Evaluation of Predictive Motor Control With Two Touchscreen Tablet-Based Tests: Reliability and Validity in School-Aged Children.

As predictive motor control is an important index of neuromotor development and maturation, we developed two touchscreen tablet-based tests of this function. Our aim was to investigate the reliability and validity of both a rapid manual interception test and a pursuit tracking test, using a sample of 124 children (62 boys and 62 girls) from two age groups (7-8-year-oldss and 9-10-year-olds). Participants performed both tablet tests with a stylus (sample rate 100 Hz) with both a visible and a temporarily invisible moving target. Confirmatory factor analyses and omega coefficients showed that both tests were univariate methods that provided a reliable assessment of the latent factor related to predictive visuomotor control. As would be expected, compared to younger children, older children performed better on both manual interception and pursuit tracking. The correlations between the latent factors of the two tests at 95% confidence intervals (-.276, -.608) suggested shared variance. Thus, the touchscreen-tablet based tests of rapid manual interception and manual pursuit tracking appear psychometrically suitable for assessing the neuromotor ability of predictive control in 7-10-year-old children.

Correction: Krízová et al. Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein. Viruses 2021, 13, 2451.

The authors wish to make the following corrections to this paper [...].

Cobalt Bis-Dicarbollide Enhances Antibiotics Action towards Staphylococcus epidermidis Planktonic Growth Due to Cell Envelopes Disruption.

The emergence of antibiotic resistance in opportunistic pathogens represents a huge problem, the solution for which may be a treatment with a combination of multiple antimicrobial agents. Sodium salt of cobalt bis-dicarbollide (COSAN.Na) is one of the very stable, low-toxic, amphiphilic boron-rich sandwich complex heteroboranes. This compound has a wide range of potential applications in the biological sciences due to its antitumor, anti-HIV-1, antimicrobial and antibiofilm activity. Our study confirmed the ability of COSAN.Na (in the concentration range 0.2-2.48 µg/mL) to enhance tetracycline, erythromycin, and vancomycin action towards Staphylococcus epidermidis planktonic growth with an additive or synergistic effect (e.g., the combination of 1.24 µg/mL COSAN.Na and 6.5 µg/mL TET). The effective inhibitory concentration of antibiotics was reduced up to tenfold most efficiently in the case of tetracycline (from 65 to 6.5 µg/mL). In addition, strong effect of COSAN.Na on disruption of the cell envelopes was determined using propidium iodide uptake measurement and further confirmed by transmission electron microscopy. The combination of amphiphilic COSAN.Na with antibiotics can therefore be considered a promising way to overcome antibiotic resistance in Gram-positive cocci.

Alteration in DNA-binding affinity of Wilms tumor 1 protein due to WT1 genetic variants associated with steroid - resistant nephrotic syndrome in children.

Approximately one third of children with steroid-resistant nephrotic syndrome (SRNS) carry pathogenic variants in one of the many associated genes. The WT1 gene coding for the WT1 transcription factor is among the most frequently affected genes. Cases from the Czech national SRNS database were sequenced for exons 8 and 9 of the WT1 gene. Eight distinct exonic WT1 variants in nine children were found. Three children presented with isolated SRNS, while the other six manifested with additional features. To analyze the impact of WT1 genetic variants, wild type and mutant WT1 proteins were prepared and the DNA-binding affinity of these proteins to the target EGR1 sequence was measured by microscale thermophoresis. Three WT1 mutants showed significantly decreased DNA-binding affinity (p.Arg439Pro, p.His450Arg and p.Arg463Ter), another three mutants showed significantly increased binding affinity (p.Gln447Pro, p.Asp469Asn and p.His474Arg), and the two remaining mutants (p.Cys433Tyr and p.Arg467Trp) showed no change of DNA-binding affinity. The protein products of WT1 pathogenic variants had variable DNA-binding affinity, and no clear correlation with the clinical symptoms of the patients. Further research is needed to clarify the mechanisms of action of the distinct WT1 mutants; this could potentially lead to individualized treatment of a so far unfavourable disease.

Fullerene Derivatives Prevent Packaging of Viral Genomic RNA into HIV-1 Particles by Binding Nucleocapsid Protein.

Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.

Inhibition of Mitochondrial Metabolism Leads to Selective Eradication of Cells Adapted to Acidic Microenvironment.

Metabolic transformation of cancer cells leads to the accumulation of lactate and significant acidification in the tumor microenvironment. Both lactate and acidosis have a well-documented impact on cancer progression and negative patient prognosis. Here, we report that cancer cells adapted to acidosis are significantly more sensitive to oxidative damage induced by hydrogen peroxide, high-dose ascorbate, and photodynamic therapy. Higher lactate concentrations abrogate the sensitization. Mechanistically, acidosis leads to a drop in antioxidant capacity caused by a compromised supply of nicotinamide adenine dinucleotide phosphate (NADPH) derived from glucose metabolism. However, lactate metabolism in the Krebs cycle restores NADPH supply and antioxidant capacity. CPI-613 (devimistat), an anticancer drug candidate, selectively eradicates the cells adapted to acidosis through inhibition of the Krebs cycle and induction of oxidative stress while completely abrogating the protective effect of lactate. Simultaneous cell treatment with tetracycline, an inhibitor of the mitochondrial proteosynthesis, further enhances the cytotoxic effect of CPI-613 under acidosis and in tumor spheroids. While there have been numerous attempts to treat cancer by neutralizing the pH of the tumor microenvironment, we alternatively suggest considering tumor acidosis as the Achilles' heel of cancer as it enables selective therapeutic induction of lethal oxidative stress.

Betulinic Acid Decorated with Polar Groups and Blue Emitting BODIPY Dye: Synthesis, Cytotoxicity, Cell-Cycle Analysis and Anti-HIV Profiling.

Betulinic acid (BA) is a potent triterpene, which has shown promising potential in cancer and HIV-1 treatment. Here, we report a synthesis and biological evaluation of 17 new compounds, including BODIPY labelled analogues derived from BA. The analogues terminated by amino moiety showed increased cytotoxicity (e.g., BA had on CCRF-CEM IC50 > 50 µM, amine 3 IC50 0.21 and amine 14 IC50 0.29). The cell-cycle arrest was evaluated and did not show general features for all the tested compounds. A fluorescence microscopy study of six derivatives revealed that only 4 and 6 were detected in living cells. These compounds were colocalized with the endoplasmic reticulum and mitochondria, indicating possible targets in these organelles. The study of anti-HIV-1 activity showed that 8, 10, 16, 17 and 18 have had IC50i > 10 µM. Only completely processed p24 CA was identified in the viruses formed in the presence of compounds 4 and 12. In the cases of 2, 8, 9, 10, 16, 17 and 18, we identified not fully processed p24 CA and p25 CA-SP1 protein. This observation suggests a similar mechanism of inhibition as described for bevirimat.

Influence of Cultivation Conditions on the Sioxanthin Content and Antioxidative Protection Effect of a Crude Extract from the Vegetative Mycelium of Salinispora tropica.

Due to their bioavailability, glycosylated carotenoids may have interesting biological effects. Sioxanthin, as a representative of this type of carotenoid, has been identified in marine actinomycetes of the genus Salinispora. This study evaluates, for the first time, the effect of cultivation temperature (T) and light intensity (LI) on the total cellular carotenoid content (TC), antioxidant activity (AA) and sioxanthin content (SX) of a crude extract (CE) from Salinispora tropica biomass in its vegetative state. Treatment-related differences in TC and SX values were statistically significantly and positively affected by T and LI, while AA was most significantly affected by T. In the S. tropica CE, TC correlated well (R2 = 0.823) with SX and somewhat less with AA (R2 = 0.777). A correlation between AA and SX was found to be less significant (R2 = 0.731). The most significant protective effect against oxidative stress was identified in the CE extracted from S. tropica biomass grown at the highest T and LI (CE-C), as was demonstrated using LNCaP and KYSE-30 human cell lines. The CE showed no cytotoxicity against LNCaP and KYSE-30 cell lines.

In Vitro Quantification of the Effects of IP6 and Other Small Polyanions on Immature HIV-1 Particle Assembly and Core Stability.

Proper assembly and disassembly of both immature and mature HIV-1 hexameric lattices are critical for successful viral replication. These processes are facilitated by several host-cell factors, one of which is myo-inositol hexaphosphate (IP6). IP6 participates in the proper assembly of Gag into immature hexameric lattices and is incorporated into HIV-1 particles. Following maturation, IP6 is also likely to participate in stabilizing capsid protein-mediated mature hexameric lattices. Although a structural-functional analysis of the importance of IP6 in the HIV-1 life cycle has been reported, the effect of IP6 has not yet been quantified. Using two in vitro methods, we quantified the effect of IP6 on the assembly of immature-like HIV-1 particles, as well as its stabilizing effect during disassembly of mature-like particles connected with uncoating. We analyzed a broad range of molar ratios of protein hexamers to IP6 molecules during assembly and disassembly. The specificity of the IP6-facilitated effect on HIV-1 particle assembly and stability was verified by K290A, K359A, and R18A mutants. In addition to IP6, we also tested other polyanions as potential assembly cofactors or stabilizers of viral particles.IMPORTANCE Various host cell factors facilitate critical steps in the HIV-1 replication cycle. One of these factors is myo-inositol hexaphosphate (IP6), which contributes to assembly of HIV-1 immature particles and helps maintain the well-balanced metastability of the core in the mature infectious virus. Using a combination of two in vitro methods to monitor assembly of immature HIV-1 particles and disassembly of the mature core-like structure, we quantified the contribution of IP6 and other small polyanion molecules to these essential steps in the viral life cycle. Our data showed that IP6 contributes substantially to increasing the assembly of HIV-1 immature particles. Additionally, our analysis confirmed the important role of two HIV-1 capsid lysine residues involved in interactions with IP6. We found that myo-inositol hexasulphate also stabilized the HIV-1 mature particles in a concentration-dependent manner, indicating that targeting this group of small molecules may have therapeutic potential.

Octahedral Molybdenum Cluster Complexes with Optimized Properties for Photodynamic Applications.

Two new octahedral molybdenum cluster complexes act as an efficient singlet oxygen supplier in the context of the photodynamic therapy of cancer cells under blue-light irradiation. These complexes integrate the {Mo6I8}4+ core with 4'-carboxybenzo-15-crown-5 or cholate apical ligands and were characterized by 1H NMR, HR ESI-MS, and CHN elemental analysis. Both complexes display high quantum yields of luminescence and singlet oxygen formation in aqueous media associated with a suitable stability against hydrolysis. They are internalized into lysosomes of HeLa cells with no dark toxicity at pharmacologically relevant concentrations and have a strong phototoxic effect under blue-light irradiation, even in the presence of fetal bovine serum. The last feature is essential for further translation to in vivo experiments. Overall, these complexes are attractive molecular photosensitizers toward photodynamic applications.

Characterization and in vitro assembly of tick-borne encephalitis virus C protein.

Tick-borne encephalitis virus (TBEV), a member of flaviviruses, represents a serious health threat by causing human encephalitis mainly in central and eastern Europe, Russia, and northeastern Asia. As no specific therapy is available, there is an urgent need to understand all steps of the TBEV replication cycle at the molecular level. One of the critical events is the packaging of flaviviral genomic RNA by TBEV C protein to form a nucleocapsid. We purified recombinant TBEV C protein and used a combination of physical-chemical approaches, such as size-exclusion chromatography, circular dichroism, NMR spectroscopies, and transmission electron microscopy, to analyze its structural stability and its ability to dimerize/oligomerize. We compared the ability of TBEV C protein to assemble in vitro into a nucleocapsid-like structure with that of dengue C protein.

PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles.

A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein-protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

PEGylated Purpurin 18 with Improved Solubility: Potent Compounds for Photodynamic Therapy of Cancer.

Purpurin 18 derivatives with a polyethylene glycol (PEG) linker were synthesized as novel photosensitizers (PSs) with the goal of using them in photodynamic therapy (PDT) for cancer. These compounds, derived from a second-generation PS, exhibit absorption at long wavelengths; considerable singlet oxygen generation and, in contrast to purpurin 18, have higher hydrophilicity due to decreased logP. Together, these properties make them potentially ideal PSs. To verify this, we screened the developed compounds for cell uptake, intracellular localization, antitumor activity and induced cell death type. All of the tested compounds were taken up into cancer cells of various origin and localized in organelles known to be important PDT targets, specifically, mitochondria and the endoplasmic reticulum. The incorporation of a zinc ion and PEGylation significantly enhanced the photosensitizing efficacy, decreasing IC50 (half maximal inhibitory compound concentration) in HeLa cells by up to 170 times compared with the parental purpurin 18. At effective PDT concentrations, the predominant type of induced cell death was apoptosis. Overall, our results show that the PEGylated derivatives presented have significant potential as novel PSs with substantially augmented phototoxicity for application in the PDT of cervical, prostate, pancreatic and breast cancer.

A simple, high-throughput stabilization assay to test HIV-1 uncoating inhibitors.

Shortly after entering the cell, HIV-1 copies its genomic RNA into double-stranded DNA in a process known as reverse transcription. This process starts inside a core consisting of an enclosed lattice of capsid proteins that protect the viral RNA from cytosolic sensors and degradation pathways. To accomplish reverse transcription and integrate cDNA into the host cell genome, the capsid shell needs to be disassembled, or uncoated. Premature or delayed uncoating attenuates reverse transcription and blocks HIV-1 infectivity. Small molecules that bind to the capsid lattice of the HIV-1 core and either destabilize or stabilize its structure could thus function as effective HIV-1 inhibitors. To screen for such compounds, we modified our recently developed FAITH assay to allow direct assessment of the stability of in vitro preassembled HIV-1 capsid-nucleocapsid (CANC) tubular particles. This new assay is a high-throughput fluorescence method based on measuring the amount of nucleic acid released from CANC complexes under disassembly conditions. The amount of disassembled CANC particles and released nucleic acid is proportional to the fluorescence signal, from which the relative percentage of CANC stability can be calculated. We consider our assay a potentially powerful tool for in vitro screening for compounds that alter HIV disassembly.

Argon plasma-treated fluorinated ethylene propylene: Growth of primary dermal fibroblasts and mesenchymal stem cells.

Surface modification is an important step in making a synthetic polymer cytocompatible. We have previously reported improved cytocompatibility of immortalized human keratinocytes (HaCaT) with the otherwise bioinert fluorinated ethylene propylene (FEP) upon treatment with argon plasma discharge. In this article, we show that FEP modified with Ar plasma with the power of 3 and 8 W for 40 and 240 s served as a suitable material for cultivation of primary human dermal fibroblasts (HDF), which showed significantly improved proliferation and spreading comparable to standard tissue culture polystyrene. We also evaluated focal adhesions formed by HDF cells on modified FEP, which were far more numerous compared to pristine FEP. Moreover, we attempted spontaneous osteogenic differentiation of adipose-derived mesenchymal stem cells modified with human telomerase reverse transcriptase on Ar plasma-modified FEP. While the spontaneous osteogenic differentiation was unsuccessful, the cells were able to adhere and differentiated on tested matrices upon the administration of osteodifferentiation medium. These combined findings suggest that the treatment of FEP with Ar plasma comprises and efficient method to enable the adhesion and proliferation of various cell types on an otherwise largely bioinert material.

Nuclear transport of nicotinamide phosphoribosyltransferase is cell cycle-dependent in mammalian cells, and its inhibition slows cell growth.

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.

The nanoscaled metal-organic framework ICR-2 as a carrier of porphyrins for photodynamic therapy.

Nanosized porphyrin-containing metal-organic frameworks (MOFs) attract considerable attention as solid-state photosensitizers for biological applications. In this study, we have for the first time synthesised and characterised phosphinate-based MOF nanoparticles, nanoICR-2 (Inorganic Chemistry Rez). We demonstrate that nanoICR-2 can be decorated with anionic 5,10,15,20-tetrakis(4-R-phosphinatophenyl)porphyrins (R = methyl, isopropyl, phenyl) by utilizing unsaturated metal sites on the nanoparticle surface. The use of these porphyrins allows for superior loading of the nanoparticles when compared with commonly used 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin. The nanoICR-2/porphyrin composites retain part of the free porphyrins photophysical properties, while the photodynamic efficacy is strongly affected by the R substituent at the porphyrin phosphinate groups. Thus, phosphinatophenylporphyrin with phenyl substituents has the strongest photodynamic efficacy due to the most efficient cellular uptake.

Phosphinatophenylporphyrins tailored for high photodynamic efficacy.

The development of effective photosensitizers is particularly attractive for photodynamic therapy of cancer. Three novel porphyrin photosensitizers functionalized with phosphinic groups were synthesized and their physicochemical, photophysical, and photobiological properties were collected. Phosphinic acid groups (R1R2POOH) attached to the porphyrin moiety (R1) contain different R2 substituents (methyl, isopropyl, phenyl in this study). The presence of phosphinic groups does not influence absorption and photophysical properties of the porphyrin units, including the O2(1Δg) productivity. In vitro studies show that these porphyrins accumulate in cancer cells, are inherently nontoxic, however, exhibit high phototoxicity upon irradiation with visible light with their phototoxic efficacy tuned by R2 substituents on the phosphorus centre. Thus, phosphinatophenylporphyrin with isopropyl substituents has the strongest photodynamic efficacy due to the most efficient cellular uptake. We demonstrate that these porphyrins are attractive candidates for photodynamic applications since their photodynamic efficacy can be easily tuned by the R2 substituent.

Conserved cysteines in Mason-Pfizer monkey virus capsid protein are essential for infectious mature particle formation.

Retrovirus assembly is driven mostly by Gag polyprotein oligomerization, which is mediated by inter and intra protein-protein interactions among its capsid (CA) domains. Mason-Pfizer monkey virus (M-PMV) CA contains three cysteines (C82, C193 and C213), where the latter two are highly conserved among most retroviruses. To determine the importance of these cysteines, we introduced mutations of these residues in both bacterial and proviral vectors and studied their impact on the M-PMV life cycle. These studies revealed that the presence of both conserved cysteines of M-PMV CA is necessary for both proper assembly and virus infectivity. Our findings suggest a crucial role of these cysteines in the formation of infectious mature particles.