Hoxb6 can interfere with somitogenesis in the posterior embryo through a mechanism independent of its rib-promoting activity.

Formation of the vertebrate axial skeleton requires coordinated Hox gene activity. Hox group 6 genes are involved in the formation of the thoracic area owing to their unique rib-promoting properties. Here we show that the linker region (LR) connecting the homeodomain and the hexapeptide is essential for Hoxb6 rib-promoting activity in mice. The LR-defective Hoxb6 protein was still able to bind a target enhancer together with Pax3, producing a dominant-negative effect, indicating that the LR brings additional regulatory factors to target DNA elements. We also found an unexpected association between Hoxb6 and segmentation in the paraxial mesoderm. In particular, Hoxb6 can disturb somitogenesis and anterior-posterior somite patterning by dysregulation of Lfng expression. Interestingly, this interaction occurred differently in thoracic versus more caudal embryonic areas, indicating functional differences in somitogenesis before and after the trunk-to-tail transition. Our results suggest the requirement of precisely regulated Hoxb6 expression for proper segmentation at tailbud stages.

Role of a polymorphism in a Hox/Pax-responsive enhancer in the evolution of the vertebrate spine.

Patterning of the vertebrate skeleton requires the coordinated activity of Hox genes. In particular, Hox10 proteins are essential to set the transition from thoracic to lumbar vertebrae because of their rib-repressing activity. In snakes, however, the thoracic region extends well into Hox10-expressing areas of the embryo, suggesting that these proteins are unable to block rib formation. Here, we show that this is not a result of the loss of rib-repressing properties by the snake proteins, but rather to a single base pair change in a Hox/Paired box (Pax)-responsive enhancer, which prevents the binding of Hox proteins. This polymorphism is also found in Paenungulata, such as elephants and manatees, which have extended rib cages. In vivo, this modified enhancer failed to respond to Hox10 activity, supporting its role in the extension of rib cages. In contrast, the enhancer could still interact with Hoxb6 and Pax3 to promote rib formation. These results suggest that a polymorphism in the Hox/Pax-responsive enhancer may have played a role in the evolution of the vertebrate spine by differently modulating its response to rib-suppressing and rib-promoting Hox proteins.

Regulatory role for a conserved motif adjacent to the homeodomain of Hox10 proteins.

Development of the vertebrate axial skeleton requires the concerted activity of several Hox genes. Among them, Hox genes belonging to the paralog group 10 are essential for the formation of the lumbar region of the vertebral column, owing to their capacity to block rib formation. In this work, we explored the basis for the rib-repressing activity of Hox10 proteins. Because genetic experiments in mice demonstrated that Hox10 proteins are strongly redundant in this function, we first searched for common motifs among the group members. We identified the presence of two small sequences flanking the homeodomain that are phylogenetically conserved among Hox10 proteins and that seem to be specific for this group. We show here that one of these motifs is required but not sufficient for the rib-repressing activity of Hox10 proteins. This motif includes two potential phosphorylation sites, which are essential for protein activity as their mutation to alanines resulted in a total loss of rib-repressing properties. Our data indicates that this motif has a significant regulatory function, modulating interactions with more N-terminal parts of the Hox protein, eventually triggering the rib-repressing program. In addition, this motif might also regulate protein activity by alteration of the protein's DNA-binding affinity through changes in the phosphorylation state of two conserved tyrosine residues within the homeodomain.

Controlling Hox gene expression and activity to build the vertebrate axial skeleton.

It has long been known that Hox genes are central players in patterning the vertebrate axial skeleton. Extensive genetic studies in the mouse have revealed that the combinatorial activity of Hox genes along the anterior-posterior body axis specifies different vertebral identities. In addition, Hox genes were instrumental for the evolutionary diversification of the vertebrate body plan. In this review, we focus on fundamental questions regarding the intricate mechanisms controlling Hox gene activity. In particular, we discuss the functional relevance of the precise timing of Hox gene activation in the embryo. Moreover, we provide insight into the epigenetic regulatory mechanisms that are likely to control this process and are responsible for the maintenance of spatially restricted Hox expression domains throughout embryonic development. We also analyze how specific features of each Hox protein may contribute to the functional diversity of Hox family. Altogether, the work reviewed here further supports the notion that the Hox program is far more complex than initially assumed. Exciting new findings will surely emerge in the years ahead.

The control of transitions along the main body axis.

Although vertebrates display a large variety of forms and sizes, the mechanisms controlling the layout of the basic body plan are substantially conserved throughout the clade. Following gastrulation, head, trunk, and tail are sequentially generated through the continuous addition of tissue at the caudal embryonic end. Development of each of these major embryonic regions is regulated by a distinct genetic network. The transitions from head-to-trunk and from trunk-to-tail development thus involve major changes in regulatory mechanisms, requiring proper coordination to guarantee smooth progression of embryonic development. In this review, we will discuss the key cellular and embryological events associated with those transitions giving particular attention to their regulation, aiming to provide a cohesive outlook of this important component of vertebrate development.

Population homogeneity for the antibody response to COVID-19 BNT162b2/Comirnaty vaccine is only reached after the second dose across all adult age ranges.

While mRNA vaccines are administrated worldwide in an effort to contain the COVID-19 pandemic, the heterogeneity of the humoral immune response they induce at the population scale remains unclear. Here, in a prospective, longitudinal, cohort-study, including 1245 hospital care workers and 146 nursing home residents scheduled for BNT162b2 vaccination, together covering adult ages from 19 to 99 years, we analyse seroconversion to SARS-CoV-2 spike protein and amount of spike-specific IgG, IgM and IgA before vaccination, and 3-5 weeks after each dose. We show that immunogenicity after a single vaccine dose is biased to IgG, heterogeneous and reduced with increasing age. The second vaccine dose normalizes IgG seroconversion in all age strata. These findings indicate two dose mRNA vaccines is required to reach population scale humoral immunity. The results advocate for the interval between the two doses not to be extended, and for serological monitoring of elderly and immunosuppressed vaccinees.

The heterogeneous ribonuclear protein C interacts with the hepatitis delta virus small antigen.

BACKGROUND: Hepatitis delta virus (HDV) is considered to be a satellite virus of the Hepatitis B virus. The genome consists of a 1679 nt ssRNA molecule in which a single ORF was identified. This ORF codes for a unique protein, the Delta antigen (HDAg). During transcription, two forms, small (S-HDAg; p24) and large (L-HDAg; p27) of this antigen are derived as a result of an editing mechanism catalyzed by cellular adenosine deaminase 1. Despite its simplicity, little is still known about the host factors that interact with the virus RNA and antigens being to modulate virus replication. METHODS: A yeast two-hybrid screening of a human liver cDNA library, using the hepatitis delta virus (HDV) small antigen (S-HDAg) as bait, was performed. Blot overlay and co-immunoprecipitation assays were used in an attempt to confirm the interaction of hnRNPC and S-HDAg. siRNA knockdown assays of hnRNPC were performed to assess the effect on HDV antigen expression. RESULTS: Thirty known proteins were identified as S-HDAg interactors in the yeast two-hybrid screening. One of the identified proteins, hnRNPC, was found to interact with S-HDAg in vitro and in vivo in human liver cells. The interaction of the two proteins is mediated by the C-terminal half of the S-HDAg which contains a RNA-binding domain (aa 98-195). HDV RNA, S-HDAg, and hnRNPC, were also found to co-localize in the nucleus of human liver cells. Knockdown of hnRNPC mRNA using siRNAs resulted in a marked decreased expression of HDV antigens. CONCLUSIONS: S-HDAg was found to interact with human liver proteins previously assigned to different functional categories. Among those involved in nucleic acid metabolism, hnRNPC was found to interact in vitro and in vivo in human liver cells. Similar to other RNA viruses, it seems plausible that hnRNPC may also be involved in HDV replication. However, further investigation is mandatory to clarify this question.

The MOVE.TE Falls Prevention and Management Program: lessons learnt in the Portuguese context.

MOVE.TE is a non-profit participatory physiotherapy platform that aims at translating knowledge in the field of physiotherapy and developing freely available evidence-based physiotherapy programmes targeting the primary care services of the Portuguese National Health service. A group of volunteer academics and clinicians collaborated at different stages and time points to create the first ever falls prevention and management programme and guidance for Physiotherapy in primary care, in Portugal. This report describes this seven-step process. In spite of many challenges, this project constitutes an example of advocacy in physiotherapy for the promotion of better healthcare for older adults.

Regulatory landscape of the Hox transcriptome.

Precise regulation of Hox gene activity is essential to achieve proper control of animal embryonic development and to avoid generation of a variety of malignancies. This is a multilayered process, including complex polycistronic transcription, RNA processing, microRNA repression, long noncoding RNA regulation and sequence-specific translational control, acting together to achieve robust quantitative and qualitative Hox protein output. For many such mechanisms, the Hox cluster gene network has turned out to serve as a paradigmatic model for their study. In this review, we discuss current knowledge of how the different layers of post-transcriptional regulation and the production of a variety of noncoding RNA species control Hox output, and how this shapes formation of developmental systems that are reproducibly patterned by complex Hox networks.

Structural and nucleic acid binding properties of hepatitis delta virus small antigen.

AIM: To further characterize the structure and nucleic acid binding properties of the 195 amino acid small delta antigen, S-HDAg, a study was made of a truncated form of S-HDAg, comprising amino acids 61-195 (∆60HDAg), thus lacking the domain considered necessary for dimerization and higher order multimerization. METHODS: Circular dichroism, and nuclear magnetic resonance experiments were used to assess the structure of ∆60HDAg. Nucleic acid binding properties were investigated by gel retardation assays. RESULTS: Results showed that the truncated ∆60HDAg protein is intrinsically disordered but compact, whereas the RNA binding domain, comprising residues 94-146, adopts a dynamic helical conformation. We also found that ∆60HDAg fails to multimerize but still contains nucleic acid binding activity, indicating that multimerization is not essential for nucleic acid binding. Moreover, in agreement with what has been previously reported for full-length protein, no apparent specificity was found for the truncated protein regarding nucleic acid binding. CONCLUSION: Taken together these results allowed concluding that ∆60HDAg is intrinsically disordered but compact; ∆60HDAg is not a multimer but is still capable of nucleic acid binding albeit without apparent specificity.

In Vivo Interaction of the Hepatitis Delta Virus Small Antigen with the ELAV-Like Protein HuR.

The small and large delta antigens (S-HDAg and L-HDAg, respectively) represent two forms of the only protein encoded by the hepatitis delta virus (HDV) RNA genome. Consequently, HDV relies, at a large extent, on the host cell machinery for replication and transcription. Until now, only a limited number of cellular proteins were identified as S-HDAg or L-HDAg partners being involved in the modulation of the virus life cycle. In an attempt to identify cellular S-HDAg-binding proteins we made use of a yeast two-hybrid approach to screen a human liver cDNA library. We were able to identify HuR, a ubiquitously expressed protein involved in RNA stabilization, as an S-HDAg partner both in vitro and in vivo. HuR was found to be overexpressed and colocalize with HDAg in human hepatoma cells. siRNA knockdown of HuR mRNA resulted in inhibition of S-HDAg and L-HDAg expression.