Advances in Nrf2 Signaling Pathway by Targeted Nanostructured-Based Drug Delivery Systems.

Nanotechnology has gained significant interest in various applications, including sensors and therapeutic agents for targeted disease sites. Several pathological consequences, including cancer, Alzheimer's disease, autoimmune diseases, and many others, are mostly driven by inflammation and Nrf2, and its negative regulator, the E3 ligase adaptor Kelch-like ECH-associated protein 1 (Keap1), plays a crucial role in maintaining redox status, the expression of antioxidant genes, and the inflammatory response. Interestingly, tuning the Nrf2/antioxidant response element (ARE) system can affect immune-metabolic mechanisms. Although many phytochemicals and synthetic drugs exhibited potential therapeutic activities, poor aqueous solubility, low bioavailability, poor tissue penetration, and, consequently, poor specific drug targeting, limit their practical use in clinical applications. Also, the therapeutic use of Nrf2 modulators is hampered in clinical applications by the absence of efficient formulation techniques. Therefore, we should explore the engineering of nanotechnology to modulate the inflammatory response via the Nrf2 signaling pathway. This review will initially examine the role of the Nrf2 signaling pathway in inflammation and oxidative stress-related pathologies. Subsequently, we will also review how custom-designed nanoscale materials encapsulating the Nrf2 activators can interact with biological systems and how this interaction can impact the Nrf2 signaling pathway and its potential outcomes, emphasizing inflammation.

Disruption of the early-life microbiota alters Peyer's patch development and germinal center formation in gastrointestinal-associated lymphoid tissue.

During postnatal development, both the maturing microbiome and the host immune system are susceptible to environmental perturbations such as antibiotic use. The impact of timing in which antibiotic exposure occurs was investigated by treating mice from days 5-9 with amoxicillin or azithromycin, two of the most commonly prescribed medications in children. Both early-life antibiotic regimens disrupted Peyer's patch development and immune cell abundance, with a sustained decrease in germinal center formation and diminished intestinal immunoglobulin A (IgA) production. These effects were less pronounced in adult mice. Through comparative analysis of microbial taxa, Bifidobacterium longum abundance was found to be associated with germinal center frequency. When re-introduced to antibiotic-exposed mice, B. longum partially rescued the immunological deficits. These findings suggest that early-life antibiotic use affects the development of intestinal IgA-producing B cell functions and that probiotic strains could be used to restore normal development after antibiotic exposure.

Opsin knockdown specifically slows phototransduction in broadband and UV-sensitive photoreceptors in Periplaneta americana.

Photoreceptors with different spectral sensitivities serve different physiological and behavioral roles. We hypothesized that such functional evolutionary optimization could also include differences in phototransduction dynamics. We recorded elementary responses to light, quantum bumps (QBs), of broadband green-sensitive and ultraviolet (UV)-sensitive photoreceptors in the cockroach, Periplaneta americana, compound eyes using intracellular recordings. In addition to control photoreceptors, we used photoreceptors from cockroaches whose green opsin 1 (GO1) or UV opsin expression was suppressed by RNA interference. In the control broadband and UV-sensitive photoreceptors average input resistances were similar, but the membrane capacitance, a proxy for membrane area, was smaller in the broadband photoreceptors. QBs recorded in the broadband photoreceptors had comparatively short latencies, high amplitudes and short durations. Absolute sensitivities of both opsin knockdown photoreceptors were significantly lower than in wild type, and, unexpectedly, their latency was significantly longer while the amplitudes were not changed. Morphologic examination of GO1 knockdown photoreceptors did not find significant differences in rhabdom size compared to wild type. Our results differ from previous findings in Drosophila melanogaster rhodopsin mutants characterized by progressive rhabdomere degeneration, where QB amplitudes were larger but phototransduction latency was not changed compared to wild type.

Protein surface topography as a tool to enhance the selective activity of a potassium channel blocker.

Tk-hefu is an artificial peptide designed based on the α-hairpinin scaffold, which selectively blocks voltage-gated potassium channels Kv1.3. Here we present its spatial structure resolved by NMR spectroscopy and analyze its interaction with channels using computer modeling. We apply protein surface topography to suggest mutations and increase Tk-hefu affinity to the Kv1.3 channel isoform. We redesign the functional surface of Tk-hefu to better match the respective surface of the channel pore vestibule. The resulting peptide Tk-hefu-2 retains Kv1.3 selectivity and displays ∼15 times greater activity compared with Tk-hefu. We verify the mode of Tk-hefu-2 binding to the channel outer vestibule experimentally by site-directed mutagenesis. We argue that scaffold engineering aided by protein surface topography represents a reliable tool for design and optimization of specific ion channel ligands.

Changes in electrophysiological properties of photoreceptors in Periplaneta americana associated with the loss of screening pigment.

Absence of screening pigment in insect compound eyes has been linked to visual dysfunction. We investigated how its loss in a white-eyed mutant (W-E) alters the photoreceptor electrophysiological properties, opsin gene expression, and the behavior of the cockroach, Periplaneta americana. Whole-cell patch-clamp recordings of green-sensitive photoreceptors in W-E cockroaches gave reduced membrane capacitance, absolute sensitivity to light, and light-induced currents. Decreased low-pass filtering increased voltage-bump amplitudes in W-E photoreceptors. Intracellular recordings showed that angular sensitivity of W-E photoreceptors had two distinct components: a large narrow component with the same acceptance angle as wild type, plus a relatively small wide component. Information processing was evaluated using Gaussian white-noise modulated light stimulation. In bright light, W-E photoreceptors demonstrated higher signal gain and signal power than wild-type photoreceptors. Expression levels of the primary UV- and green-sensitive opsins were lower and the secondary green-sensitive opsin significantly higher in W-E than in wild-type retinae. In behavioral experiments, W-E cockroaches were significantly less active in dim green light, consistent with the relatively low light sensitivity of their photoreceptors. Overall, these differences can be related to the loss of screening pigment function and to a compensatory decrease in the rhabdomere size in W-E retinae.

Behavioral responses to visual overstimulation in the cockroach Periplaneta americana L.

In the visual systems of insects, different types of photoreceptors contribute to specialized visual channels that mediate distinct functions and behaviors. Large compound eyes of Periplaneta americana contain photoreceptors of two spectral classes, broadband green-sensitive photoreceptors and narrow-band UV-sensitive photoreceptors. Here, we investigated how visual stimulation by UV and green light affects locomotor, resting, and grooming behaviors in P. americana under conditions when light avoidance is not possible. We show that green but not UV light stimulates locomotor activity, inducing paradoxical positive masking. Duration of resting and grooming decreased with increasing light intensity, consistent with development of behavioral stress in response to visual overstimulation. A reaction of full immobility is described under UV light and at higher intensities of green light, with relative periods of immobility and grooming strongly negatively correlated. Low-intensity UV was more effective than low-intensity green light in suppressing grooming and inducing immobility. Our results suggest that locomotor activity in P. americana is mainly regulated by green-sensitive photoreceptors, and that dim UV light can trigger behavioral immobility, whereas both wavelengths induce stress-like reactions at high intensities. Considering the intrinsic UV sensitivity of green-sensitive photoreceptors, the contrasting behavioral responses indicate antagonistic interactions between UV and green visual channels.

Spatial structure of TLR4 transmembrane domain in bicelles provides the insight into the receptor activation mechanism.

Toll-like receptors (TLRs) play a key role in the innate and adaptive immune systems. While a lot of structural data is available for the extracellular and cytoplasmic domains of TLRs, and a model of the dimeric full-length TLR3 receptor in the active state was build, the conformation of the transmembrane (TM) domain and juxtamembrane regions in TLR dimers is still unclear. In the present work, we study the transmembrane and juxtamembrane parts of human TLR4 receptor using solution NMR spectroscopy in a variety of membrane mimetics, including phospholipid bicelles. We show that the juxtamembrane hydrophobic region of TLR4 includes a part of long TM α-helix. We report the dimerization interface of the TM domain and claim that long TM domains with transmembrane charged aminoacids is a common feature of human toll-like receptors. This fact is analyzed from the viewpoint of protein activation mechanism, and a model of full-length TLR4 receptor in the dimeric state has been proposed.

Towards standardization of next-generation sequencing of FFPE samples for clinical oncology: intrinsic obstacles and possible solutions.

BACKGROUND: Next generation sequencing has a potential to revolutionize the management of cancer patients within the framework of precision oncology. Nevertheless, lack of standardization decelerated entering of the technology into the clinical testing space. Here we dissected a number of common problems of NGS diagnostics in oncology and introduced ways they can be resolved. METHODS: DNA was extracted from 26 formalin fixed paraffin embedded (FFPE) specimens and processed with the TrueSeq Amplicon Cancer Panel (Illumina Inc, San Diego, California) targeting 48 cancer-related genes and sequenced in single run. Sequencing data were comparatively analyzed by several bioinformatics pipelines. RESULTS: Libraries yielded sufficient coverage to detect even low prevalent mutations. We found that the number of FFPE sequence artifacts significantly correlates with pre-normalization concentration of libraries (rank correlation -0.81; p < 1e-10), thus, contributing to sample-specific variant detection cut-offs. Surprisingly, extensive validation of EGFR mutation calls by a combination of aligners and variant callers resulted in identification of two false negatives and one false positive that were due to complexity of underlying genomic change, confirmed by Sanger sequencing. Additionally, the study of the non-EGFR amplicons revealed 33 confirmed unique mutations in 17 genes, with TP53 being the most frequently mutated. Clinical relevance of these finding is discussed. CONCLUSIONS: Reporting of entire mutational spectrum revealed by targeted sequencing is questionable, at least until the clinically-driven guidelines on reporting of somatic mutations are established. The standardization of sequencing protocols, especially their data analysis components, requires assay-, disease-, and, in many cases, even sample-specific customization that could be performed only in cooperation with clinicians.