Epidemiological and Clinical Characteristics of Patients Admitted to a Secondary Hospital with Suspected MPOX Virus Infection: Is HIV Playing a Role?

MPOX (monkeypox) is a zoonotic viral disease, endemic in some Central and West African countries. However, in May 2022, cases began to be reported in non-endemic countries, demonstrating community transmission. Since the beginning of the outbreak, different epidemiological and clinical behaviors have been observed. We conducted an observational study at a secondary hospital in Madrid to characterize suspected and confirmed cases of MPOX epidemiologically and clinically. Besides the general descriptive analysis, we compared data between HIV-positive and HIV-negative subjects; 133 patients were evaluated with suspected MPOX, of which 100 were confirmed. Regarding positive cases, 71.0% were HIV positive, and 99.0% were men with a mean age of 33. In the previous year, 97.6% reported having sex with men, 53.6% used apps for sexual encounters, 22.9% practiced chemsex, and 16.7% went to saunas. Inguinal adenopathies were significantly higher in MPOX cases (54.0% vs. 12.1%, p < 0.001), as the involvement of genital and perianal area (57.0% vs. 27.3% and 17.0% vs. 1.0%, p = 0.006 and p = 0.082 respectively). Pustules were the most common skin lesion (45.0%). In HIV-positive cases, only 6.9% had a detectable viral load, and the mean CD4 count was 607.0/mm3. No significant differences were observed in the disease course, except for a greater tendency towards the appearance of perianal lesions. In conclusion, the MPOX 2022 outbreak in our area has been related to sexual intercourse among MSM, with no severe clinical cases nor apparent differences in HIV and non-HIV patients.

An extensible vector toolkit and parts library for advanced engineering of plant genomes.

Plant biotechnology is rife with new advances in transformation and genome engineering techniques. A common requirement for delivery and coordinated expression in plant cells, however, places the design and assembly of transformation constructs at a crucial juncture as desired reagent suites grow more complex. Modular cloning principles have simplified some aspects of vector design, yet many important components remain unavailable or poorly adapted for rapid implementation in biotechnology research. Here, we describe a universal Golden Gate cloning toolkit for vector construction. The toolkit chassis is compatible with the widely accepted Phytobrick standard for genetic parts, and supports assembly of arbitrarily complex T-DNAs through improved capacity, positional flexibility, and extensibility in comparison to extant kits. We also provision a substantial library of newly adapted Phytobricks, including regulatory elements for monocot and dicot gene expression, and coding sequences for genes of interest such as reporters, developmental regulators, and site-specific recombinases. Finally, we use a series of dual-luciferase assays to measure contributions to expression from promoters, terminators, and from cross-cassette interactions attributable to enhancer elements in certain promoters. Taken together, these publicly available cloning resources can greatly accelerate the testing and deployment of new tools for plant engineering.

Analyzing Plant Gene Targeting Outcomes and Conversion Tracts with Nanopore Sequencing.

The high-throughput molecular analysis of gene targeting (GT) events is made technically challenging by the residual presetabce of donor molecules. Large donor molecules restrict primer placement, resulting in long amplicons that cannot be readily analyzed using standard NGS pipelines or qPCR-based approaches such as ddPCR. In plants, removal of excess donor is time and resource intensive, often requiring plant regeneration and weeks to months of effort. Here, we utilized Oxford Nanopore Amplicon Sequencing (ONAS) to bypass the limitations imposed by donor molecules with 1 kb of homology to the target and dissected GT outcomes at three loci in Nicotiana benthamia leaves. We developed a novel bioinformatic pipeline, Phased ANalysis of Genome Editing Amplicons (PANGEA), to reduce the effect of ONAS error on amplicon analysis and captured tens of thousands of somatic plant GT events. Additionally, PANGEA allowed us to collect thousands of GT conversion tracts 5 days after reagent delivery with no selection, revealing that most events utilized tracts less than 100 bp in length when incorporating an 18 bp or 3 bp insertion. These data demonstrate the usefulness of ONAS and PANGEA for plant GT analysis and provide a mechanistic basis for future plant GT optimization.

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis.

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10 bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

Multiplexed heritable gene editing using RNA viruses and mobile single guide RNAs.

An in planta gene editing approach was developed wherein Cas9 transgenic plants are infected with an RNA virus that expresses single guide RNAs (sgRNAs). The sgRNAs are augmented with sequences that promote cell-to-cell mobility. Mutant progeny are recovered in the next generation at frequencies ranging from 65 to 100%; up to 30% of progeny derived from plants infected with a virus expressing three sgRNAs have mutations in all three targeted loci.

Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.

BACKGROUND: The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease. Previously, we generated soybean lines with knockout mutations within fatty acid desaturase 2-1A (FAD2-1A) and FAD2-1B genes, resulting in oil with increased levels of monounsaturated oleic acid (18:1) and decreased levels of linoleic (18:2) and linolenic acid (18:3). Here, we stack mutations within FAD2-1A and FAD2-1B with mutations in fatty acid desaturase 3A (FAD3A) to further decrease levels of linolenic acid. Mutations were introduced into FAD3A by directly delivering TALENs into fad2-1a fad2-1b soybean plants. RESULTS: Oil from fad2-1a fad2-1b fad3a plants had significantly lower levels of linolenic acid (2.5 %), as compared to fad2-1a fad2-1b plants (4.7 %). Furthermore, oil had significantly lower levels of linoleic acid (2.7 % compared to 5.1 %) and significantly higher levels of oleic acid (82.2 % compared to 77.5 %). Transgene-free fad2-1a fad2-1b fad3a soybean lines were identified. CONCLUSIONS: The methods presented here provide an efficient means for using sequence-specific nucleases to stack quality traits in soybean. The resulting product comprised oleic acid levels above 80 % and linoleic and linolenic acid levels below 3 %.

¿Conocemos el bienestar psicológico de nuestro personal sanitario? / Do we know the psychological well-being of our health personnel?

Introducción: En los últimos años se ha producido un aumento de la enfermedad psíquica con graves repercusiones en el mundo laboral (aumento de los accidentes y del absentismo por enfermedad común, descensos en la productividad, mala calidad de los servicios) y en la calidad de vida de los trabajadores (problemas físicos, psíquicos, bajo nivel de satisfacción, etc.). El personal sanitario es uno de los más afectados. Hay numerosos estudios sobre síndrome de burnout, estresores en personal sanitario, mobbing, etc., pero no hay datos sobre la percepción que tiene el personal sanitario en su bienestar psicológico. Con el propósito de confirmar la hipótesis sobre la existencia de probables alteraciones psiquiátricas menores en personal sanitario, nos planteamos este trabajo de investigación. Objetivo: Identificar los posibles casos de alteraciones psiquiátricas menores en el personal sanitario. Analizar los factores sociodemográficos y laborales de riesgo en los casos de probables alteraciones psíquicas. Describir la disposición de los síntomas (somáticos, ansiedad e insomnio, depresión y disfunción social), en los casos de alteraciones psíquicas. Material y método: Estudio descriptivo transversal, realizado en una muestra de personal sanitario del Área 10 de Madrid. La recogida de información se realizó mediante cuestionarios autoadministrados. Para medir la variable respuesta (bienestar psicológico) se utilizó el cuestionario de Salud General de Goldberg en la versión de 28 ítems (GHQ-28). Resultado: La prevalencia de malestar psíquico para el total de la muestra es del 18,2%, con diferencias significativas que superan el punto de corte en las variables: lugar de trabajo (hospital), sexo (mujeres), edad (entre 36 y 50 años), categoría profesional (auxiliar de enfermería), contrato (fijo), turno (rotatorio) y llevar trabajando (entre 11 y 15 años). Conclusiones: Es necesario incluir cuestionarios psicosociales en la vigilancia de la salud, que puedan objetivar enfermedades no recogidas de otra forma y permitan tener un indicador de salud mental para elaborar medidas preventivas adecuadas. Una mayor atención a los procesos organizativos y una cultura de "cuidar al cuidador" puede contribuir a disminuir el problema creciente de distrés psicológico en el personal sanitario

Introduction: In the last few years, psychological distress among the population has increased with serious repercussions for employers (an increase in accidents and absenteeism due to common illnesses, lower productivity, poor quality of services) and for workers' quality of life (physical and psychological problems, low levels of satisfaction, etc.). Health workers are among the collectives most affected by this increase. Numerous studies have been published on burnout syndrome, stressors among health workers, and mobbing, among other phenomena, but data on health workers' perception of their psychological well-being are lacking. Objective: To identify possible cases of minor psychiatric disturbances among health workers. To analyze the sociodemographic and occupational risk factors in workers with psychiatric disturbances. To describe the type of symptoms (somatic, anxiety and insomnia, depression and social dysfunction) in workers with psychiatric alterations. Material and method: We performed a cross-sectional descriptive study in a sample of the health workers in Area 10 of Madrid. Information was gathered through self-administered questionnaires. To measure the response variable (psychological well-being), the 28-item Goldberg General Health Questionnaire was used (GHQ-28). Result: The prevalence of psychological distress for the sample as a whole was 18.2%. Significant differences and scores exceeding the cut-off point indicating vulnerability to psychiatric disturbances were found for the following variables: place of work (hospital), gender (women), age (between 36 and 50 years), professional status (nurse's aide), contract (permanent), shift (rotating), and years of service (between 11 and 15 years). Conclusions: Psychosocial questionnaires should be included in health surveillance. These questionnaires could identify alterations undetected by other measures and would allow appropriate preventive measures to be designed. Greater attention to organizational factors and a culture of "caring for the carer" could help to reduce the growing problem of psychological distress among health workers