Este método nos permite explorar la composición y dinámica de las comunidades microbianas del tracto digestivo. Conclusiones: Esta es una era prometedora para la investigación ecológica sobre equinodermos, donde los avances de las herramientas moleculares han permitido un nivel de detalle sin precedentes, resolviendo desafíos de larga data en la comprensión de sus interacciones tróficas, composición de la dieta y relaciones huésped-microbiota, y abriendo nuevas vías de investigación. en estudios ecológicos.

Importancia biológica de los telómeros / Biological importance of telomeres

Se exponen los hallazgos históricos y la importancia biológica de los telómeros en la vida celular y en los aspectos genéticos del ADN humano. (AU)

The discovery and the biological importance of the telomeres are exposed. (AU)

Innovative insights into extrachromosomal circular DNAs in gynecologic tumors and reproduction

Originating but free from chromosomal DNA, extrachromosomal circular DNAs (eccDNAs) are organized in circular form and have long been found in unicellular and multicellular eukaryotes. Their biogenesis and function are poorly understood as they are characterized by sequence homology with linear DNA, for which few detection methods are available. Recent advances in high-throughput sequencing technologies have revealed that eccDNAs play crucial roles in tumor formation, evolution, and drug resistance as well as aging, genomic diversity, and other biological processes, bringing it back to the research hotspot. Several mechanisms of eccDNA formation have been proposed, including the breakage-fusion-bridge (BFB) and translocation-deletion-amplification models. Gynecologic tumors and disorders of embryonic and fetal development are major threats to human reproductive health. The roles of eccDNAs in these pathological processes have been partially elucidated since the first discovery of eccDNA in pig sperm and the double minutes in ovarian cancer ascites. The present review summarized the research history, biogenesis, and currently available detection and analytical methods for eccDNAs and clarified their functions in gynecologic tumors and reproduction. We also proposed the application of eccDNAs as drug targets and liquid biopsy markers for prenatal diagnosis and the early detection, prognosis, and treatment of gynecologic tumors. This review lays theoretical foundations for future investigations into the complex regulatory networks of eccDNAs in vital physiological and pathological processes.

Prenatal diagnosis and outcome of pregnancy for women with high risks by screening of fetal free DNA from peripheral blood samples / 中华医学遗传学杂志

OBJECTIVE@#To analyze the results of prenatal diagnosis and outcome of pregnancy for women with a high risk for fetal aneuploidies.@*METHODS@#A total of 747 cases of prenatal diagnosis by amniocentesis due to high risks by non-invasive prenatal testing (NIPT) were selected from January 2015 to March 2022 in the Drum Tower Hospital Affiliated to Nanjing University Medical School. The amniotic fluid samples were subjected to chromosomal karyotyping and/or chromosomal microarray analysis. All cases were followed up by searching the birth information or telephone calls, and the results were recorded. 2 test or F test were used for comparing the difference between the groups.@*RESULTS@#Among the 747 pregnant women with a high risk by NIPT, 387 were true positives, and the overall positive predictive value (PPV) was 51.81%. The PPVs for trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13) and sex chromosome aneuploidies (SCA) were 80.24% (199/248), 60% (48/80), 14% (7/50) and 38.97% (106/272), respectively. The PPV for T21 was significantly higher than T18 and T13 (χ2 = 85.216, P < 0.0001). The PPV for other chromosomal aneuploidies and copy number variations (CNVs) were 11.11% (5/45) and 40.74% (22/52), respectively. The PPV for increased X chromosomes was significantly higher than X chromosome decreases (64.29% vs. 22.22%, χ2 = 5.530, P < 0.05). The overall PPV for elder women (≥ 35 years old) was significantly higher than younger women (69.35% vs. 42.39%, χ2 = 49.440, P < 0.0001). For T21 and T18, the PPV of Z ≥ 10 group was significantly higher than that for 3 ≤ Z < 5 group or 5 ≤ Z < 10 group (P < 0.05). Among 52 cases with a high risk for CNVs, the PPV for the ≤ 5 Mb group was significantly higher than the 5 Mb < CNVs < 10 Mb or > 10 Mb groups (60% vs. 30%60% vs. 23.53%, P < 0.05). Among the 387 true positive cases, 322 had opted for induced labor, 53 had delivered with no abnormal growth and development, and 12 were lost during the follow-up.@*CONCLUSION@#The PPVs for common chromosomal aneuploidies are related to the age and Z value of the pregnant women, which were higher in the elder group and higher Z value group. In addition, the PPV is associated with high risk types. The PPV for T21 was higher than T18 and T13, and that for 45,X was lower than 47,XXX, 47,XYY or 47,XXY syndrome. NIPT therefore has relatively high PPVs for the identification of chromosomal CNVs.

Association between telomere length in the DNA of peripheral blood leukocytes and the propofol dose in anesthesia induction: an observational study

Abstract Introduction: Propofol is a widely used anesthetic and its dose is closely related to aging. Telomere length (TL) is a unique heritable trait, and emerging as a biomarker of aging, health and disease. Telomerase RNA component (TERC) plays an important role in maintaining TL. We proposed a hypothesis that propofol dose in general anesthesia can be predicted by measuring TL before operation, which greatly reduced the risk of anesthesia, especially the elderly. Methods: The association between the propofol dose in anesthesia induction and: TL in the DNA of peripheral blood leukocytes; body weight; sex; difference of the Bispectral Index (BIS) before and after anesthesia induction in patients was evaluated by multivariable linear regression analyses. The mutation at the 5'end or 3'end of TERC was detected. We recruited 100 patients of elective surgery. Results: We found that propofol dose in anesthesia induction was clearly correlated significantly with TL (r = 0.78, p < 0.001), body weight (r = 0.84, p = 0.004), sex (r = 0.83, p= 0.84, p = 0.004), sex (r = 0.83, p = 0.004), and difference of BIS before and after anesthesia induction (r = 0.85, p = 0.029). By comparing the absolute values of standardized regression coefficients (0.58, 0.21, 0.19, and 0.12) of the four variables, it can be seen that TL contributes the most to the propofol dose in anesthesia induction. However, the mutation at the 5' end or 3' end of TERC was not found. Conclusions: These findings provide preliminary evidence that the propofol dose in anesthesia induction was clearly correlated with genetically determined TL. TL may be a promising predictor of the propofol dose, which is beneficial to improve the safety of anesthesia and reduce perioperative complications.

Diagnóstico micológico de paracoccidioidomicosis en un hospital de área no endémica: metodología clásica y molecular / Mycological diagnosis of paracoccidioidomycosis in a hospital from a nonendemic area: classical and molecular methods

Introducción. La paracoccidioidomicosis es una micosis sistémica y endémica en Latinoamérica. El cambio climático y el movimiento migratorio del huésped enfatizan la necesidad de optimizar el diagnóstico de esta infección. Objetivo. Evaluar la implementación de la detección de ADN de Paracoccidioides spp. al diagnóstico micológico de pacientes con sospecha de paracoccidioidomicosis. Materiales y métodos. Estudio retrospectivo con datos de laboratorio de pacientes con sospecha de paracoccidioidomicosis en un hospital de área no endémica. Resultados. Se analizaron los resultados de las muestras de 19 pacientes con sospecha clínica de paracoccidioidomicosis. El 90 % de los pacientes había nacido o visitado un área endémica de esta micosis en Latinoamérica. En 14 pacientes varones adultos se confirmó paracoccidioidomicosis por diagnóstico convencional. El examen directo fue positivo en 12 pacientes con enfermedad comprobada y en 4 de ellos se obtuvo crecimiento del hongo. Se detectaron anticuerpos contra Paracoccidioides spp. en ocho pacientes con la enfermedad. Se realizó PCR anidada con muestras de 14 pacientes para detectar ADN de Paracoccidioides spp. En 9 de los 10 pacientes con diagnóstico convencional de paracoccidioidomicosis se obtuvo una prueba de PCR positiva. Conclusiones. La implementación de técnicas moleculares para detectar ADN de Paracoccidioides spp. complementa el diagnóstico convencional de paracoccidioidomicosis y permite instaurar el tratamiento antifúngico, sobre todo en los casos clínicos donde no se observa la presencia del hongo en las muestras clínicas. La migración actual de poblaciones humanas dificulta el diagnóstico de paracoccidioidiomicosis y otras infecciones endémicas, por lo que se requiere optimizar el diagnostico micológico en los laboratorios clínicos para tratar pacientes con este tipo micosis desatendida.

Introduction. Paracoccidioidomycosis is a systemic mycosis endemic in Latin America. Climate change and host migration emphasize the need to optimize this infection diagnosis. Objective. To evaluate the implementation of Paracoccidioides spp. DNA detection in the mycological diagnosis of patients with suspected paracoccidioidomycosis. Materials and methods. It is a retrospective study with laboratory data from patients with clinical suspicion of paracoccidioidomycosis, who consulted a university hospital from a non-endemic area. Results. We analyzed the laboratory results of samples from 19 patients with suspected paracoccidioidomycosis. Seventeen out of 19 patients were born in or had visited an endemic area in Latin America. Fourteen adult male patients were confirmed to have paracoccidioidomycosis by conventional diagnosis: the direct examination was positive in 12 samples while fungal growth was found only in 4. Anti-Paracoccidioides spp. antibodies were detected in 10 patients, 8 of them with proven paracoccidioidomycosis. Nested PCR for Paracoccidioides spp. detection was performed on clinical samples from 14 patients, and positive results were obtained for 9 out of 10 patients with the conventional diagnosis of paracoccidioidomycosis. Conclusions. The incorporation of molecular techniques to detect Paracoccidioides spp. DNA complements the conventional diagnosis of paracoccidioidomycosis. This tool allows the prescription of antifungal treatment in those cases where the fungus is not observed in the clinical samples. Current human migrations difficult the mycological diagnosis of paracoccidioidomycosis and other fungal infections. For this reason, it is necessary to improve mycological diagnosis in clinical laboratories to adequately treat patients with this neglected mycosis.

Genotipificación del gen RHD en ADN fetal libre en plasma de embarazadas RhD negativo / Genotyping of the RHD gene in free fetal DNA in plasma of RhD negative pregnant women

Objetivo: Determinar el grupo RhD fetal a través del estudio del gen RHD en ADN fetal que se encuentra libre en plasma de embarazadas RhD negativo. Método: Se analizó la presencia de los genes RHD, SRY y BGLO en ADNfl obtenido de plasma de 51 embarazadas RhD negativo no sensibilizadas, utilizando una qPCR. Los resultados del estudio genético del gen RHD se compararon con el estudio del grupo sanguíneo RhD realizado por método serológico en muestras de sangre de cordón, y los resultados del estudio del gen SRY fueron cotejados con el sexo fetal determinado por ecografía. Se calcularon la sensibilidad, la especificidad, los valores predictivos y la capacidad discriminativa del método estandarizado. Resultados: El gen RHD estaba presente en el 72,5% de las muestras y el gen SRY en el 55,5%, coincidiendo en un 100% con los resultados del grupo RhD detectado en sangre de cordón y con el sexo fetal confirmado por ecografía, respectivamente. Conclusiones: Fue posible deducir el grupo sanguíneo RhD del feto mediante el estudio del ADN fetal que se encuentra libre en el plasma de embarazadas con un método molecular no invasivo desarrollado y validado para este fin. Este test no invasivo puede ser utilizado para tomar la decisión de administrar inmunoglobulina anti-D solo a embarazadas RhD negativo que portan un feto RhD positivo.

Objective: To determine the fetal RhD group through the study of the RHD gene in fetal DNA found free in plasma of RhD negative pregnant women. Method: The presence of the RHD, SRY and BGLO genes in fetal DNA obtained from plasma of 51 non-sensitized RhD negative pregnant women was analyzed using qPCR. The results of the genetic study of the RHD gene were compared with the RhD blood group study performed by serological method in cord blood samples, and the results of the SRY gene study were compared with the fetal sex determined by ultrasound. Sensitivity, specificity, predictive values and discriminative capacity of the standardized method were calculated. Results: The RHD gene was present in 72.5% of the samples and the SRY gene in 55.5%, coinciding 100% with the results of the RhD group detected in cord blood, and with the fetal sex confirmed by ultrasound, respectively. Conclusions: It was possible to deduce the RhD blood group of the fetus through the study of fetal DNA found free in the plasma of pregnant women with a non-invasive molecular method developed and validated for this purpose. This non-invasive test can be used to make the decision to administer anti-D immunoglobulin only to RhD-negative pregnant women carrying an RhD-positive fetus.

Chromosomal integration of large DNA fragments in microorganisms: a review / 生物工程学报

The modern bio-fermentation industry requires design and creation of efficient microbial cell factories for directed conversion of raw materials to target products. The main criteria for assessing the performance of microbial cell factories are their product synthesis capacity and stability. Due to the deficiencies of plasmids in gene expression such as instability and being easy to lose, integration of genes into chromosome is often a better choice for stable expression in microbial hosts. To this end, chromosomal gene integration technology has received much attention and has developed rapidly. In this review, we summarize the recent research progresses of chromosomal integration of large DNA fragments in microorganisms, illustrate the principles and features of various technologies, highlight the opportunity brought by the CRISPR-associated transposon systems, and prospect future research direction of this technology.

Biomanufacturing driven by engineered organisms (2022) / 生物工程学报

This article summarizes the reviews and original research papers published in Chinese Journaol of Biotechnology in the area of biomanufacturing driven by engineered organisms in the year of 2022. The enabling technologies including DNA sequencing, DNA synthesis, and DNA editing as well as regulation of gene expression and in silico cell modeling were highlighted. This was followed by discussing the biomanufacturing of biocatalytics products, amino acids and its derivatives, organic acids, natural products, antibiotics and active peptides, functional polysaccharides, and functional proteins. Lastly, the technologies for utilizing C1 compounds and biomass as well as synthetic microbial consortia were discussed. The aim of this article was to help the readers to gain insights into this rapidly developing field from the journal point of view.

Levels of neutrophil extracellular traps in neonates with acute respiratory distress syndrome / 中国当代儿科杂志

OBJECTIVES@#To study the changes in cell free-DNA (cf-DNA), a marker of neutrophil extracellular traps (NETs), in neonates with acute respiratory distress syndrome (ARDS), and to evaluate its relationship with the severity and early diagnosis of ARDS.@*METHODS@#The neonates diagnosed with ARDS in the Affiliated Hospital of Jiangsu University from January 2021 to June 2022 were enrolled in the prospective study. The neonates were divided into mild, moderate, and severe ARDS groups based on the oxygen index (OI) (4≤OI<8, 8≤OI<16, and OI≥16, respectively). The control group was selected from jaundice neonates who were observed in the neonatal department of the hospital during the same period, and they had no pathological factors causing neonatal jaundice. Peripheral blood samples were collected on day 1, day 3, and day 7 after admission for the ARDS group, and on the day of admission for the control group. Serum cf-DNA levels were measured using a fluorescence enzyme-linked immunosorbent assay. Serum interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) levels were measured using enzyme-linked immunosorbent assay. A Pearson correlation analysis was used to evaluate the correlation of serum cf-DNA levels with serum IL-6 and TNF-α levels.@*RESULTS@#A total of 50 neonates were enrolled in the ARDS group, including 15 neonates with mild ARDS, 25 with moderate ARDS, and 10 with severe ARDS. Twenty-five neonates were enrolled in the control group. Compared with the control group, the serum levels of cf-DNA, IL-6, and TNF-α in all ARDS groups were significantly increased (P<0.05). Compared with the mild ARDS group, the serum levels of cf-DNA, IL-6, and TNF-α in the moderate and severe ARDS groups were significantly increased (P<0.05), and the increase was more significant in the severe ARDS group (P<0.05). The serum levels of cf-DNA, IL-6, and TNF-α in all ARDS groups were significantly increased on day 3 after admission and significantly decreased on day 7 after admission compared with those on day 1 after admission (P<0.05). The Pearson correlation analysis showed that there was a positive correlation between serum cf-DNA levels and IL-6 levels as well as TNF-α levels in 50 neonates with ARDS (P<0.05).@*CONCLUSIONS@#There is an excessive expression of NETs in neonates with ARDS, and dynamic monitoring of serum cf-DNA levels has certain clinical value in evaluating the severity and early diagnosis of ARDS in neonates.

Analysis of prognostic factors of extranodal NK/T-cell lymphoma treated with pegaspargase/L-asparaginase: a multicenter retrospective study / 中华血液学杂志

Objective: To explore the prognostic factors of extracellular NK/T cell lymphoma (ENKTL) treated with pegaspargase/L-asparaginase. Methods: The clinical data of 656 ENKTL patients diagnosed at 11 medical centers in the Huaihai Lymphoma Working Group from March 2014 to April 2021 were retrospectively analyzed. The patients were randomly divided into two groups: a training set (460 cases) and a validation set (196 cases) at 7∶3, and the prognostic factors of the patients were analyzed. A prognostic scoring system was established, and the predictive performance of different models was compared. Results: Patients' median age was 46 (34, 57) years, with 456 males (69.5% ) and 561 nasal involvement (85.5% ). 203 patients (30.9% ) received a chemotherapy regimen based on L-asparaginase combined with anthracyclines, and the 5-year overall survival rate of patients treated with P-GEMOX regimen (pegaspargase+gemcitabine+oxaliplatin) was better than those treated with SMILE regimen (methotrexate+dexamethasone+cyclophosphamide+L-asparaginase+etoposide) (85.9% vs 63.8% ; P=0.004). The results of multivariate analysis showed that gender, CA stage, the Eastern Cooperative Oncology Group performance status (ECOG PS) score, HGB, and EB virus DNA were independent influencing factors for the prognosis of ENKTL patients (P<0.05). In this study, the predictive performance of the prognostic factors is superior to the international prognostic index, Korean prognostic index, and prognostic index of natural killer lymphoma. Conclusion: Gender, CA stage, ECOG PS score, HGB, and EB virus DNA are prognostic factors for ENKTL patients treated with pegaspargase/L-asparaginase.

Neutrophil extracellular trap increase the risk of sepsis: a two-sample, one-way Mendelian randomization study / 中华危重病急救医学

OBJECTIVE@#To investigate the causal relationship between neutrophil extracellular trap (NET) and sepsis based on Mendelian randomization analysis.@*METHODS@#The genome wide association study (GWAS) dataset for the NET biomarker myeloperoxidase (MPO)-DNA complex based on Donkel et al. 's Rotterdam study (RS) and GWAS dataset for identifying sepsis from the UK biobank were selected to screen single nucleotide polymorphisms (SNPS) associated with MPO-DNA complex as instrumental variable (IV) for genetic variation, using MPO-DNA complex as exposure factor. Potential causal associations between MPO-DNA complex and the risk of occurrence of sepsis, 28-day death from sepsis, need for intensive care due to sepsis, and 28-day death from sepsis requiring intensive care were analyzed using a two-sample, one-way Mendelian randomization analysis primary analysis method of inverse analysis of variance (IVW). Potential pleiotropy was assessed using the MR Egger regression intercept test. Sensitivity analysis was performed using the "leave one out" test.@*RESULTS@#The GWAS data were obtained from a European population of both sexes, and the screening criteria was based on the three main assumptions of Mendelian randomization, resulting in 22 SNP entering the Mendelian randomization analysis. The results of the Mendelian randomization causal association effect analysis using the IVW method showed that for every standard deviation increase in the level of the MPO-DNA complex, the risk of sepsis increased by approximately 18% [odds ratio (OR) = 1.18, 95% confidence interval (95%CI) was 1.07-1.29, P < 0.001], the risk of 28-day death from sepsis increased by approximately 51% (OR = 1.51, 95%CI was 1.27-1.81, P < 0.001), an increase of approximately 38% in the risk of occurrence of needing intensive care due to sepsis (OR = 1.38, 95%CI was 1.12-1.70, P = 0.002), and an increase of approximately 125% in the risk of 28-day death from sepsis requiring intensive care (OR = 2.25, 95%CI was 1.21-4.18, P = 0.01). MR Egger regression intercept test suggested that there was no horizontal pleiotropy in the included SNP, and the MR-PRESSO test did not find outliers. Sensitivity analysis suggested that the results of Mendelian randomization were robust.@*CONCLUSIONS@#Rising NET can increase the risk of sepsis onset, progression and death as derived from Mendelian randomization analysis.

Dual role of lipids for genome stability and pluripotency facilitates full potency of mouse embryonic stem cells

While Mek1/2 and Gsk3β inhibition ("2i") supports the maintenance of murine embryonic stem cells (ESCs) in a homogenous naïve state, prolonged culture in 2i results in aneuploidy and DNA hypomethylation that impairs developmental potential. Additionally, 2i fails to support derivation and culture of fully potent female ESCs. Here we find that mouse ESCs cultured in 2i/LIF supplemented with lipid-rich albumin (AlbuMAX) undergo pluripotency transition yet maintain genomic stability and full potency over long-term culture. Mechanistically, lipids in AlbuMAX impact intracellular metabolism including nucleotide biosynthesis, lipid biogenesis, and TCA cycle intermediates, with enhanced expression of DNMT3s that prevent DNA hypomethylation. Lipids induce a formative-like pluripotent state through direct stimulation of Erk2 phosphorylation, which also alleviates X chromosome loss in female ESCs. Importantly, both male and female "all-ESC" mice can be generated from de novo derived ESCs using AlbuMAX-based media. Our findings underscore the importance of lipids to pluripotency and link nutrient cues to genome integrity in early development.

Fibrillarin promotes homologous recombination repair by facilitating the recruitment of recombinase RAD51 to DNA damage sites / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Eukaryotic organisms constantly face a wide range of internal and external factors that cause damage to their DNA. Failure to accurately and efficiently repair these DNA lesions can result in genomic instability and the development of tumors (Canela et al., 2017). Among the various forms of DNA damage, DNA double-strand breaks (DSBs) are particularly harmful. Two major pathways, non-homologous end joining (NHEJ) and homologous recombination (HR), are primarily responsible for repairing DSBs (Katsuki et al., 2020; Li and Yuan, 2021; Zhang and Gong, 2021; Xiang et al., 2023). NHEJ is an error-prone repair mechanism that simply joins the broken ends together (Blunt et al., 1995; Hartley et al., 1995). In contrast, HR is a precise repair process. It involves multiple proteins in eukaryotic cells, with the RAD51 recombinase being the key player, which is analogous to bacterial recombinase A (RecA) (Shinohara et al., 1992). The central event in HR is the formation of RAD51-single-stranded DNA (ssDNA) nucleoprotein filaments that facilitate homology search and DNA strand invasion, ultimately leading to the initiation of repair synthesis (Miné et al., 2007; Hilario et al., 2009; Ma et al., 2017).

Molecular dynamics simulation reveals DNA-specific recognition mechanism via c-Myb in pseudo-palindromic consensus of mim-1 promoter / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study aims to gain insight into the DNA-specific recognition mechanism of c-Myb transcription factor during the regulation of cell early differentiation and proliferation. Therefore, we chose the chicken myeloid gene, mitochondrial import protein 1 (mim-1), as a target to study the binding specificity between potential dual-Myb-binding sites. The c-Myb-binding site in mim-1 is a pseudo-palindromic sequence AACGGTT, which contains two AACNG consensuses. Simulation studies in different biological scenarios revealed that c-Myb binding with mim-1 in the forward strand (complex F) ismore stable than that inthereverse strand (complex R). The principal component analysis (PCA) dynamics trajectory analyses suggested an opening motion of the recognition helices of R2 and R3 (R2R3), resulting in the dissociation of DNA from c-Myb in complex R at 330 K, triggered by the reduced electrostatic potential on the surface of R2R3. Furthermore, the DNA confirmation and hydrogen-bond interaction analyses indicated that the major groove width of DNA increased in complex R, which affected on the hydrogen-bond formation ability between R2R3 and DNA, and directly resulted in the dissociation of DNA from R2R3. The steered molecular dynamics (SMD) simulation studies also suggested that the electrostatic potential, major groove width, and hydrogen bonds made major contribution to the DNA‍-specific recognition. In vitro trials confirmed the simulation results that c-Myb specifically bound to mim-1 in the forward strand. This study indicates that the three-dimensional (3D) structure features play an important role in the DNA-specific recognition mechanism by c-Myb besides the AACNG consensuses, which is beneficial to understanding the cell early differentiation and proliferation regulated by c-Myb, as well as the prediction of novel c-Myb-binding motifs in tumorigenesis.

Plasmid DNA extraction by double magnetic beads method and its application / 生物工程学报

Plasmids are the most commonly used gene carriers in the field of gene synthesis and sequencing. However, the main problems faced by traditional plasmid DNA extraction technology are low extraction throughput and high production cost, so they cannot meet the growing demand. In this study, a double-magnetic-bead method (DMBM) for plasmid extraction was developed based on the principle of plasmid extraction. The effects of the input of magnetic beads, the size of plasmid DNA fragments, and the volume of bacterial on plasmid DNA extraction were explored. In addition, the quality, throughput, and cost of plasmid DNA extraction were also compared between this technique and the commercial plasmid DNA extraction kits. The results showed that the DMBM can meet the needs of extracting plasmid DNA with different cell densities and fragment lengths. Moreover, the sensitivity and quality of plasmid extraction by the DMBM method were both superior to those of the centrifugal adsorption column method. In addition, this technique could be applied on a 96-channel automated nucleic acid extractor, resulting in higher purity of the extracted plasmid DNA, 80% reduction in extraction time, and 57.1% reduction in cost. It also reduces manual operations, achieving high-throughput and low-cost plasmid DNA extraction, thus may facilitate gene synthesis and sequencing.

A novel CRISPR/Cas9-hLacI donor adapting system for dsDNA-templated gene editing / 生物工程学报

During the gene editing process mediated by CRISPR/Cas9, precise genome editing and gene knock-in can be achieved by the homologous recombination of double-stranded DNA (dsDNA) donor template. However, the low-efficiency of homologous recombination in eukaryotic cells hampers the development and application of this gene editing strategy. Here, we developed a novel CRISPR/Cas9-hLacI donor adapting system (DAS) to enhance the dsDNA-templated gene editing, taking the advantage of the specific binding of the LacI repressor protein and the LacO operator sequence derived for the Escherichia coli lactose operon. The codon-humanized LacI gene was fused as an adaptor to the Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus lugdunensis Cas9 (SlugCas9-HF) genes, and the LacO operator sequence was used as the aptamer and linked to the dsDNA donor template by PCR. The Cas9 nuclease activity after the fusion and the homology-directed repair (HDR) efficiency of the LacO-linked dsDNA template were firstly examined using surrogate reporter assays with the corresponding reporter vectors. The CRISPR/Cas9-hLacI DASs mediated genome precise editing were further checked, and we achieved a high efficiency up to 30.5% of precise editing at the VEGFA locus in HEK293T cells by using the CRISPR/SlugCas9-hLacI DAS. In summary, we developed a novel CRISPR/Cas9-hLacI DAS for dsDNA-templated gene editing, which enriches the CRISPR/Cas9-derived gene editing techniques and provides a novel tool for animal molecular design breeding researches.