Radical Scavenging Mechanisms of Phenolic Compounds: A Quantitative Structure-Property Relationship (QSPR) Study.

Due to their antioxidant properties, secondary plant metabolites can scavenge free radicals such as reactive oxygen species and protect foods from oxidation processes. Our aim was to study structural influences, like basic structure, number of hydroxyl groups and number of Bors criteria on the outcome of the oxygen radical absorbance capacity (ORAC) assay. Furthermore, similarities and differences to other in vitro antioxidant assays were analyzed by principal component analysis. Our studies confirmed that the antioxidant behavior in the ORAC assay is dominated by the number and types of substituents and not by the Bors criteria, as long as no steric hindrance occurs. For example, morin (MOR) with five hydroxyl groups and two Bors criteria reached an area under the curve of (3.64 ± 0.08) × 105, which was significantly higher than quercetin-7-D-glucoside (QGU7) (P < 0.001), and thus the highest result. Principal component analysis showed different dependencies regarding structural properties of Folin-Ciocalteu (FC)- and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-assays or 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)- and ORAC-assays, respectively. Therefore, we conclude that they are based on different reaction mechanisms. The number of hydroxyl groups showed a stronger influence on the antioxidant activity than the Bors criteria. Due to these differences, the correlation of these rapid tests to specific applications should be validated.

Efficacy of Vancomycin and Meropenem in Central Nervous System Infections in Children and Adults: Current Update.

The current antimicrobial therapy of bacterial infections of the central nervous system (CNS) in adults and pediatric patients is faced with many pitfalls as the drugs have to reach necessary levels in serum and cross the blood-brain barrier. Furthermore, several studies report that different factors such as the structure of the antimicrobial agent, the severity of disease, or the degree of inflammation play a significant role. Despite the available attempts to establish pharmacokinetic (PK) modeling to improve the required dosing regimen for adults and pediatric patients, conclusive recommendations for the best therapeutic strategies are still lacking. For instance, bacterial meningitis, the most common CNS infections, and ventriculitis, a severe complication of meningitis, are still associated with 10% and 30% mortality, respectively. Several studies report on the use of vancomycin and meropenem to manage meningitis and ventriculitis; therefore, this review aims to shed light on the current knowledge about their use in adults and pediatric patients. Consequently, studies published from 2015 until mid-July 2021 are included, and data about the study population, levels of drugs in serum and cerebrospinal fluid (CSF), and measured PK data in serum and CSF are provided. The overall aim is to provide the readers a recent reference that summarizes the pitfalls and success of the current therapy and emphasizes the importance of performing more studies to improve the clinical outcome of the current therapeutical approach.

Could the new dairy policy affect milk allocation to infants in Kenya? A best-worst scaling approach.

Milk is an important food item in the diet of Kenyans, especially infants. During the last two decades, the dairy sector in Kenya has witnessed important growth in production and improvements in milk quality. The informal marketing channel still prevails, and the Kenya Dairy Board, the regulator of the dairy sector, is currently introducing new regulations to increase registration and licensing of smallholder producers and dairy business operators, improve product hygiene and quality, and safeguard the health of consumers. These new regulations encompass, among others, the requirement to pasteurize milk before it is sold and adopt traceability processes and quality tests; most of these will probably result in higher milk prices at retail level. Using the best-worst scaling approach in this study, we analyzed the potential effects of milk price increase on household milk purchase and allocation to infants (6-48 months of age). The results indicate that an increase in milk price will decrease milk allocation to and intake by children. Households will replace the lost infant milk intake by fruits or porridge that might not be of equivalent nutritional value to milk. Any reforms to policies and regulatory systems aimed at streamlining the dairy sector should account for impacts on milk prices, responsiveness of consumers to price variations and infant nutrition. We recommend that regulatory and development agencies consider interventions that do not increase price for consumers and facilitate access to affordable and safe milk for children and entire households.

Cholestasis impairs hepatic lipid storage via AMPK and CREB signaling in hepatitis B virus surface protein transgenic mice.

Clinical studies demonstrated that nonalcoholic steatohepatitis is associated with liver-related outcomes in chronic hepatitis B. Furthermore, primary biliary fibrosis and biliary atresia occurred in patients with HBV infection. Interestingly, hepatitis B virus surface protein (HBs) transgenic mice spontaneously develop hepatic steatosis. Our aim is to investigate the effect of Abcb4 knockout-induced cholestasis on liver steatosis in HBs transgenic mice. Hybrids of HBs transgenic and Abcb4-/- mice were bred on the BALB/c genetic background. Lipid synthesis, storage, and catabolism as well as proteins and genes that control lipid metabolism were analyzed using HPTLC, qPCR, western blot, electrophoretic mobility shift assay (EMSA), lipid staining, and immunohistochemistry. Hepatic neutral lipid depots were increased in HBs transgenic mice and remarkably reduced in Abcb4-/- and HBs/Abcb4-/- mice. Similarly, HPTLC-based quantification analyses of total hepatic lipid extracts revealed a significant reduction in the amount of triacylglycerols (TAG), while the amount of free fatty acids (FFA) was increased in Abcb4-/- and HBs/Abcb4-/- in comparison to wild-type and HBs mice. PLIN2, a lipid droplet-associated protein, was less expressed in Abcb4-/- and HBs/Abcb4-/-. The expression of genes-encoding proteins involved in TAG synthesis and de novo lipogenesis (Agpat1, Gpat1, Mgat1, Dgat1, Dgat2, Fasn, Hmgcs1, Acc1, Srebp1-c, and Pparγ) was suppressed, and AMPK and CREB were activated in Abcb4-/- and HBs/Abcb4-/- compared to wild-type and HBs mice. Simulating cholestatic conditions in cell culture resulted in AMPK and CREB activation while FASN and PLIN2 were reduced. A concurrent inhibition of AMPK signaling revealed normal expression level of FASN and PLIN2, suggesting that activation of AMPK-CREB signaling regulates hepatic lipid metabolism, i.e. synthesis and storage, under cholestatic condition. In conclusions, in vivo and mechanistic in vitro data suggest that cholestasis reduces hepatic lipid storage via AMPK and CREB signaling. The results of the current study could be the basis for novel therapeutic strategies as NASH is a crucial factor that can aggravate chronic liver diseases.

Anti-nociceptive action of peripheral mu-opioid receptors by G-beta-gamma protein-mediated inhibition of TRPM3 channels.

Opioids, agonists of µ-opioid receptors (µORs), are the strongest pain killers clinically available. Their action includes a strong central component, which also causes important adverse effects. However, µORs are also found on the peripheral endings of nociceptors and their activation there produces meaningful analgesia. The cellular mechanisms downstream of peripheral µORs are not well understood. Here, we show in neurons of murine dorsal root ganglia that pro-nociceptive TRPM3 channels, present in the peripheral parts of nociceptors, are strongly inhibited by µOR activation, much more than other TRP channels in the same compartment, like TRPV1 and TRPA1. Inhibition of TRPM3 channels occurs via a short signaling cascade involving Gßγ proteins, which form a complex with TRPM3. Accordingly, activation of peripheral µORs in vivo strongly attenuates TRPM3-dependent pain. Our data establish TRPM3 inhibition as important consequence of peripheral µOR activation indicating that pharmacologically antagonizing TRPM3 may be a useful analgesic strategy.

Complex Photochemistry within the Green-Absorbing Channelrhodopsin ReaChR.

Channelrhodopsins (ChRs) are light-activated ion channels widely employed for photostimulation of excitable cells. This study focuses on ReaChR, a chimeric ChR variant with optimal properties for optogenetic applications. We combined electrophysiological recordings with infrared and UV-visible spectroscopic measurements to investigate photocurrents and photochemical properties of ReaChR. Our data imply that ReaChR is green-light activated (λmax = 532 nm) with a non-rhodopsin-like action spectrum peaking at 610 nm for stationary photocurrents. This unusual spectral feature is associated with photoconversion of a previously unknown light-sensitive, blue-shifted photocycle intermediate L (λmax = 495 nm), which is accumulated under continuous illumination. To explain the complex photochemical reactions, we propose a symmetrical two-cycle-model based on the two C15=N isomers of the retinal cofactor with either syn- or anti-configuration, each comprising six consecutive states D, K, L, M, N, and O. Ion conduction involves two states per cycle, the late M- (M2) with a deprotonated retinal Schiff base and the consecutive green-absorbing N-state that both equilibrate via reversible reprotonation. In our model, a fraction of the deprotonated M-intermediate of the anti-cycle may be photoconverted-as the L-state-back to its inherent dark state, or to its M-state pendant (M') of the syn-cycle. The latter reaction pathway requires a C13=C14, C15=N double-isomerization of the retinal chromophore, whereas the intracircular photoconversion of M back to D involves only one C13=C14 double-bond isomerization.

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2.

A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.

Contrasting invertebrate immune defense behaviors caused by a single gene, the Caenorhabditis elegans neuropeptide receptor gene npr-1.

BACKGROUND: The invertebrate immune system comprises physiological mechanisms, physical barriers and also behavioral responses. It is generally related to the vertebrate innate immune system and widely believed to provide nonspecific defense against pathogens, whereby the response to different pathogen types is usually mediated by distinct signalling cascades. Recent work suggests that invertebrate immune defense can be more specific at least at the phenotypic level. The underlying genetic mechanisms are as yet poorly understood. RESULTS: We demonstrate in the model invertebrate Caenorhabditis elegans that a single gene, a homolog of the mammalian neuropeptide Y receptor gene, npr-1, mediates contrasting defense phenotypes towards two distinct pathogens, the Gram-positive Bacillus thuringiensis and the Gram-negative Pseudomonas aeruginosa. Our findings are based on combining quantitative trait loci (QTLs) analysis with functional genetic analysis and RNAseq-based transcriptomics. The QTL analysis focused on behavioral immune defense against B. thuringiensis, using recombinant inbred lines (RILs) and introgression lines (ILs). It revealed several defense QTLs, including one on chromosome X comprising the npr-1 gene. The wildtype N2 allele for the latter QTL was associated with reduced defense against B. thuringiensis and thus produced an opposite phenotype to that previously reported for the N2 npr-1 allele against P. aeruginosa. Analysis of npr-1 mutants confirmed these contrasting immune phenotypes for both avoidance behavior and nematode survival. Subsequent transcriptional profiling of C. elegans wildtype and npr-1 mutant suggested that npr-1 mediates defense against both pathogens through p38 MAPK signaling, insulin-like signaling, and C-type lectins. Importantly, increased defense towards P. aeruginosa seems to be additionally influenced through the induction of oxidative stress genes and activation of GATA transcription factors, while the repression of oxidative stress genes combined with activation of Ebox transcription factors appears to enhance susceptibility to B. thuringiensis. CONCLUSIONS: Our findings highlight the role of a single gene, npr-1, in fine-tuning nematode immune defense, showing the ability of the invertebrate immune system to produce highly specialized and potentially opposing immune responses via single regulatory genes.

Optogenetic acidification of synaptic vesicles and lysosomes.

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation. Under physiological conditions, glutamatergic vesicles are nearly full, as additional vesicle acidification with pHoenix only slightly increased the quantal size. By contrast, we found that incompletely filled vesicles exhibited a lower release probability than full vesicles, suggesting preferential exocytosis of vesicles with high transmitter content. Our subcellular targeting approach can be transferred to other organelles, as demonstrated for a pHoenix variant that allows light-activated acidification of lysosomes.

Properties and functions of TRPM1 channels in the dendritic tips of retinal ON-bipolar cells.

An increase in light intensity induces a depolarization in retinal ON-bipolar cells via a reduced glutamate release from presynaptic photoreceptor cells. The underlying transduction cascade in the dendritic tips of ON-bipolar cells involves mGluR6 glutamate receptors signaling to TRPM1 proteins that are an indispensable part of the transduction channel. Several other proteins are recognized to participate in the transduction machinery. Deficiency in many of these leads to congenital stationary night blindness, because rod bipolar cells, a subgroup of ON-bipolar cells, constitute the main route for sensory information under scotopic conditions. Here, we review the current knowledge about TRPM1 ion channels and how their activity is regulated within the postsynaptic compartment of ON-bipolar cells. The functional properties of TRPM1 channels in the dendritic compartment are not well understood as they differ substantially from those of recombinant TRPM1 channels. Critical evaluation of possible explanations of these discrepancies indicates that some key components of this transduction pathway might still not be known. The continued exploration of this pathway will yield further clinically useful insights.

Biophysics of Channelrhodopsin.

Channelrhodopsins (ChRs) are directly light-gated ion channels that function as sensory photoreceptors in flagellated green algae, allowing these algae to identify optimal light conditions for growth. In neuroscience, ChRs constitute the most versatile tools for the light-induced activation of selected cells or cell types with unprecedented precision in time and space. In recent years, many ChR variants have been discovered or engineered, and countless electrical and spectroscopic studies of these ChRs have been carried out, both in host cells and on purified recombinant proteins. With significant support from a high-resolution 3D structure and from molecular dynamics calculations, scientists are now able to develop models that conclusively explain ChR activation and ion conductance on the basis of chromophore isomerization, structural changes, proton transfer reactions, and water rearrangement on timescales ranging from femtoseconds to minutes.

Epidemiological traits of the malaria-like parasite Polychromophilus murinus in the Daubenton's bat Myotis daubentonii.

BACKGROUND: The great diversity of bat haemosporidians is being uncovered with the help of molecular tools. Yet most of these studies provide only snapshots in time of the parasites discovered. Polychromophilus murinus, a malaria-like blood parasite, specialised on temperate-zone bats is a species that is being 'rediscovered'. This study describes the infection dynamics over time and between host sex and age classes. METHODS: For three years we followed the members of three breeding colonies of Myotis daubentonii in Western Switzerland and screened them for the prevalence and parasitemia of P. murinus using both molecular tools and traditional microscopy. In order to identify more susceptible classes of hosts, we measured, sexed and aged all individuals. During one year, we additionally measured body temperature and haematocrit values. RESULTS: Juvenile bats demonstrated much higher parasitemia than any other age class sampled, suggesting that first exposure to the parasite is very early in life during which infections are also at their most intense. Moreover, in subadults there was a clear negative correlation between body condition and intensity of infection, whereas a weak positive correlation was observed in adults. Neither body temperature, nor haematocrit, two proxies used for pathology, could be linked to intensities of infection. CONCLUSION: If both weaker condition and younger age are associated with higher infection intensity, then the highest selection pressure exerted by P. murinus should be at the juvenile stage. Confusion over the identities and nomenclature of malarial-like parasites requires that molecular barcodes are coupled to accurate morphological descriptions.

Conversion of channelrhodopsin into a light-gated chloride channel.

The field of optogenetics uses channelrhodopsins (ChRs) for light-induced neuronal activation. However, optimized tools for cellular inhibition at moderate light levels are lacking. We found that replacement of E90 in the central gate of ChR with positively charged residues produces chloride-conducting ChRs (ChloCs) with only negligible cation conductance. Molecular dynamics modeling unveiled that a high-affinity Cl(-)-binding site had been generated near the gate. Stabilizing the open state dramatically increased the operational light sensitivity of expressing cells (slow ChloC). In CA1 pyramidal cells, ChloCs completely inhibited action potentials triggered by depolarizing current injections or synaptic stimulation. Thus, by inverting the charge of the selectivity filter, we have created a class of directly light-gated anion channels that can be used to block neuronal output in a fully reversible fashion.

Fruit flies learn to avoid odours associated with virulent infection.

While learning to avoid toxic food is common in mammals and occurs in some insects, learning to avoid cues associated with infectious pathogens has received little attention. We demonstrate that Drosophila melanogaster show olfactory learning in response to infection with their virulent intestinal pathogen Pseudomonas entomophila. This pathogen was not aversive to taste when added to food. Nonetheless, flies exposed for 3 h to food laced with P. entomophila, and scented with an odorant, became subsequently less likely to choose this odorant than flies exposed to pathogen-laced food scented with another odorant. No such effect occurred after an otherwise identical treatment with an avirulent mutant of P. entomophila, indicating that the response is mediated by pathogen virulence. These results demonstrate that a virulent pathogen infection can act as an aversive unconditioned stimulus which flies can associate with food odours, and thus become less attracted to pathogen-contaminated food.

Ion selectivity and competition in channelrhodopsins.

Channelrhodopsins are light-gated ion channels of green algae. They are widely used for the analysis of neuronal networks using light in the emerging field of optogenetics. Under steady-state light conditions, the two open states, O1 and O2, mediate the photocurrents with different ion conductance and selectivity. To understand the conducting process as well as its optogenetic applications, it is important to study ion binding and transport of this promiscuous cation channel. Here, we present an enzyme kinetic algorithm that allowed us to calculate the ion composition of the initial and steady-state photocurrents for multication media. The approach is based on current-voltage relations determined for the individual ions H(+), Na(+), Ca(2+), and Mg(2+). We identify and quantify the widely different competition of the ions in wild-type channelrhodopsin-2 and two high-performing channelrhodopsin variants CatCh+ and C1V1. Both variants show enhanced Ca(2+) conductance, but only CatCh+ displays high steady-state Ca(2+) currents at neutral pH due to reduced H+ competition and low inactivation. We demonstrate that for optogenetic applications, one should always take into account that the variable equilibria of the two open states depend on light intensity, voltage, and the ionic composition of the medium.

Bimodal activation of different neuron classes with the spectrally red-shifted channelrhodopsin chimera C1V1 in Caenorhabditis elegans.

The C. elegans nervous system is particularly well suited for optogenetic analyses of circuit function: Essentially all connections have been mapped, and light can be directed at the neuron of interest in the freely moving, transparent animals, while behavior is observed. Thus, different nodes of a neuronal network can be probed for their role in controlling a particular behavior, using different optogenetic tools for photo-activation or -inhibition, which respond to different colors of light. As neurons may act in concert or in opposing ways to affect a behavior, one would further like to excite these neurons concomitantly, yet independent of each other. In addition to the blue-light activated Channelrhodopsin-2 (ChR2), spectrally red-shifted ChR variants have been explored recently. Here, we establish the green-light activated ChR chimera C1V1 (from Chlamydomonas and Volvox ChR1's) for use in C. elegans. We surveyed a number of red-shifted ChRs, and found that C1V1-ET/ET (E122T; E162T) works most reliable in C. elegans, with 540-580 nm excitation, which leaves ChR2 silent. However, as C1V1-ET/ET is very light sensitive, it still becomes activated when ChR2 is stimulated, even at 400 nm. Thus, we generated a highly efficient blue ChR2, the H134R; T159C double mutant (ChR2-HR/TC). Both proteins can be used in the same animal, in different neurons, to independently control each cell type with light, enabling a further level of complexity in circuit analyses.

Color-tuned channelrhodopsins for multiwavelength optogenetics.

Channelrhodopsin-2 is a light-gated ion channel and a major tool of optogenetics. It is used to control neuronal activity via blue light. Here we describe the construction of color-tuned high efficiency channelrhodopsins (ChRs), based on chimeras of Chlamydomonas channelrhodopsin-1 and Volvox channelrhodopsin-1. These variants show superb expression and plasma membrane integration, resulting in 3-fold larger photocurrents in HEK cells compared with channelrhodopsin-2. Further molecular engineering gave rise to chimeric variants with absorption maxima ranging from 526 to 545 nm, dovetailing well with maxima of channelrhodopsin-2 derivatives ranging from 461 to 492 nm. Additional kinetic fine-tuning led to derivatives in which the lifetimes of the open state range from 19 ms to 5 s. Finally, combining green- with blue-absorbing variants allowed independent activation of two distinct neural cell populations at 560 and 405 nm. This novel panel of channelrhodopsin variants may serve as an important toolkit element for dual-color cell stimulation in neural circuits.

Structural model of channelrhodopsin.

Channelrhodopsins (ChRs) are light-gated cation channels that mediate ion transport across membranes in microalgae (vectorial catalysis). ChRs are now widely used for the analysis of neural networks in tissues and living animals with light (optogenetics). For elucidation of functional mechanisms at the atomic level, as well as for further engineering and application, a detailed structure is urgently needed. In the absence of an experimental structure, here we develop a structural ChR model based on several molecular computational approaches, capitalizing on characteristic patterns in amino acid sequences of ChR1, ChR2, Volvox ChRs, Mesostigma ChR, and the recently identified ChR of the halophilic alga Dunaliella salina. In the present model, we identify remarkable structural motifs that may explain fundamental electrophysiological properties of ChR2, ChR1, and their mutants, and in a crucial validation of the model, we successfully reproduce the excitation energy predicted by absorption spectra.

Rectification of the channelrhodopsin early conductance.

We analyzed the nonlinear current-voltage relationships of the early conducting state of channelrhodopsin-2 expressed in Xenopus oocytes and human embryonic kidney cells with respect to changes of the electrochemical gradients of H(+), Na(+)/K(+), and Ca(2+)/Mg(2+). Several models were tested for wild-type ChR2 and mutations at positions E90, E123, H134, and T159. Voltage-gating was excluded as cause for the nonlinearity. However, a general enzyme kinetic model with one predominant binding site yielded good fits throughout. The empty site with an apparent charge number of about -0.3 and strong external cation binding causes some inward rectification of the uniport function. Additional inward rectification is due to asymmetric competition from outside between the transported ion species. Significant improvement of the fits was achieved by introducing an elastic voltage-divider formed by the voltage-sensitive barriers.

Neocortical excitation/inhibition balance in information processing and social dysfunction.

Severe behavioural deficits in psychiatric diseases such as autism and schizophrenia have been hypothesized to arise from elevations in the cellular balance of excitation and inhibition (E/I balance) within neural microcircuitry. This hypothesis could unify diverse streams of pathophysiological and genetic evidence, but has not been susceptible to direct testing. Here we design and use several novel optogenetic tools to causally investigate the cellular E/I balance hypothesis in freely moving mammals, and explore the associated circuit physiology. Elevation, but not reduction, of cellular E/I balance within the mouse medial prefrontal cortex was found to elicit a profound impairment in cellular information processing, associated with specific behavioural impairments and increased high-frequency power in the 30-80 Hz range, which have both been observed in clinical conditions in humans. Consistent with the E/I balance hypothesis, compensatory elevation of inhibitory cell excitability partially rescued social deficits caused by E/I balance elevation. These results provide support for the elevated cellular E/I balance hypothesis of severe neuropsychiatric disease-related symptoms.