Size-Dependent Effects of Gold Nanoparticles Uptake on Maturation and Antitumor Functions of Human Dendritic Cells In Vitro

Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP₁₀ and GNP₅₀, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP₁₀ was more prominent. However, GNP₁₀ inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP₅₀ promoted Th17 polarization. Such effects of GNP₁₀ correlated with a stronger inhibition of LPS-induced changes in Ca²⁺ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP₁₀ inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs'' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP₁₀ could potentially induce more adverse DC-mediated immunological effects, compared to GNP₅₀.     

Molecular sexing assays in 114 mammalian species: In silico sequence reanalysis and a unified graphical visualization of diagnostic tests

Molecular‐based methods for identifying sex in mammals have a wide range of applications, from embryo manipulation to ecological studies. Various sex‐specific or homologous genes can be used for this purpose, PCR amplification being a common method. Over the years, the number of reported tests and the range of tested species have increased greatly. The aim of the present analysis was to retrieve PCR‐based sexing assays for a range of mammalian species, gathering the gene sequences from either the articles or online databases, and visualize the molecular design in a uniform manner. For nucleotide alignment and diagnostic test visualization, the following genomic databases and tools were used: NCBI, Ensembl Nucleotide BLAST, ClustalW2, and NEBcutter V2.0. In the 45 gathered articles, 59 different diagnostic tests based on eight different PCR‐based methods were developed for 114 mammalian species. Most commonly used genes for the analysis were ZFX, ZFY, AMELX, and AMELY. The tests were most commonly based on sex‐specific insertions and deletions (SSIndels) and sex‐specific sequence polymorphisms (SSSP). This review provides an overview of PCR‐based sexing methods developed for mammals. This information will facilitate more efficient development of novel molecular sexing assays and reuse of previously developed tests. Development of many novel and improvement of previously developed tests is also expected with the rapid increase in the quantity and quality of available genetic information.     

The SARS-CoV-2 Nsp3 macrodomain reverses PARP9/DTX3L-dependent ADP-ribosylation induced by interferon signaling

SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.     

Synthesis and structure–activity relationships of 2- and/or 4-halogenated 13β- and 13α-estrone derivatives as enzyme inhibitors of estrogen biosynthesis

Ring A halogenated 13α-, 13β-, and 17-deoxy-13α-estrone derivatives were synthesised with N-halosuccinimides as electrophile triggers. Substitutions occurred at positions C-2 and/or C-4. The potential inhibitory action of the halogenated estrones on human aromatase, steroid sulfatase, or 17β-hydroxysteroid dehydrogenase 1 activity was investigated via in vitro radiosubstrate incubation. Potent submicromolar or low micromolar inhibitors were identified with occasional dual or multiple inhibitory properties. Valuable structure–activity relationships were established from the comparison of the inhibitory data obtained. Kinetic experiments performed with selected compounds revealed competitive reversible inhibition mechanisms against 17β-hydroxysteroid dehydrogenase 1 and competitive irreversible manner in the inhibition of the steroid sulfatase enzyme.     

Discovery of pyrrolopyridazines as novel DGAT1 inhibitors

A new structural class of DGAT1 inhibitors was discovered and the structure–activity relationship was explored. The pyrrolotriazine core of the original lead molecule was changed to a pyrrolopyridazine core providing an increase in potency. Further exploration resulted in optimization of the propyl group at C7 and the discovery that the ester at C6 could be replaced by five-membered heterocyclic rings. The analogs prepared have DGAT1 IC₅₀ values ranging from >10 μM to 48 nM.     

The NKCC1 ion transporter modulates microglial phenotype and inflammatory response to brain injury in a cell-autonomous manner

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

This study shows that inflammatory responses of microglial cells are markedly influenced by the ion transporter, NKCC1; blockade or genetic deletion of microglial NKCC1 has broad cell-autonomous effects, leading to changes in morphology, membrane conductance, process recruitment after injury, and cytokine production, with worsened neurological outcome after stroke.     

Comparative immunohistochemical study of tissue integration of macroporous and laminar surgical meshes

Intraperitoneal surgical mesh implantation is required for laparoscopic ventral hernia repair. Composite meshes are well known in animal models and human practice. The aim of our study is to compare the biological behaviour of two different textured silicone-covered polypropylene meshes. Transmural abdominal wall defect was created in 40 rabbits and treated as follows: In 20 animals a polypropylene mesh with a laminar silicone covering (LSPP) and in the rest a macroporous textured mesh knitted of silicone-impregnated polypropylene filaments (MSPP) was applied. One and three weeks after implantation we evaluated the intraperitoneal adhesion formation of the mesh macroscopically, histologically and immunohistochemically to detect the reactive cells, especially inflammatory, endothelial and mesothelial cells, as well as their proliferative activity, and with Scanning Electron microscopy to visualize the surface of the meshes. The adhesion formation caused by the composites showed no statistical difference after one week although in the three weeks old samples the LSPP adhesion was significantly weaker than that of MSPP. As complications, serome formation in both groups, fistulas, abscesses, and sc. haematoma in the LSPP group were found. Only in MSPP containing tissues was the decrease of Ki-67 positive proliferating cells significant. A significant increase in VEGF expressing cells was observed only in MSPP containing three week old samples, suggesting better regulation of vascular growth in tissues surrounding the implants. In one week old specimens we observed an irregular proliferation of cytokeratin containing mesothelial cells in both group. The intraperitoneal surface of MSPP mesh was covered with neoperitoneum, while it was not regularly seen on LSPP mesh after three week.