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The most severe form of α-thalassemia results from loss of all four copies of α-globin. Postnatally, patients face challenges similar to β-thalassemia, including severe anemia and erythrotoxicity due to the imbalance of β-globin and α-globin chains. Despite progress in genome editing treatments for β-thalassemia, there is no analogous curative option for α-thalassemia. To address this, we designed a Cas9/AAV6-mediated genome editing strategy that integrates a functional α-globin gene into the β-globin locus in α-thalassemia patient-derived hematopoietic stem and progenitor cells (HSPCs). Incorporation of a truncated erythropoietin receptor transgene into the α-globin integration cassette significantly increased erythropoietic output from edited HSPCs and led to the most robust production of α-globin, and consequently hemoglobin tetramers. By directing edited HSPCs toward increased production of clinically relevant erythroid cells, this approach has the potential to mitigate the limitations of current treatments for the hemoglobinopathies, including low genome editing and low engraftment rates.

Background: Circular RNAs (circRNAs) play multifaceted roles to precisely control expression of broad gene networks. These highly stable molecules are often accumulated in the mammalian brain and thought to serve as "memory molecules" that govern the long process of aging. Mounting evidence demonstrated circRNA dysregulation in the postmortem brains of Alzheimer's disease (AD). However, whether and how circRNAs dysregulation underlies AD disease progression remain unexplored. Methods: Poly(A)‐tailing/RNase R digestion was coupled with a recently published computational algorithm called CARP to examine circRNA landscapes alteration and identify functional circRNA‐pathways progressively dysregulated in the cerebral cortex of the 5xFAD mouse model of AD between 5‐ and 7‐month, a critical window for the transition of starting pathology to full manifestation. We have also employed comprehensive biochemical approaches to identify circRNA interactome, as well as short hairpin RNA‐based genetic approaches to manipulate circRNAs and their key regulators to pinpoint their mechanistic contributions to AD. Results: We discovered genome‐wide alterations of brain circRNAs within the above age window critical for AD progression. Further analysis revealed co‐operation of distinct circRNAs and age‐dependent switch of circRNA isoforms derived from internal alternative splicing and alternative back splicing, which offered new means for regulating miRNA‐mRNA pathways in the 5xFAD cortex. In particular, we focused on conserved circRNAs that were progressively downregulated with increased severity of dementia in human AD brains. During circRNA biogenesis, we identified several previously established AD‐affected splicing factors, such as hnRNPL, regulate the production of AD‐associated circRNAs. Mechanistically, we found these circRNAs contained binding sites for many miRNAs and demonstrated its activity in sponging several AD‐relevant miRNAs to regulate their target mRNAs indicated in AD pathogenesis. Furthermore, we identified multiple circRNA‐interacting RNA binding proteins associated with AD, including the cleavage and polyadenylation factor 6 (CPSF6). In fact, altered polyadenylation efficiency was observed, which results in aberrant upregulation of many known CPSF6 target mRNAs indicated in AD pathogenesis. Conclusions: Together, our results unveiled pathological brain circRNA landscape alterations within a critical window for phenotypic progression in a mouse model of AD and identified novel molecular mechanisms underlying dysregulation of conserved circRNA pathways that contribute to AD pathogenesis. [ABSTRACT FROM AUTHOR]

Background: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease.Methods: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months.Results: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption.Conclusions: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease. [ABSTRACT FROM AUTHOR]

In bacteria, RNA-binding proteins of the RsmA/CsrA family act as post-transcriptional regulators that modulate translation initiation at target transcripts. The Pseudomonas aeruginosa genome contains two phenazine biosynthetic (phz) gene clusters, phzA1-G1 (phz1) and phzA2-G2 (phz2), each of which is responsible for phenazine-1-carboxylic acid (PCA) biosynthesis. In the present study, we show that RsmA exhibits differential gene regulation on two phz clusters in P. aeruginosa M18 at the post-transcriptional level. Based on the sequence analysis, four GGA motifs, the potential RsmA binding sites, are found on the 5′-untranslated region (UTR) of the phz2 transcript. Studies with a series of lacZ reporter fusions, and gel mobility shift assays suggest that the third GGA motif (S3), located 21 nucleotides upstream of the Shine-Dalgarno (SD) sequence, is involved in direct RsmA-mediated activation of phz2 expression. We therefore propose a novel model in which the binding of RsmA to the target S3 results in the destabilization of the stem-loop structure and the enhancement of ribosome access. This model could be fully supported by RNA structure prediction, free energy calculations, and nucleotide replacement studies. In contrast, various RsmA-mediated translation repression mechanisms have been identified in which RsmA binds near the SD sequence of target transcripts, thereby blocking ribosome access. Similarly, RsmA is shown to negatively regulate phz1 expression. Our new findings suggest that the differential regulation exerted by RsmA on the two phz clusters may confer an advantage to P. aeruginosa over other pseudomonads containing only a single phz cluster in their genomes. [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 pandemic and the vaccination effort that is ongoing has created an unmet need for accessible, affordable, flexible and precise platforms for monitoring the induction, specificity and maintenance of virus-specific immune responses. Herein we validate a multiplex (Luminex-based) assay capable of detecting SARS-CoV-2-specific antibodies irrespective of host species, antibody isotype, and specimen type (e.g. plasma, serum, saliva or blood spots). The well-established precision of Luminex-based assays provides the ability to follow changes in antibody levels over time to many antigens, including multiple permutations of the most common SARS-CoV-2 antigens. This platform can easily measure antibodies known to correlate with neutralization activity as well as multiple non-SARS-CoV-2 antigens such as vaccines ( e.g . Tetanus toxoid) or those from frequently encountered agents (influenza), which serve as stable reference points for quantifying the changing SARS-specific responses. All of the antigens utilized in our study can be made in-house, many in E. coli using readily available plasmids. Commercially sourced antigens may also be incorporated and newly available antigen variants can be rapidly produced and integrated, making the platform adaptable to the evolving viral strains in this pandemic., Competing Interests: The authors have declared that no conflicts of interest exists.

Objective: To explore the role in the pathogenesis of nasal polyp (NP) by comparison of eosinophil major basic protein (MBP), and neutrophil elastase (NE) in nasal polyps (NP) epithelium, stroma and the secretion of expression., Method: Immunohistochemical detection of 30 cases of patients with chronic sinusitis (CRS) NP epithelium NE stroma and the expression and secretion of MBP., Result: 1. There were significant differences of the expression of NE and MBP in epithelial tissue, stroma and secretion compared with the control group (P < 0.05); 2. There was not significant difference of the expression of NE and MBP between epithelial tissue and stroma (P > 0.05), while there was significant difference between epithelial tissue and the secretion (P < 0.05); 3. There were significant differences of the average positive expression of MBP and NE among epithelial tissue, stroma and secretion (P < 0.05); 4. MBP and NE were usually degranulated in secretion, while usually located in eosinophils (Eos) and neutrophils (Neu) in epithelial and mesenchymal; 5. There were abundant expression of MBP and NE in epithelial shedding regional, while small amounts of expression in stroma and integrated epithelial; 6. Electron microscopy could show the characteristics of electron density of MBP and NE particles., Conclusion: MBP and NE collaborated to cause pathological effects on the occurrence of NP.