Molecular mechanism of synovial joint site specification and induction in developing vertebrate limbs.

The vertebrate appendage comprises three primary segments, the stylopod, zeugopod and autopod, each separated by joints. The molecular mechanisms governing the specification of joint sites, which define segment lengths and thereby limb architecture, remain largely unknown. Existing literature suggests that reciprocal gradients of retinoic acid (RA) and fibroblast growth factor (FGF) signaling define the expression domains of the putative segment markers Meis1, Hoxa11 and Hoxa13. Barx1 is expressed in the presumptive joint sites. Our data demonstrate that RA-FGF signaling gradients define the expression domain of Barx1 in the first presumptive joint site. When misexpressed, Barx1 induces ectopic interzone-like structures, and its loss of function partially blocks interzone development. Simultaneous perturbations of RA-FGF signaling gradients result in predictable shifts of Barx1 expression domains along the proximo-distal axis and, consequently, in the formation of repositioned joints. Our data suggest that during early limb bud development in chick, Meis1 and Hoxa11 expression domains are overlapping, whereas the Barx1 expression domain resides within the Hoxa11 expression domain. However, once the interzone is formed, the expression domains are refined and the Barx1 expression domain becomes congruent with the border of these two putative segment markers.

Axial skeleton anterior-posterior patterning is regulated through feedback regulation between Meis transcription factors and retinoic acid.

Vertebrate axial skeletal patterning is controlled by co-linear expression of Hox genes and axial level-dependent activity of HOX protein combinations. MEIS transcription factors act as co-factors of HOX proteins and profusely bind to Hox complex DNA; however, their roles in mammalian axial patterning remain unknown. Retinoic acid (RA) is known to regulate axial skeletal element identity through the transcriptional activity of its receptors; however, whether this role is related to MEIS/HOX activity remains unknown. Here, we study the role of Meis in axial skeleton formation and its relationship to the RA pathway in mice. Meis elimination in the paraxial mesoderm produces anterior homeotic transformations and rib mis-patterning associated to alterations of the hypaxial myotome. Although Raldh2 and Meis positively regulate each other, Raldh2 elimination largely recapitulates the defects associated with Meis deficiency, and Meis overexpression rescues the axial skeletal defects in Raldh2 mutants. We propose a Meis-RA-positive feedback loop, the output of which is Meis levels, that is essential to establish anterior-posterior identities and patterning of the vertebrate axial skeleton.

ISG20L2: an RNA nuclease regulating T cell activation.

ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.

Tigecycline Opposes Bortezomib Effect on Myeloma Cells Decreasing Mitochondrial Reactive Oxygen Species Production.

Multiple myeloma is an incurable plasma cell malignancy. Most patients end up relapsing and developing resistance to antineoplastic drugs, like bortezomib. Antibiotic tigecycline has activity against myeloma. This study analyzed tigecycline and bortezomib combination on cell lines and plasma cells from myeloma patients. Apoptosis, autophagic vesicles, mitochondrial mass, mitochondrial superoxide, cell cycle, and hydrogen peroxide were studied by flow cytometry. In addition, mitochondrial antioxidants and electron transport chain complexes were quantified by reverse transcription real-time PCR (RT-qPCR) or western blot. Cell metabolism and mitochondrial activity were characterized by Seahorse and RT-qPCR. We found that the addition of tigecycline to bortezomib reduces apoptosis in proportion to tigecycline concentration. Supporting this, the combination of both drugs counteracts bortezomib in vitro individual effects on the cell cycle, reduces autophagy and mitophagy markers, and reverts bortezomib-induced increase in mitochondrial superoxide. Changes in mitochondrial homeostasis and MYC upregulation may account for some of these findings. These data not only advise to avoid considering tigecycline and bortezomib combination for treating myeloma, but caution on the potential adverse impact of treating infections with this antibiotic in myeloma patients under bortezomib treatment.

Previously diagnosed cancer and mortality after ST-segment elevation acute myocardial infarction treated with primary angioplasty.

OBJECTIVE: We investigated if a previous cancer diagnosis influences the outcome of patients with STEMI treated with primary coronary intervention (PCI). BACKGROUND: ST-segment myocardial infarction (STEMI) and a history of cancer can coexist because both have a high incidence and prevalence. METHODS: Prospective cohort observational study, The primary end-point was total mortality. RESULTS: We included 917 patients, 53 of them (5.8%) were cancer survivors. During follow-up (median, 643 days [interquartile range, 258 to 1,015 days]), 100 patients died, 88 (10.2%) patients without a cancer diagnosis and 12 (22.6%) patients with a previous cancer diagnosis, which was significantly different (log-rank test = 8.4, p = .004). Cancer patients were older (73.4 (11.5) vs. 65.2 (13.8) years, p < .001), with a lower prevalence of previous stroke (1.1% vs. 2.2%, p = .002). Their hemoglobin concentration was also lower (13.4 (2.1) vs. 14.4 (1.7) g/L, p = .001). A trend towards a lower use of coronary stents in cancer survivors was noted (p = .061). Cancer was associated with a high probability of death (HR = 2.37, 95% confidence interval [CI] 1.30-4.34, p = .005). When confounding variables were included, this association was no longer significant (HR = 1.63, 95% CI 0.84-3.18, p = .150). CONCLUSIONS: Patients with a previous cancer diagnosis who had an acute STEMI treated by primary PCI did not seem to have a worse prognosis. The difference in the crude mortality rate can be explained by the baseline differences between both groups. Previous cancer diagnosis should not be included in the clinical decision process when a patient is having an acute STEMI.

Olfactory and gustatory dysfunctions as a clinical presentation of mild-to-moderate forms of the coronavirus disease (COVID-19): a multicenter European study.

OBJECTIVE: To investigate the occurrence of olfactory and gustatory dysfunctions in patients with laboratory-confirmed COVID-19 infection. METHODS: Patients with laboratory-confirmed COVID-19 infection were recruited from 12 European hospitals. The following epidemiological and clinical outcomes have been studied: age, sex, ethnicity, comorbidities, and general and otolaryngological symptoms. Patients completed olfactory and gustatory questionnaires based on the smell and taste component of the National Health and Nutrition Examination Survey, and the short version of the Questionnaire of Olfactory Disorders-Negative Statements (sQOD-NS). RESULTS: A total of 417 mild-to-moderate COVID-19 patients completed the study (263 females). The most prevalent general symptoms consisted of cough, myalgia, and loss of appetite. Face pain and nasal obstruction were the most disease-related otolaryngological symptoms. 85.6% and 88.0% of patients reported olfactory and gustatory dysfunctions, respectively. There was a significant association between both disorders (p < 0.001). Olfactory dysfunction (OD) appeared before the other symptoms in 11.8% of cases. The sQO-NS scores were significantly lower in patients with anosmia compared with normosmic or hyposmic individuals (p = 0.001). Among the 18.2% of patients without nasal obstruction or rhinorrhea, 79.7% were hyposmic or anosmic. The early olfactory recovery rate was 44.0%. Females were significantly more affected by olfactory and gustatory dysfunctions than males (p = 0.001). CONCLUSION: Olfactory and gustatory disorders are prevalent symptoms in European COVID-19 patients, who may not have nasal symptoms. The sudden anosmia or ageusia need to be recognized by the international scientific community as important symptoms of the COVID-19 infection.

Post-translational add-ons mark the path in exosomal protein sorting.

Extracellular vesicles (EVs) are released by cells to the extracellular environment to mediate inter-cellular communication. Proteins, lipids, nucleic acids and metabolites shuttled in these vesicles modulate specific functions in recipient cells. The enrichment of selected sets of proteins in EVs compared with global cellular levels suggests the existence of specific sorting mechanisms to specify EV loading. Diverse post-translational modifications (PTMs) of proteins participate in the loading of specific elements into EVs. In this review, we offer a perspective on PTMs found in EVs and discuss the specific role of some PTMs, specifically Ubiquitin and Ubiquitin-like modifiers, in exosomal sorting of protein components. The understanding of these mechanisms will provide new strategies for biomedical applications. Examples include the presence of defined PTM marks on EVs as novel biomarkers for the diagnosis and prognosis of certain diseases, or the specific import of immunogenic components into EVs for vaccine generation.

Gradients, waves and timers, an overview of limb patterning models.

The vertebrate limb represents one of the oldest and most studied models in developmental and regenerative biology. Starting with classical experimental embryology and regenerative studies, its relevance in understanding biological mechanisms has expanded through the molecular biology era and now leads systems biology approaches in organogenesis. Limb patterning is organized along three main orthogonal axes; proximo-distal (P-D), antero-posterior (A-P) and dorso-ventral (D-V). Considerable heterogeneity has been found for the mechanisms involved in patterning these three axes, including signal gradients, cell-intrinsic timers and Turing-type signalling wave formation. Here we concentrate on reviewing patterning mechanisms along the P-D and A-P axes, in which different mechanisms converge and interact to pattern segmented structures.

Coordination of limb development by crosstalk among axial patterning pathways.

Vertebrate limb development relies on the activity of signaling centers that promote growth and control patterning along three orthogonal axes of the limb bud. The apical ectodermal ridge, at the distal rim of the limb bud ectoderm, produces WNT and FGF signals, which promote limb bud growth and progressive distalization. The zone of polarizing activity, a discrete postero-distal mesenchymal domain, produces SHH, which stimulates growth and organizes patterning along the antero-posterior axis. The dorsal and ventral ectoderms produce, respectively, WNT7A and BMPs, which induce dorso-ventral limb fates. Interestingly, these signaling centers and the mechanisms they instruct interact with each other to coordinate events along the three axes. We review here the main interactions described between the three axial systems of the developing limb and discuss their relevance to proper limb growth and patterning.

C-Functionalized Cationic Diazaoxatriangulenes: Late-Stage Synthesis and Tuning of Physicochemical Properties.

A series of nine C-functionalized cationic diazaoxatriangulene (DAOTA) dyes have been successfully synthesized and fully characterized, including X-ray structural analysis of four derivatives. The introduction of electron-withdrawing or -donating functions enables the tuning of both electro- and photochemical properties with, for instance, two consecutive (reversible) reductions or oxidations observed for nitro or amino derivatives, respectively. The substituents also impacted on the optical properties, with absorption maxima varying from λ=528 to 640 nm and fluorescence being shifted from the yellow to the red range, up to λ=656 nm.

Diffusible signals and epigenetic timing cooperate in late proximo-distal limb patterning.

Developing vertebrate limbs initiate proximo-distal patterning by interpreting opposing gradients of diffusible signaling molecules. We report two thresholds of proximo-distal signals in the limb bud: a higher threshold that establishes the upper-arm to forearm transition; and a lower one that positions a later transition from forearm to hand. For this last transition to happen, however, the signal environment seems to be insufficient, and we show that a timing mechanism dependent on histone acetylation status is also necessary. Therefore, as a consequence of the time dependence, the lower signaling threshold remains cryptic until the timing mechanism reveals it. We propose that this timing mechanism prevents the distal transition from happening too early, so that the prospective forearm has enough time to expand and form a properly sized segment. Importantly, the gene expression changes provoked by the first transition further regulate proximo-distal signal distribution, thereby coordinating the positioning of the two thresholds, which ensures robustness. This model is compatible with the most recent genetic analyses and underscores the importance of growth during the time-dependent patterning phase, providing a new mechanistic framework for understanding congenital limb defects.

Sp6 and Sp8 transcription factors control AER formation and dorsal-ventral patterning in limb development.

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. The induction of the AER is a complex process that relies on integrated interactions among the Fgf, Wnt, and Bmp signaling pathways that operate within the ectoderm and between the ectoderm and the mesoderm of the early limb bud. The transcription factors Sp6 and Sp8 are expressed in the limb ectoderm and AER during limb development. Sp6 mutant mice display a mild syndactyly phenotype while Sp8 mutants exhibit severe limb truncations. Both mutants show defects in AER maturation and in dorsal-ventral patterning. To gain further insights into the role Sp6 and Sp8 play in limb development, we have produced mice lacking both Sp6 and Sp8 activity in the limb ectoderm. Remarkably, the elimination or significant reduction in Sp6;Sp8 gene dosage leads to tetra-amelia; initial budding occurs, but neither Fgf8 nor En1 are activated. Mutants bearing a single functional allele of Sp8 (Sp6-/-;Sp8+/-) exhibit a split-hand/foot malformation phenotype with double dorsal digit tips probably due to an irregular and immature AER that is not maintained in the center of the bud and on the abnormal expansion of Wnt7a expression to the ventral ectoderm. Our data are compatible with Sp6 and Sp8 working together and in a dose-dependent manner as indispensable mediators of Wnt/ßcatenin and Bmp signaling in the limb ectoderm. We suggest that the function of these factors links proximal-distal and dorsal-ventral patterning.

GATA4 loss in the septum transversum mesenchyme promotes liver fibrosis in mice.

UNLABELLED: The zinc finger transcription factor GATA4 controls specification and differentiation of multiple cell types during embryonic development. In mouse embryonic liver, Gata4 is expressed in the endodermal hepatic bud and in the adjacent mesenchyme of the septum transversum. Previous studies have shown that Gata4 inactivation impairs liver formation. However, whether these defects are caused by loss of Gata4 in the hepatic endoderm or in the septum transversum mesenchyme remains to be determined. In this study, we have investigated the role of mesenchymal GATA4 activity in liver formation. We have conditionally inactivated Gata4 in the septum transversum mesenchyme and its derivatives by using Cre/loxP technology. We have generated a mouse transgenic Cre line, in which expression of Cre recombinase is controlled by a previously identified distal Gata4 enhancer. Conditional inactivation of Gata4 in hepatic mesenchymal cells led to embryonic lethality around mouse embryonic stage 13.5, likely as a consequence of fetal anemia. Gata4 knockout fetal livers exhibited reduced size, advanced fibrosis, accumulation of extracellular matrix components and hepatic stellate cell (HSC) activation. Haploinsufficiency of Gata4 accelerated CCl4 -induced liver fibrosis in adult mice. Moreover, Gata4 expression was dramatically reduced in advanced hepatic fibrosis and cirrhosis in humans. CONCLUSIONS: Our data demonstrate that mesenchymal GATA4 activity regulates HSC activation and inhibits the liver fibrogenic process.

A reevaluation of X-irradiation-induced phocomelia and proximodistal limb patterning.

Phocomelia is a devastating, rare congenital limb malformation in which the long bones are shorter than normal, with the upper portion of the limb being most severely affected. In extreme cases, the hands or fingers are attached directly to the shoulder and the most proximal elements (those closest to the shoulder) are entirely missing. This disorder, previously known in both autosomal recessive and sporadic forms, showed a marked increase in incidence in the early 1960s due to the tragic toxicological effects of the drug thalidomide, which had been prescribed as a mild sedative. This human birth defect is mimicked in developing chick limb buds exposed to X-irradiation. Both X-irradiation and thalidomide-induced phocomelia have been interpreted as patterning defects in the context of the progress zone model, which states that a cell's proximodistal identity is determined by the length of time spent in a distal limb region termed the 'progress zone'. Indeed, studies of X-irradiation-induced phocomelia have served as one of the two major experimental lines of evidence supporting the validity of the progress zone model. Here, using a combination of molecular analysis and lineage tracing in chick, we show that X-irradiation-induced phocomelia is fundamentally not a patterning defect, but rather results from a time-dependent loss of skeletal progenitors. Because skeletal condensation proceeds from the shoulder to fingers (in a proximal to distal direction), the proximal elements are differentially affected in limb buds exposed to radiation at early stages. This conclusion changes the framework for considering the effect of thalidomide and other forms of phocomelia, suggesting the possibility that the aetiology lies not in a defect in the patterning process, but rather in progenitor cell survival and differentiation. Moreover, molecular evidence that proximodistal patterning is unaffected after X-irradiation does not support the predictions of the progress zone model.

Two new targeted alleles for the comprehensive analysis of Meis1 functions in the mouse.

Meis1 is a highly conserved transcription factor that is activated in a regionally restricted manner from early stages of development. Meis1 belongs to the three amino acid loop extension (TALE) homeodomain family. Together with Pbx1, Meis1 plays a major role as a Hox cofactor, and therefore, plays an essential role in the development of several embryonic organs and systems, including limbs, heart, blood, and vasculature. In addition, Meis1 is required for the development of Hox-free embryonic regions and interacts with non-Hox homeodomain and non-homeodomain transcription factors. During post-natal life Meis1 is involved in adult cardiomyocyte homeostasis and has been associated with pre-disposition to human neural and cardiac pathologies. Given the relevance of this transcription factor, we have developed two new Meis1 gene knockin models; a direct ECFP knockin insertion that allows the direct identification of Meis1-expressing cells in living tissues, and a CreERT2 insertion that allows the inducible genetic tracing of Meis1-expressing cells in a time-controlled manner. Importantly, these two alleles represent the first Meis1 mutations in which Meis1 protein production is completely eliminated. These newly targeted Meis1 alleles will be valuable tools to further our understanding of the role of this critical transcription factor during development and disease.

The new standardised nomenclature in Otorhinolaryngology by SEORL-CCC - ANAESTHESIA (SEDAR). Executive version 2024.

PURPOSE: After the publication of the new standardized nomenclature for the specialty of Otorhinolaryngology in 2021, a joint adaptation was carried out with the Spanish Society of Anesthesiology, Resuscitation and Pain Therapy (SEDAR), creating an executive version. In this version, the Anesthesia groups are added for those procedures that require it and, in addition, the number of acts is reduced to facilitate its implementation in the daily basis healthcare activity. The aim of this article is to update the definitive executive version of the nomenclature for the specialty of Otolaryngology. METHODS: The nomenclature published in 2021 was updated, reducing the number of acts and procedures. For this purpose, a grouping of procedures similar in description and in order and ranking has been made. Those procedures that have been grouped together have received a new description that reflects all the acts included in order to facilitate its coding but respecting the essence of the proposal of the complete version of the 2021 nomenclature. Subsequently, the private medicine committee of SEDAR has assigned the anaesthetic act for those procedures that may require it. In addition, a provisional code has been assigned for those acts that are new with respect to the latest version approved by the OMC, which allows their numerical identification. RESULTS: The executive version of the nomenclature presents a total of 234 medical acts, compared to 395 listed in the 2021 version, which are distributed by OMC classification groups and ENT subspecialties. One-hundred and fourteen procedures maintain the original OMC code, with some modifications in the description of the medical act. Other procedures also performed by ENT but listed elsewhere were kept with their same description and group and assigned OMC codes. The remaining 120 procedures are new proposals made by the scientific society and its subspecialty committees. CONCLUSIONS: The executive version of the new nomenclature of Otorhinolaryngology proposed by the SEORL-CCC and SEDAR updates the one from 2021 and is the only one valid in our specialty for its use in the private healthcare daily practice. The reduction of medical procedures, without losing richness or modifications of the surgical groups, and the allocation of the anesthesia scales, facilitates its implementation, and provides the highest standards of quality and clinical timelines.

Spatial enhancer activation influences inhibitory neuron identity during mouse embryonic development.

The mammalian telencephalon contains distinct GABAergic projection neuron and interneuron types, originating in the germinal zone of the embryonic basal ganglia. How genetic information in the germinal zone determines cell types is unclear. Here we use a combination of in vivo CRISPR perturbation, lineage tracing and ChIP-sequencing analyses and show that the transcription factor MEIS2 favors the development of projection neurons by binding enhancer regions in projection-neuron-specific genes during mouse embryonic development. MEIS2 requires the presence of the homeodomain transcription factor DLX5 to direct its functional activity toward the appropriate binding sites. In interneuron precursors, the transcription factor LHX6 represses the MEIS2-DLX5-dependent activation of projection-neuron-specific enhancers. Mutations of Meis2 result in decreased activation of regulatory enhancers, affecting GABAergic differentiation. We propose a differential binding model where the binding of transcription factors at cis-regulatory elements determines differential gene expression programs regulating cell fate specification in the mouse ganglionic eminence.

Mother-to-child Chagas disease transmission: The challenge of detection and prevention in areas without the risk of vectorial transmission.

Chagas disease (CD) is caused by the parasite Trypanosoma cruzi. Although it is endemic in many Latin American (LA) countries, mother-to-child transmission has caused it to expand to other countries and continents. In places where vector transmission is controlled or absent, the epidemiological importance of T. cruzi transmission of the infected mother to her child during pregnancy or childbirth (i.e., perinatal CD) increases. In countries where CD is not endemic, CD screening should be performed in pregnant or fertile women who are native to LA countries or whose mothers are native to LA countries. Diagnosis is established by detecting anti-T. cruzi IgG antibodies in a serum or plasma sample. Antiparasitic treatment cannot be offered during pregnancy, and since the majority of infected newborns are asymptomatic at birth, a diagnosis is made by direct observation or concentration (microhematocrit) or by using molecular testing techniques. Once the infected child receives a diagnosis, it is essential to offer treatment (benznidazole/nifurtimox) as soon as possible, with good tolerance and effectiveness in the first year of life. Even if the diagnosis is negative at birth, the newborn must be followed up for at least the first 9 months of life.