Detection of SARS-CoV-2 in aerosols in long term care facilities and other indoor spaces with known COVID-19 outbreaks.

Coronavirus outbreaks are likely to occur in crowded and congregate indoor spaces, and their effects are most severe in vulnerable long term care facilities (LTCFs) residents. Public health officers benefit from tools that allow them to control COVID-19 outbreaks in vulnerable settings such as LTCFs, but which could be translated in the future to control other known and future virus outbreaks. This study aims to develop and test a methodology based on detection of SARS-CoV-2 in aerosol samples collected with personal pumps that could be easily implemented by public health officers. The proposed methodology was used to investigate the levels of SARS-CoV-2 in aerosol in indoor settings, mainly focusing on LTCFs, suffering COVID-19 outbreaks, or in the presence of known COVID-19 cases, and targeting the initial days after diagnosis. Aerosol samples (N = 18) were collected between November 2020 and March 2022 in Castelló (Spain) from LTCFs, merchant ships and a private home with recently infected COVID-19 cases. Sampling was performed for 24-h, onto 47 mm polytetrafluoroethylene (PTFE) and quartz filters, connected to personal pumps at 2 and 4 L/min respectively. RNA from filters was extracted and SARS-CoV-2 was determined by detection of regions N1 and N2 of the nucleocapsid gene alongside the E gene using RT-PCR technique. SARS-CoV-2 genetic material was detected in 87.5% samples. Concentrations ranged ND-19,525 gc/m3 (gene E). No genetic traces were detected in rooms from contacts that were isolated as a preventative measure. Very high levels were also measured at locations with poor ventilation. Aerosol measurement conducted with the proposed methodology provided useful information to public health officers and contributed to manage and control 12 different COVID-19 outbreaks. SARS-CoV-2 was detected in aerosol samples collected during outbreaks in congregate spaces. Indoor aerosol sampling is a useful tool in the early detection and management of COVID-19 outbreaks and supports epidemiological investigations.

Correction to: Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.

In the original publication of the article, some symbols in Figure 3 were not correctly aligned with the image.

Similarities and differences in tissue distribution of DLK1 and DLK2 during E16.5 mouse embryogenesis.

DLK1 and DLK2 are transmembrane proteins belonging to the EGF-like repeat-containing family that function as non-canonical NOTCH inhibitory ligands. DLK1 is usually downregulated after embryo development and its distribution in some adult and embryonic tissues has been described. However, the expression and role of DLK2 in embryo and adult tissues remains unclear. To better understand the relevance of both proteins during embryo development, we analyzed the expression pattern of DLK1 and DLK2 in 16.5-day-old mouse embryos (E16.5) and evaluated the possible relationship between these two proteins in embryo tissues and cell types. We found that DLK1 and DLK2 proteins exhibited a broad distribution pattern, which was detected in developing mouse organs from each of the three germ layers: ectoderm (brain, salivary glands), mesoderm (skeletal muscle, vertebral column, kidney, cartilage), and endoderm (thymus, lung, pancreas, intestine, liver). The expression pattern of DLK1 and DLK2 indicates that both proteins could play a synergistic role during cell differentiation. This study provides additional information for understanding temporal and site-specific effects of DLK1 and DLK2 during embryo morphogenesis and cell differentiation.

Delta-like protein 1 in the pituitary-adipose axis in the adult male mouse.

With the aim of studying delta-like protein 1 (DLK1) with respect to the relationship between adipocyte leptin and adenohypophyseal hormones, we carried out an immunohistochemical study analysing the presence of receptors for these hormones in the pituitary and adipose cells of male wild-type (WT) mice (Dlk1+/+ ) compared to knockout (KO) mice (Dlk1-/- ). The mRNA expression of these molecules was also determined using the reverse transcriptase-polymerase chain reaction. The results obtained showed that, in WT adipose cells, all of the adenohypophyseal hormone receptors were present, with a higher mRNA expression for growth hormone (GH) receptor and thyroid-stimulating hormone (TSH) receptor. Of the total cells in the anterior pituitary lobe, 17.09±0.9% were leptin receptor (LEPR) immunoreactive (-IR), mainly in GH-IR and prolactin (PRL)-IR cells (41.5±3.8%; 13.5±1.7%, respectively). In Dlk1-/- mice, adipocyte cells showed a significant increase in the TSH receptor mRNA expression level. Moreover, the percentage of LEPR-IR GH cells showed a statistically significant increase compared to controls, from 41.5±3.8% to 53.1±4.0%. By contrast, only 3.0±0.6% of LEP-IR anterior pituitary cells were detected in Dlk1 KO mice, as opposed to 6.8±1.1% observed in WT mice. The results suggest that relationships exist between adipocytes and pituitary GH, PRL and TSH cells, in addition to an influence with respect to the synthesis and release of pituitary leptin, particularly in PRL cells.

Reduced salivary gland size and increased presence of epithelial progenitor cells in DLK1-deficient mice.

DLK1 (PREF1, pG2, or FA1) is a transmembrane and secreted protein containing epidermal growth factor-like repeats. Dlk1 expression is abundant in many tissues during embryonic and fetal development and is believed to play an important role in the regulation of tissue differentiation and fetal growth. After birth, Dlk1 expression is abolished in most tissues but is possibly reactivated to regulate stem cell activation and responses to injury. We have recently reported that DLK1 regulates many aspects of salivary gland organogenesis. Here, we have extended our studies of the salivary gland phenotype of Dlk1 knock-out mice. We have observed that salivary glands are smaller and weigh significantly less in both Dlk1 knock-out males and females compared with gender and age-matched wild-type mice and regardless of the natural sexual dimorphism in rodent salivary glands. This reduced size correlates with a reduced capacity of Dlk1-deficient mice to secrete saliva after stimulation with pilocarpine. However, histological and ultrastructural analyses of both adult and developing salivary gland tissues have revealed no defects in Dlk1 ((-/-)) mice, indicating that genetic compensation accounts for the relatively mild salivary phenotype in these animals. Finally, despite their lack of severe anomalies, we have found that salivary glands from Dlk1-deficient mice present a higher amount of CK14-positive epithelial progenitors at various developmental stages, suggesting a role for DLK1 in the regulation of salivary epithelial stem cell balance.

Role of the non-canonical notch ligand delta-like protein 1 in hormone-producing cells of the adult male mouse pituitary.

To better understand the role of the non-canonical Notch ligand delta-like protein 1 (DLK1), in hormone-producing cells, we studied the cell distribution and subcellular localisation of DLK1 in the pituitary of male adult 129/SvJ mice, and analysed the variations in the hormone-producing cells associated with the lack of this gene in Dlk1 knockout mice. The results obtained showed the presence of DLK1-immunoreactive (ir) cells in all hormone-producing cells of the anterior pituitary. Immunoelectron microscopy showed DLK1-ir in the rough endoplasmic reticulum and inside secretory vesicles, suggesting that DLK1 is released together with pituitary hormones. Moreover, we found that prolactin (PRL)-DLK1-ir cells are in intimate contact with follicle-stimulating hormone (FSH)-ir-DLK1-negative cells. In Dlk1 knockout mice, we detected a significantly lower number of gowth hormone (GH)-ir cells, a reduction in the FSH and PRL immunostaining intensity, and a significant decrease in FSH mRNA expression compared to wild-type mice. An increase in pituitary GH mRNA expression and serum leptin levels was also found. These findings provide evidence supporting several regulatory functions of DLK1 in the pituitary gland.

Efecto de la Desnutrición Oculta Prenatal Sobre la Histología del Esplenio Callosal / Effect of Mild Protein Prenatal Malnutrition in the Callosal Splenium Histology

Ratas malnutridas prenatalmente con una dieta isocalórica y baja en proteínas, presentaron un menor diámetro axonal promedio en el esplenio callosal que los animales control, tanto de las fibras mielínicas como amielínicas. También se observó una mayor densidad axonal promedio, con respecto a los controles. Estas observaciones sugieren que: 1) las conexiones cortico-corticales (interhemisféricas) son vulnerables a la malnutrición proteica; y 2) lo anterior tendría incidencia en la velocidad de conducción interhemisférica, en particular con lo que dice relación con las conexiones visuales.

Adult rats malnourished prenatally with a low-protein, isocaloric diet showed smaller median fiber diameter of myelinated and unmyelinated fibers and a higher axonal density in the callosal splenium than controls. These findings suggest (i) that cortico-cortical (interhemispheric) connections are vulnerable to protein malnutrition; and (ii) this may affect interhemispheric conduction velocity, particularly in visual connections.

Specific regions of the extracellular domain of dlk, an EGF-like homeotic protein involved in differentiation, participate in intramolecular interactions.

The level of expression of dlk, an EGF-like protein possessing six EGF-like repeats in its extracellular region, is critical for 3T3-L1 fibroblasts to differentiate into adipocytes in response to IGF1. The mechanism of action of dlk is not well understood, but its localization on the cell membrane suggests that dlk may function as a receptor, as a ligand or as a regulatory protein modulating the binding, the signaling, or the expression of other molecules involved in cell differentiation and growth. In this work, we demonstrate, by using the Yeast Two-Hybrid system, that dlk interacts with itself through specific regions of its extracellular domain. The strongest interactions were observed between specific EGF-like repeats and between a non EFG-like region where unknown proteases act to generate soluble forms of dlk. These observations suggest that the interaction between two membrane dlk molecules belonging to the same or to different cells, or the interaction between soluble and membrane dlk variants, may be important to regulate dlk function.

The role of the epidermal growth factor-like protein dlk in cell differentiation.

This review focuses on the current knowledge about the function of the EGF-like homeotic protein dlk. dlk is a transmembrane protein that possesses six Epidermal Growth Factor-like sequences at the extracellular domain, a single transmembrane domain and a short intracellular tail. Because of its overall structure and amino acid homology, dlk belongs to the EGF-like homeotic protein family. This family includes proteins such as the Notch receptor and its homologues, as well as Notch ligands, such as Delta, Serrate, and their mammalian homologues Dll1, Dll2 and Dll3 and Jagged 1 and Jagged 2. (For a recent review see Fleming, 1998). dlk is highly expressed by preadipose cell lines, and neuroendocrine tumors, such as pheochromocytomas and neuroblastomas. dlk has been involved in several differentiation processes, such as adipogenesis, hematopoiesis and B cell lymphopoiesis, and neuroendocrine differentiation, including the differentiation of pancreas and the adrenal gland. The extracellular region of dlk can be released by action of an unknown protease and this soluble dlk variant accumulates in the amniotic fluid and is able to inhibit adipocyte differentiation in vitro. Recent evidence indicates, however, that membrane-associated dlk variants play a positive role in the differentiation process. These findings suggest that dlk plays an important role in differentiation and tumorigenesis of several cellular types.

Potencialidades y limitaciones del uso de recursos multimediales en la enseñanza de anatomía veterinaria / Potentiality and limitations in the use of multimedial resources in the teaching of veterinary anatomy

El presente trabajo persigue el propósito de demostrar las potencialidades de los recursos multimediales utilizados como herramientas complementarias a los materiales usados de manera tradicional, durante el proceso de enseñanza-aprendizaje de Anatomía Veterinaria. Asimismo, pretende evidenciar el impacto que dichos soportes producen en la motivación de los alumnos, frente a las tareas áulicas, así como en instancias de aprendizaje autónomo. La experiencia se lleva a cabo en el marco de un proyecto de investigación educativa, desarrollado en la Universidad Nacional del Litoral (Argentina) por docentes de la cátedra de Anatomía Veterinaria, en articulación con docentes de la cátera homónima de la Universidad de Zaragoza (España). A través de la investigación, se procura evaluar los resultados de aprendizaje de los alumnos ante esta innovación didáctica y metodológica. Las entidades educativas involucradas interactúan entre sí mediante la comunicación electrónica. En tal sentido, se destaca que la producción de los recursos multimediales es desarrollada en Zaragoza y la etapa experimental de dicha investigación se lleva a cabo en ambos contextos educativos

Adipocyte differentiation is modulated by secreted delta-like (dlk) variants and requires the expression of membrane-associated dlk.

Previous studies demonstrate that the delta-like (dlk) and preadipocyte factor 1 (Pref-1) genes encode similar proteins. Pref-1 is downregulated during adipocyte differentiation, and expression of ectopic Pref-1 inhibits adipogenesis. We explored whether dlk functions similarly to Pref-1 and studied the role of alternately spliced dlk variants encoding membrane-associated or -secreted forms. We also studied whether enforced downregulation of dlk/Pref-1 may enhance the differentiation response of non-committed cells. Ectopic expression of a potentially secreted dlk variant, conditioned media from dlk expressing cells or several individual epidermal-growth-factor-dlk peptides inhibited 3T3-L1 differentiation. This demonstrates that dlk and Pref-1 are functionally equivalent. dlk gene mRNA encoding for secreted variants decreased much faster than total dlk gene mRNA during differentiation of 3T3-L1 cells. In fact, total dlk or membrane-associated dlk protein expression increased during the first hours of differentiation. Cells sorted for lowest levels of dlk protein diminished or lost their ability to differentiate. These data suggest that membrane and secreted dlk protein variants play opposite roles in the control of adipogenesis. In addition, enforced downregulation of dlk protein expression in the weakly adipogenic Balb/c 3T3 cell line dramatically enhanced adipogenesis in response to insulin. These results indicate that dlk protein not only participates in processes leading to inhibition of adipogenesis but that the control of its expression and different spliced variants is essential for the adipogenic response to extracellular signals.

Notch-1 expression levels in 3T3-L1 cells influence ras signaling and transformation by oncogenic ras.

Notch proteins participate in interactions between several cell types involved on the specification of numerous cell fates during development. We previously showed that enforced downregulation of Notch-1 expression prevented adipogenesis of 3T3-L1 cells. Since adipogenesis of 3T3-L1 cells can be induced by oncogenic ras, we studied whether this was also the case in 3T3-L1 cells with decreased levels of Notch-1 expression. We found that oncogenic ras induces transformation and not differentiation of 3T3-L1 cells with diminished levels of Notch-1. This result suggests that Notch-1 is implicated in the interpretation of signals leading to activation of p21 Ras.

Effect of diabetes on the tissular Zn/Cu ratio.

One of the parameters related to the development of coronary disease in diabetic patients is the tissular Zn/Cu ratio. We evaluated the levels of Zn and Cu, and the Zn/Cu ratio in insulin target tissues in diabetic and normoglucemic growing Wistar rats in order to determine the influence of diabetes and the disease evolution period. Diabetes was induced chemically by administration of streptozotocin. In order to determine the influence of the duration of diabetes on the Zn/Cu ratio, three time periods were studied: 7, 21 and 60 days. The animals were subsequently sacrificed and the target tissues (liver, adipose tissue, and skeletal muscle) were removed. Zn and Cu levels were measured by AAS after wet mineralization. STZ-induced diabetes modified the tissular Zn and Cu content. There was a significant decrease (p < 0.01) in liver and adipose tissue, but not in skeletal muscle. The in adipose tissue and skeletal muscle, but not in liver, effects were dependent on the duration of diabetes. The Cu content was higher in the liver of diabetic rats (p < 0.1) and lower in adipose tissue (p < 0.1) and skeletal muscle (NS). Tissular Cu levels also were affected significantly by the duration of diabetes. The Zn/Cu ratio showed a generalized decrease, except in skeletal muscle. This decrease was dependent on the presence of diabetes mellitus and the duration of the disease (p < 0.01).

Sequences surrounding the Src-homology 3 domain of phospholipase Cgamma-1 increase the domain's association with Cbl.

SH3 domains are protein modules that interact with proline-rich polypeptide fragments. Cbl is an adapter-like protein known to interact with several SH3 domains, including the PLCgamma1 SH3 domain and the Grb2 amino terminal SH3 domain. Here we explore whether sequences surrounding the PLCgamma1 SH3 domain core sequence (aa.796-851) can affect the binding to Cbl, a target used as a prototypical ligand. Consistent with previous reports, our results demonstrated a weak binding of Cbl to GST fusion proteins that strictly encompass the structural core of the PLCgamma1 SH3 domain but a high-avidity binding to the Grb2 amino-terminal SH3 domain. Inclusion of amino acids immediately flanking the PLCgamma1 SH3 core domain, however, substantially increased binding of Cbl to a level comparable to that of the Grb2 amino-terminal SH3 domain. The interaction of this extended PLCgamma1 SH3 domain fusion protein with Cbl was shown to depend entirely upon the interaction of the domain with a proline-rich motif in Cbl, ruling out the possibility that amino acids adjacent to the core SH3 domain of PLCgamma1 provide independent Cbl binding. These data suggest that sequences surrounding the SH3 domain of PLCgamma1 may contribute to or stabilize the association of the domain with the target protein, thus increasing its binding efficiency.

Modulated expression of the epidermal growth factor-like homeotic protein dlk influences stromal-cell-pre-B-cell interactions, stromal cell adipogenesis, and pre-B-cell interleukin-7 requirements.

A close relationship exists between adipocyte differentiation of stromal cells and their capacity to support hematopoiesis. The molecular basis for this is unknown. We have studied whether dlk, an epidermal growth factor-like molecule that intervenes in adipogenesis and fetal liver hematopoiesis, affects both stromal cell adipogenesis and B-cell lymphopoiesis in an established pre-B-cell culture system. Pre-B-cell cultures require both soluble interleukin-7 (IL-7) and interactions with stromal cells to promote cell growth and prevent B-cell maturation or apoptosis. We found that BALB/c 3T3 fibroblasts express dlk and function as stromal cells. Transfection of these cells with antisense dlk decreased dlk expression and increased insulin-induced adipocytic differentiation. When antisense transfectants were used as stroma, IL-7 was no longer required to support the growth of pre-B cells and prevent maturation or apoptosis. Antisense dlk transfectants of S10 stromal cells also promoted pre-B-cell growth in the absence of IL-7. These results show that modulation of dlk on stromal cells can influence their adipogenesis and the IL-7 requirements of the pre-B cells growing in contact with them. These results indicate that dlk influences differentiation signals directed both to the stromal cells and to the lymphocyte precursors, suggesting that dlk may play an important role in the bone marrow hematopoietic environment.

[Cartilaginous tumors of the larynx. A report of 3 cases]. / Tumores cartilaginosos de la laringe. A propósito de tres casos.

Cartilaginous tumours of the larynx are very seldom encountered. Worldwide literature report until now no more than 250 cases. The greatest number of cases (72%) are benign and localized on cricoid cartilage. They are characterized by its slow pace spread and chondrosarcomata shows scant tendency to metastasize, prevailing, instead, local increase. Best treatment resulted the conservative surgery. We report 3 cases (1 chondroma and 2 low ranked chondrosarcomata) diagnosed and treated in our Hospital in the last 15 years. Clinical and anatomopathologic features are exposed and the management as well, and also the postoperative course of each patient.

The amino-terminal Src homology 2 domain of phospholipase C gamma 1 is essential for TCR-induced tyrosine phosphorylation of phospholipase C gamma 1.

TCR engagement activates phospholipase C gamma 1 (PLC gamma 1) via a tyrosine phosphorylation-dependent mechanism. PLC gamma 1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLC gamma 1 SH2 domains in PLC gamma 1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLC gamma 1 in response to TCR/CD3 perturbation. In addition, the PLC gamma 1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36-38), which has previously been recognized to interact with PLC gamma 1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLC gamma 1. Furthermore, binding of p36-38 to PLC gamma 1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLC gamma 1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLC gamma 1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLC gamma 1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLC gamma 1 with a tyrosine kinase.