A novel cis-regulatory element regulates αD and αA-globin gene expression in chicken erythroid cells.

Background: Cis-regulatory elements (CREs) play crucial roles in regulating gene expression during erythroid cell differentiation. Genome-wide erythroid-specific CREs have not been characterized in chicken erythroid cells, which is an organism model used to study epigenetic regulation during erythropoiesis. Methods: Analysis of public genome-wide accessibility (ATAC-seq) maps, along with transcription factor (TF) motif analysis, CTCF, and RNA Pol II occupancy, as well as transcriptome analysis in fibroblasts and erythroid HD3 cells, were used to characterize erythroid-specific CREs. An α-globin CRE was identified, and its regulatory activity was validated in vitro and in vivo by luciferase activity and genome-editing assays in HD3 cells, respectively. Additionally, circular chromosome conformation capture (UMI-4C) assays were used to distinguish its role in structuring the α-globin domain in erythroid chicken cells. Results: Erythroid-specific CREs displayed occupancy by erythroid TF binding motifs, CTCF, and RNA Pol II, as well as an association with genes involved in hematopoiesis and cell differentiation. An α-globin CRE, referred to as CRE-2, was identified as exhibiting enhancer activity over αD and αA genes in vitro and in vivo. Induction of terminal erythroid differentiation showed that α-globin CRE-2 is required for the induction of αD and αA. Analysis of TF binding motifs at α-globin CRE-2 shows apparent regulation mediated by GATA-1, YY1, and CTCF binding. Conclusion: Our findings demonstrate that cell-specific CREs constitute a key mechanism that contributes to the fine-tuning gene regulation of erythroid cell differentiation and provide insights into the annotation and characterization of CREs in chicken cells.

Prelamin A processing, accumulation and distribution in normal cells and laminopathy disorders.

Lamin A is part of a complex structural meshwork located beneath the nuclear envelope and is involved in both structural support and the regulation of gene expression. Lamin A is initially expressed as prelamin A, which contains an extended carboxyl terminus that undergoes a series of post-translational modifications and subsequent cleavage by the endopeptidase ZMPSTE24 to generate lamin A. To facilitate investigations of the role of this cleavage in normal and disease states, we developed a monoclonal antibody (PL-1C7) that specifically recognizes prelamin A at the intact ZMPSTE24 cleavage site, ensuring prelamin A detection exclusively. Importantly, PL-1C7 can be used to determine prelamin A localization and accumulation in cells where lamin A is highly expressed without the use of exogenous fusion proteins. Our results show that unlike mature lamin A, prelamin A accumulates as discrete and localized foci at the nuclear periphery. Furthermore, whereas treatment with farnesylation inhibitors of cells overexpressing a GFP-prelamin A fusion protein results in the formation of large nucleoplasmic clumps, these aggregates are not observed upon similar treatment of cells expressing endogenous prelamin A or in cells lacking ZMPSTE24 expression and/or activity. Finally, we show that specific laminopathy-associated mutations exhibit both positive and negative effects on prelamin A accumulation, indicating that these mutations affect prelamin A processing efficiency in different manners.

Paraspecific neutralization of the venom of African species of cobra by an equine antiserum against Naja melanoleuca: a comparative study.

Venoms of snakes belonging to the same Genera tend to share biochemical, toxinological and antigenic characteristics. Accordingly, paraspecific neutralization of venom lethality by experimental antisera and commercial antivenoms has been reported. We studied the spectrum of neutralization of lethality of an experimental monovalent equine antiserum against the strongly neurotoxic African forest cobra (Naja melanoleuca) when tested against venoms of most species of African Naja, both neuro and cytotoxic as described by some authors. We report a comparison of the median lethal doses (LD50) of the venoms and the paraspecific median effective doses (ED50) of the antiserum calculated using three methods: Spearman-Kärber and Probit (currently recommended by the World Health Organization), and non-linear regression. An ample--but not complete--spectrum of paraspecific neutralization of lethality was observed against both spitting and non-spitting species of African Naja with a clearly more efficient neutralization of the more potent venoms, the implications of which are discussed. The median lethal and effective doses calculated by the three methods are remarkably consistent and may warrant consideration of non-linear regression methods for the calculation of venom lethality and antivenom potency by venom/antivenom researchers and producers.

Characterization of a new polyvalent antivenom (Antivipmyn Africa) against African vipers and elapids.

As a response to the antivenom shortage in Sub-Saharan Africa, evident for well over a decade, we developed a new polyvalent anti-ophidian antivenom (Antivipmyn((R)) Africa) designed for use in the region. We report a detailed characterization of its biochemical composition (protein content and profiling by size-exclusion chromatography and electrophoresis) as well as the specific and para-specific neutralization potencies (as median effective dose in the mouse lethality test). Additionally, we studied the neutralization of hemorrhagic, anti-hemostatic and necrotic activities of Echis ocellatus venom, responsible for a majority of severe envenomations in the continent according to existing epidemiological data. The antivenom is currently under production and has already been employed in the field in a pragmatic Phase III clinical trial in the Republic of Benin. It is a purified lyophilized polyvalent equine F(ab')(2)-based product obtained by immunization with the venoms of eleven species of African snakes of the Genera Echis, Bitis, Naja and Dendroaspis. The criteria for its design are discussed, particularly in terms of the implementation of realistic public health policies targeting mostly rural populations in the continent.