Breaking out of the cycle: Including quiescence in cell cycle classification.

Single-cell transcriptomics has unveiled a vast landscape of cellular heterogeneity in which the cell cycle is a significant component. We trained a high-resolution cell cycle classifier (ccAFv2) using single cell RNA-seq (scRNA-seq) characterized human neural stem cells. The ccAFv2 classifies six cell cycle states (G1, Late G1, S, S/G2, G2/M, and M/Early G1) and a quiescent-like G0 state, and it incorporates a tunable parameter to filter out less certain classifications. The ccAFv2 classifier performed better than or equivalent to other state-of-the-art methods even while classifying more cell cycle states, including G0. We showcased the versatility of ccAFv2 by successfully applying it to classify cells, nuclei, and spatial transcriptomics data in humans and mice, using various normalization methods and gene identifiers. We provide methods to regress the cell cycle expression patterns out of single cell or nuclei data to uncover underlying biological signals. The classifier can be used either as an R package integrated with Seurat (https://github.com/plaisier-lab/ccafv2_R) or a PyPI package integrated with scanpy (https://pypi.org/project/ccAFv2/). We proved that ccAFv2 has enhanced accuracy, flexibility, and adaptability across various experimental conditions, establishing ccAFv2 as a powerful tool for dissecting complex biological systems, unraveling cellular heterogeneity, and deciphering the molecular mechanisms by which proliferation and quiescence affect cellular processes.

Efficient genome editing in erythroid cells unveils novel MYB target genes and regulatory functions.

Targeted genome editing holds great promise in biology. However, efficient genome modification, including gene knock-in (KI), remains an unattained goal in multiple cell types and loci due to poor transfection efficiencies and low target genes expression, impeding the positive selection of recombined cells. Here, we describe a genome editing approach to achieve efficient gene targeting using hard to transfect erythroid cell lines. We demonstrate robust fluorescent protein KI efficiency in low expressed transcription factor (TF) genes (e.g., Myb or Zeb1). We further show the ability to target two independent loci in individual cells, exemplified by MYB-GFP and NuMA-Cherry double KI, allowing multicolor labeling of regulatory factors at physiological endogenous levels. Our KI tagging approach allowed us to perform genome-wide TF analysis at increased signal-to-noise ratios, and highlighted previously unidentified MYB target genes and pathways. Overall, we establish a versatile CRISPR-Cas9-based platform, offering attractive opportunities for the dissection of the erythroid differentiation process.

Antiseptic-Loaded Casein Hydrogels for Wound Dressings.

Chronic wound treatment accounts for a substantial percentage of the medical expenses worldwide. Improving and developing novel wound care systems can potentially help to handle this problem. Wound dressings loaded with antiseptics may be an important tool for wound care, as they inhibit bacterial growth at the wound site. The goal of the present work was to investigate the potential of using casein hydrogel dressings loaded with two antiseptic drugs, Octiset® or polyhexanide, to treat chronic wounds. Casein-based hydrogels are inexpensive and have several properties that make them suitable for biomedical applications. Two types of casein were used: casein sodium salt and acid casein, with the formulations being labelled CS and C, respectively. The hydrogels were characterised with respect to their physical properties (swelling capacity, water content, morphology, mechanical resistance, and stability), before and after sterilisation, and they showed adequate values for the intended application. The hydrogels of both formulations were able to sustain controlled drug-release for, at least, 48 h. They were demonstrated to be non-irritant, highly haemocompatible, and non-cytotoxic, and revealed good antimicrobial properties against Staphylococcus aureus and Pseudomonas aeruginosa. Steam-heat sterilisation did not compromise the material's properties. The in vivo performance of C hydrogel loaded with Octiset® was evaluated in a case study with a dog. The efficient recovery of the wounds confirms its potential as an alternative for wound treatment. To our knowledge, this is the first time that wound dressings loaded with Octiset®, one of the most efficient drugs for wound treatment, were prepared and tested.

Productiveness and Berry Quality of New Wine Grape Genotypes Grown under Drought Conditions in a Semi-Arid Wine-Producing Mediterranean Region.

One alternative for adapting viticulture to high temperatures and the scarcity of water is the development of new varieties adapted to such conditions. This work describes six new genotypes, derived from "Monastrell" × "Cabernet Sauvignon" (MC16, MC19, MC72, MC80) and "Monastrell" × "Syrah" (MS104, MS49) crosses, grown under deficit irrigation and rainfed conditions in a semi-arid wine-producing area (Murcia, southeastern Spain). The effect of genotype, year, and irrigation treatment on the phenological, productiveness, morphological, and grape quality data was evaluated. The study material was obtained and selected as part of a breeding program run by the Instituto Murciano de Investigación y Desarollo Agrario y Medioambiental (IMIDA). The results obtained show that under rainfed conditions, the values for productive variables decreased, while those referring to the phenolic content increased. Notable variation in the parameters evaluated was also seen for the different genotypes studied. The behavior of the genotypes MC80 and MS104 under rainfed conditions was noteworthy. In addition to maintaining very adequate yields, phenolic contents, must pH, and total acidity values, MC80 fell into the best 'phenolic quality group' and MS104 returned a low º°Baumé value, ideal for the production of low-alcohol-content wines. These genotypes could favor the development of sustainable quality viticulture in dry and hot areas.

Evaluation and Pre-selection of New Grapevine Genotypes Resistant to Downy and Powdery Mildew, Obtained by Cross-Breeding Programs in Spain.

The need to develop an environmentally friendly, sustainable viticulture model has led to numerous grapevine improvement programmes aiming to increase resistance to downy and powdery mildew. The success of such programmes relies on the availability of protocols that can quantify the resistance/susceptibility of new genotypes, and on the existence of molecular markers of resistance loci that can aid in the selection process. The present work assesses the degree of phenotypic resistance/susceptibility to downy and powdery mildew of 28 new genotypes obtained from crosses between "Monastrell" and "Regent." Three genotypes showed strong combined resistance, making them good candidates for future crosses with other sources of resistance to these diseases (pyramiding). In general, laboratory and glasshouse assessments of resistance at the phenotype level agreed with the resistance expected from the presence of resistance-associated alleles of simple sequence repeat (SSR) markers for the loci Rpv3 and Ren3 (inherited from "Regent"), confirming their usefulness as indicators of likely resistance to downy and powdery mildew, respectively, particularly so for downy mildew.

Simulated patient and role play methodologies for communication skills and empathy training of undergraduate medical students.

BACKGROUND: Verbal and non-verbal communication, as well as empathy are central to patient-doctor interactions and have been associated with patients' satisfaction. Non-verbal communication tends to override verbal messages. The aim of this study was to analyze how medical students use verbal and non-verbal communication using two different educational approaches, student role play (SRP) and actor simulated patient (ASP), and whether the non-verbal behaviour is different in the two different poses. METHODS: Three raters evaluated 20 students playing the doctor role, 10 in the SRP group and 10 in the ASP group. The videos were analyzed with the Calgary-Cambridge Referenced Observation Guide (CCG) and, for a more accurate evaluation of non-verbal communication, we also evaluated signs of nervousness, and posture. Empathy was rated with the CARE questionnaire. Independent Mann Whitney U tests and Qhi square tests were performed for statistical analysis. RESULTS: From the 6 main tasks of the CCG score, we obtained higher scores in the ASP group for the task 'Gathering information' (p = 0.0008). Concerning the 17 descriptors of the CCG, the ASP group obtained significantly better scores for 'Exploration of the patients' problems to discover the biomedical perspective' (p = 0.007), 'Exploration of the patients' problems to discover background information and context' (p = 0.0004) and for 'Closing the session - Forward planning' (p = 0.02). With respect to non-verbal behaviour items, nervousness was significantly higher in the ASP group compared to the SRP group (p < 0.0001). Concerning empathy, no differences were found between the SRP and ASP groups. CONCLUSIONS: Medical students displayed differentiated verbal and non-verbal communication behaviour during the two communication skills training methodologies. These results show that both methodologies have certain advantages and that more explicit non-verbal communication training might be necessary in order to raise students' awareness for this type of communication and increase doctor-patient interaction effectiveness.

Prediction of Muscat aroma in table grape by analysis of rose oxide.

Aroma is an important quality characteristic in Muscat grapes and constitutes a major concern for viticulturist and grapevine breeders. For this reason, Muscat aroma variability was characterised in a segregating progeny and in a collection of table grapes, to assess the usefulness of the presence or absence of rose oxide for predicting Muscat genotypes. Simple tasting and an analysis of free and bound aroma compounds, including rose oxide, linalool oxide, linalool, α-terpineol, citronellol, nerol, geraniol, benzyl alcohol and 2-phenylethanol, were carried out. The association between Muscat score and the compounds considered as active odorants according to their odour activity values was also evaluated. The results obtained pointed to a highly significant correlation between the presence/absence of rose oxide in grapes and the presence/absence of Muscat aroma. Thus, this analysis could be a useful tool for identifying Muscat cultivars in a more objective way than sensory analysis.

A 48 SNP set for grapevine cultivar identification.

BACKGROUND: Rapid and consistent genotyping is an important requirement for cultivar identification in many crop species. Among them grapevine cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. Simple sequence repeat (SSR) markers have been preferred until now because of their high level of polymorphism, their codominant nature and their high profile repeatability. However, the rapid application of partial or complete genome sequencing approaches is identifying thousands of single nucleotide polymorphisms (SNP) that can be very useful for such purposes. Although SNP markers are bi-allelic, and therefore not as polymorphic as microsatellites, the high number of loci that can be multiplexed and the possibilities of automation as well as their highly repeatable results under any analytical procedure make them the future markers of choice for any type of genetic identification. RESULTS: We analyzed over 300 SNP in the genome of grapevine using a re-sequencing strategy in a selection of 11 genotypes. Among the identified polymorphisms, we selected 48 SNP spread across all grapevine chromosomes with allele frequencies balanced enough as to provide sufficient information content for genetic identification in grapevine allowing for good genotyping success rate. Marker stability was tested in repeated analyses of a selected group of cultivars obtained worldwide to demonstrate their usefulness in genetic identification. CONCLUSIONS: We have selected a set of 48 stable SNP markers with a high discrimination power and a uniform genome distribution (2-3 markers/chromosome), which is proposed as a standard set for grapevine (Vitis vinifera L.) genotyping. Any previous problems derived from microsatellite allele confusion between labs or the need to run reference cultivars to identify allele sizes disappear using this type of marker. Furthermore, because SNP markers are bi-allelic, allele identification and genotype naming are extremely simple and genotypes obtained with different equipments and by different laboratories are always fully comparable.

Isoschizomers and amplified fragment length polymorphism for the detection of specific cytosine methylation changes.

Different molecular techniques have been developed to study either the global level of methylated cytosines or methylation at specific gene sequences. One of them is a modification of the Amplified Fragment Length Polymorphism (AFLP) technique that has been used to study methylation of anonymous CCGG sequences in different fungi, plant and animal species. The main variation of this technique is based on the use of isoschizomers with different methylation sensitivity (such as HpaII and MspI) as a frequent cutter restriction enzyme. For each sample, AFLP analysis is performed using both EcoRI/HpaII and EcoRI/MspI digested samples. Comparative analysis between EcoRI/HpaII and EcoRI/MspI fragment patterns allows the identification of two types of polymorphisms: (1) "Methylation-insensitive polymorphisms" that show common EcoRI/HpaII and EcoRI/MspI patterns but are detected as polymorphic amplified fragments among samples; and (2) "Methylation-sensitive polymorphisms" that are associated with amplified fragments differing in their presence or absence or in their intensity between EcoRI/HpaII and EcoRI/MspI patterns. This chapter describes a detailed protocol of this technique and discusses modifications that can be applied to adjust the technology to different species of interest.

The kelp macrocystis pyrifera as nutritional supplement for goats

The present study was aimed to evaluate Macrocystis pyrifera (MP) meal as a nutritional supplement for goats. There is an increasing interest to look at nutritional alternatives to guarantee a continuous supply of good quality forage for goats, in many communities around the world. Given its abundance and chemical composition, the algae M. pyrifera is an important potential resource as animal feed. Three diets with 10, 20, and 30% of MP meal concentrations and a control diet, with no algae, were evaluated. Four rumen cannulated goats, housed individually in metabolism cages, were used. The experimental design was a 4 x 4 Latin-Square. Feed and water intake, excreted urine and faeces, were measured. Digestibility in vivo, dry matter (DM) disappearance, and the metabolic variables of pH and ammoniacal nitrogen in the rumen, were determined. There was no significant difference in the feed intake but there was in water intake and urine excreted. No significant difference in digestibility in vivo among diets (P>0.05) was found. A significant difference (P<0.05) for degradability in situ was found for the algae diets containing 10 and 30 percent MP concentrations at 96 hours of sampling (78.3 and 82.2 percent). The raw algae in situ digestibility was 77 percent. A potential degradability of 87.3 percent was obtained with 30 percent MP diet. The highest effective degradation was obtained at an estimated rate of 0.02 h-1. Ruminal pH was higher (P<0.05) in all MP treatments (10 percent MP: 6.83, 20 percent MP: 6.85, 30 percent MP: 6.91). As suggested by the results, M. pyrifera represents a good unconventional feeding as a nutritional supplement for goats.

El objetivo de este trabajo fue evaluar Macrocystis pyrifera (MP) como suplemento alimenticio para cabras. Hay un interés creciente en buscar alternativas alimenticias para garantizar el suministro continuo de forraje de buena calidad para cabras en muchas comunidades del mundo. Dada su abundancia y composición química, el alga M. pyrifera es un recurso potencial importante como pienso para ganado. Se evaluaron tres dietas con concentraciones de 10, 20, y 30 por ciento de harina de MP, y una dieta control, sin las algas. Se utilizaron cuatro cabras canuladas dispuestas individualmente en jaulas metabólicas. El diseño experimental fue un Cuadrado Latino 4 x 4. El alimento y agua consumidos, la orina y las heces excretadas fueron medidas. Se determinaron la digestibilidad in vivo, la desaparición in situ de la materia seca y las variables metabólicas pH y nitrógeno amoniacal en rumen. No se encontró diferencia significativa en el alimento consumido, ni en la digestibilidad in vivo entre las dietas (P>0,05), pero si hubo diferencia significativa en el consumo de agua y la orina excretada (P<0,05). Se encontró diferencia significativa (P<0,05) para la digestibilidad in situ en las dietas que contenían el 10 y 30% del alga a la hora 96 del muestreo (78,3 y el 82,2 por ciento). La digestibilidad in situ del alga fue de 77 por ciento. Se obtuvo una degradabilidad potencial de 87,3 por ciento con la dieta que contiene 30 por ciento de MP. La mayor degradación efectiva se obtuvo a una tasa estimada de 0,02 h-1. El pH ruminal fue más alto (P<0,05) en todos los tratamientos con MP (10 por ciento de MP: 6,83, 20 por ciento de MP: 6,85, 30 por ciento de MP: 6,91). Los resultados obtenidos sugieren que M. pyrifera representa un buen suplemento nutricional no convencional para cabras.

Shrimp head meal in laying hen rations and its effects on fresh and stored egg quality

Para conocer el efecto de la harina (HCC) de cabezas de camarón (Penaeus spp.) sobre las variables productivas, la calidad física y evaluación sensorial del huevo fresco y almacenado, se utilizaron 120 gallinas Leghorn blancas de 42 semanas de edad, distribuidas en 5 tratamientos (0, 10, 15, 20 y 25 por ciento de HCC) con 4 repeticiones cada uno. A los 28 días del ensayo se recolectaron huevos de cada tratamiento para evaluar la calidad física a los 0, 15 y 30 días de almacenamiento a 4 y 20ºC, con un diseño factorial de 5x3x2. No hubo diferencias estadísticas (P>0,05) en las variables productivas. El peso promedio del huevo fue mayor (64,08g) con 15 por ciento de HCC, sin encontrarse efecto por el tiempo de almacenamiento. Las Unidades Haugh (UH) de los huevos almacenados a 20ºC por 15 y 30 días disminuyeron, pero no a 4ºC. El color de la yema fue menor en los tratamientos con HCC respecto al testigo y se vio afectado por el tiempo de almacenamiento a 20ºC. El peso del cascarón no varió con las condiciones de almacenamiento ni los tratamientos. En grosor de cascarón y en la evaluación sensorial (sabor y color de la yema), no hubo diferencias (P>0,05). Se concluye que las diferencias encontradas se debieron al tiempo y temperatura de almacenamiento y no a la inclusión de HCC en las raciones para gallinas ponedoras

Molecular genetics of berry colour variation in table grape.

The genetics and biochemistry of anthocyanins and flavonol biosynthesis and their role in plant organ pigmentation is well established in model species. However, the genetic basis of colour variation is species specific and understanding this variation is very relevant in many fruit and flower crop species. Among grape cultivars, there is a wide genetic variation for berry colour ranging from yellow-green ("white" cultivars) to dark blue berries. Berry colour results from the synthesis and accumulation of anthocyanins in the berry skin, which in plants is commonly regulated by transcription factors belonging to the MYB and bHLH families. In this work, we aimed to identify the major genetic determinants of berry colour variation in a large collection of table grape cultivars and somatic variants. The genetic analyses of berry colour in a few grape segregating progenies had previously identified a single locus on linkage group 2 responsible for colour variation. Furthermore, somatic variation for berry skin colour in cultivar Italia had been associated with the presence of a Gret1 retrotransposon in the promoter region of VvmybA1, a Myb gene whose expression is associated to skin colouration. The results show that VvmybA1 is the gene underlying the mapped locus controlling berry colour in grape. Additionally, the molecular analyses indicate that genetic and somatic berry colour variation can be associated to molecular variation at VvmybA1 in more than 95% of the analyzed cultivars. Thus, VvmybA1 is a major determinant of berry colour variation in table grape and its instability is the major cause of somatic variation for this trait.

Regulation of flowering time by FVE, a retinoblastoma-associated protein.

The initiation of flowering in plants is controlled by environmental and endogenous signals. Molecular analysis of this process in Arabidopsis thaliana indicates that environmental control is exerted through the photoperiod and vernalization pathways, whereas endogenous signals regulate the autonomous and gibberellin pathways. The vernalization and autonomous pathways converge on the negative regulation of FLC, a gene encoding a MADS-box protein that inhibits flowering. We cloned FVE, a component of the autonomous pathway that encodes AtMSI4, a putative retinoblastoma-associated protein. FVE interacted with retinoblastoma protein in immunoprecipitation assays, and FLC chromatin was enriched in acetylated histones in fve mutants. We conclude that FVE participates in a protein complex repressing FLC transcription through a histone deacetylation mechanism. Our data provide genetic evidence of a new developmental function of these conserved proteins and identify a new genetic mechanism in the regulation of flowering.