Involvement of ferryl in the reaction between nitrite and the oxy forms of globins

The reaction between nitrite and the oxy forms of globins has complex autocatalytic kinetics with several branching steps and evolves through chain reactions mediated by reactive species (including radicals) such as hydrogen peroxide, ferryl and nitrogen dioxide, starting with a lag phase, after which it proceeds onto an autocatalytic phase. Reported here are UV–Vis spectra collected upon stopped-flow mixing of myoglobin with a supraphysiological excess of nitrite. The best fit to the experimental data follows an A → B → C reaction scheme involving the formation of a short-lived intermediate identified as ferryl. This is consistent with a mechanism where nitrite binds to oxy myoglobin to generate an undetectable ferrous-peroxynitrate intermediate, whose decay leads to nitrate and ferryl. The ferryl is then reduced to met by the excess nitrite. DFT calculations reveal an essentially barrierless reaction between nitrite and the oxy heme, with a notable outer-sphere component; the resulting metastable ferrous-peroxynitrate adduct is found to feature a very low barrier towards nitrate liberation, with ferryl as a final product—in good agreement with experiment.     

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.     

The pharmacological activation of adenosine A1 and A3 receptors does not modulate the long- or short-term repopulating ability of hematopoietic stem and multipotent progenitor cells in mice

This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A₁ and A₃ receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N ⁶-cyclopentyladenosine (CPA), an adenosine A₁ receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N ⁶-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A₃ receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.     

Therapies targeting cancer stem cells: Current trends and future challenges

Traditional therapies against cancer, chemo- and radiotherapy, have multiple limitations that lead to treatment failure and cancer recurrence. These limitations are related to systemic and local toxicity, while treatment failure and cancer relapse are due to drug resistance and self-renewal, properties of a small population of tumor cells called cancer stem cells (CSCs). These cells are involved in cancer initiation, maintenance, metastasis and recurrence. Therefore, in order to develop efficient treatments that can induce a long-lasting clinical response preventing tumor relapse it is important to develop drugs that can specifically target and eliminate CSCs. Recent identification of surface markers and understanding of molecular feature associated with CSC phenotype helped with the design of effective treatments. In this review we discuss targeting surface biomarkers, signaling pathways that regulate CSCs self-renewal and differentiation, drug-efflux pumps involved in apoptosis resistance, microenvironmental signals that sustain CSCs growth, manipulation of miRNA expression, and induction of CSCs apoptosis and differentiation, with specific aim to hamper CSCs regeneration and cancer relapse. Some of these agents are under evaluation in preclinical and clinical studies, most of them for using in combination with traditional therapies. The combined therapy using conventional anticancer drugs with CSCs-targeting agents, may offer a promising strategy for management and eradication of different types of cancers.     

Kinetics and persistence of cellular and humoral immune responses to SARS‐CoV‐2 vaccine in healthcare workers with or without prior COVID‐19

SARS‐CoV‐2 vaccines are highly efficient against severe forms of the disease, hospitalization and death. Nevertheless, insufficient protection against several circulating viral variants might suggest waning immunity and the need for an additional vaccine dose. We conducted a longitudinal study on the kinetics and persistence of immune responses in healthcare workers vaccinated with two doses of BNT162b2 mRNA vaccine with or without prior SARS‐CoV‐2 infection. No new infections were diagnosed during follow‐up. At 6 months, post‐vaccination or post‐infection, despite a downward trend in the level of anti‐S IgG antibodies, the neutralizing activity does not decrease significantly, remaining higher than 75% (85.14% for subjects with natural infection, 88.82% for vaccinated after prior infection and 78.37% for vaccinated only). In a live‐virus neutralization assay, the highest neutralization titres were present at baseline and at 6 months follow‐up in persons vaccinated after prior infection. Anti‐S IgA levels showed a significant descending trend in vaccinated subjects (p < 0.05) after 14 weeks. Cellular immune responses are present even in vaccinated participants with declining antibody levels (index ratio 1.1–3) or low neutralizing activity (30%–40%) at 6 months, although with lower T‐cell stimulation index (p = 0.046) and IFN‐γ secretion (p = 0.0007) compared to those with preserved humoral responses.     

Role of P-Selectin Cytoplasmic Domain in Granular Targeting In Vivo and in Early Inflammatory Responses

P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (ΔCT mice). The deletion did not affect the sorting of P-selectin into α-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The ΔCT–P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the ΔCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.     

Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development

A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late‐larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally‐regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). We demonstrate here that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions – glue secretion during pupariation – they remain dynamic and physiologically active up until the execution phase of PCD. We have used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68‐2, vha36‐1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7–8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early‐ to mid‐prepupal period.