Extracts from Veronica species (speedwells) are known for the various biological activities they show, such as cytotoxic, antimicrobial, anti-inflammatory, and antioxidant activities. Also, the plants from this genus are known as medicinal plants used in traditional medicine worldwide. Phenolic compounds are specialized metabolites that contribute to biological activity the most. Therefore, the aim of this research is identification and quantification of phenolic compounds present in three Veronica species (Veronica anagallis-aquatica L., Veronica persica Poir., and Veronica polita Fr.) using the liquid chromatography-mass spectrometry (LC-MS/MS) technique. All extracts were tested for antioxidant activity with two methods: DPPH (2,2-diphenyl-1-picrylhydrazyl) and ORAC (oxygen radical absorbance capacity). Also, standards for compounds that were detected in the highest amount in all species were also tested for antioxidant activity. Three different solvents (pure methanol, 80% ethanol, and water) were used for the extraction of phenolic components and their comparison in order to test their antioxidant activity as a final goal. The main compounds present in the tested Veronica extracts were: p-hydroxybenzoic acid, vanillic acid, caffeic acid, gentisic acid, and apigenin. V. anagallis–aquatica contained the highest amount of phenolic components in comparison with the two other tested species, V. persica and V. polita. Caffeic acid showed the highest antioxidant activity in both studied methods with an IC50 value for DPPH activity of 1.99 µg/mL. For the plant extracts, in general, methanolic/ethanolic extracts show
antioxidant activity, DPPH, LC-MS/MS technique, ORAC, polyphenols, Veronica
Bioequivalence, Cutaneous pharmacokinetics, Dermal open flow microperfusionTopical, Lidocaine, Prilocaine
Herpes B virus (BV) is a zoonotic virus and belongs to the genus Simplexvius, the same genus as human herpes simplex virus (HSV). BV typically establishes asymptomatic infection in its natural hosts, macaque monkeys. However, in humans, BV infection causes serious neurological diseases and death. As such, BV research can only be conducted in a high containment level facility (i.e., biosafety level [BSL] 4), and the mechanisms of BV entry have not been fully elucidated. In this study, we generated a pseudotyped vesicular stomatitis virus (VSV) expressing BV glycoproteins using G-complemented VSV∆G system, which we named VSV/BVpv. We found that four BV glycoproteins (i.e., gB, gD, gH, and gL) were required for the production of a high-titer VSV/BVpv. Moreover, VSV/BVpv cell entry was dependent on the binding of gD to its cellular receptor nectin-1. Pretreatment of Vero cells with endosomal acidification inhibitors did not affect the VSV/BVpv infection. The result indicated that VSV/BVpv entry occurred by direct fusion with the plasma membrane of Vero cells and suggested that the entry pathway was similar to that of native HSV. Furthermore, we developed a VSV/BVpv-based chemiluminescence reduction neutralization test (CRNT), which detected the neutralization antibodies against BV in macaque plasma samples with high sensitivity and specificity. Crucially, the VSV/BVpv generated in this study can be used under BSL-2 condition to study the initial entry process through gD–nectin-1 interaction and the direct fusion of BV with the plasma membrane of Vero cells.
The efficacy of nanostructured lipid carriers (NLC) for drug delivery strongly depends on their stability and cell uptake. Both properties are governed by their compositions and internal structure. To test the effect of the lipid composition of NLC on cell uptake and stability, three kinds of liquid lipids with different degrees of unsaturation are employed. After ensuring homogeneous size distributions, the thermodynamic characteristics, stability, and mixing properties of NLC are characterized. Then the rates and predominant pathways of cell uptake are determined. Although the same surfactant is used in all cases, different uptake rates are observed. This finding contradicts the view that the surface properties of NLC are dominated by the surfactant. Instead, the uptake rates are explained by the structure of the nanocarrier. Depending on the mixing properties, some liquid lipids remain inside the nanocarrier, while other liquid lipids are present on the surface. Nanocarriers with liquid lipids on the surface are taken up more readily by the cells. This shows that the engineering of efficient lipid nanocarriers requires a delicate balance of interactions between all components of the nanocarrier on the molecular level.
cellular interactions and uptake, coarse-grained molecular dynamics simulations, molecular interactions, nanocarrier structural organization, nanostructured lipid carriers, physical stability
The transition from pregnancy to lactation is characterized by major metabolic, phys-iological, and hormonal changes. In early lactation, dairy cows experience a negative energy balance (NEB) as the energy required for milk synthesis exceeds feed intake. Thus, body reserves from muscle and adipose tissue are mobilized and used for generating energy through various conversion processes in the liver or mammary gland. Excessive body fat mobilization, particularly in over-conditioned dairy cows, negatively affects liver function and is associated with an increased incidence of metabolic diseases including ketosis and fatty liver. Bile acids (BA) are synthesized from cholesterol in the liver, conjugated with the amino acids glycine or taurine, and stored in the gallbladder. To aid in the digestion of die-tary fats and fat-soluble vitamins, they are released into the intestine, where they are modi-fied by the intestinal microbiome before being absorbed through the intestinal mucosa and transported back to the liver via the portal vein. Furthermore, BA are hormone-like signaling molecules found in various tissues, including adipose tissue (AT), being able to trigger re-ceptor-mediated signaling cascades. Changes in BA concentrations, depending on hepatic synthesis, intestinal biotransformation, and tissue metabolism, have been reported to influ-ence glucose-, lipid- and energy metabolism, including body fat content in humans. The aim of this dissertation was to investigate and characterize the influence of lactation-induced and condition-dependent body fat mobilization on the BA metabolism in dairy cows. The exper-imental design included the examination of a feeding-induced (15-7 weeks before calving) high body condition (HBCS, N = 19) and normal body condition (NBCS, N = 19) in multipa-rous cows around calving. Using a targeted metabolomics approach, 20 BA were analyzed in the liver, in subcutaneous adipose tissue (scAT) and in serum at -7, 1, 3, and 12 weeks rela-tive to calving. In addition, the mRNA abundance of BA-associated enzymes, transporters, and receptors were examined in the liver and scAT. This study shows that animals with high body condition had lower concentrations of several BA within the liver, serum, and scAT and an increased hepatic mRNA expression of enzymes involved in an alternative, generally less relevant synthesis pathway. These results suggest that an increased mobilization of AT affects the BA metabolism in dairy cows. With the onset of lactation, BA concentrations increased in both groups, which was accompanied by an increased hepatic expression of the key enzyme CYP7A1, probably an adaptation to the increased energy demand due to milk synthesis. Although BA have been detected in peripheral bovine tissues in other studies, their origin and function remain largely unexplored. We detected BA in scAT; however, en-zymatic conditions (mRNA data) may exclude de novo synthesis. The detection of the mRNA abundance of specific transporters and receptors in scAT suggests that BA may play a role in signaling cascades in scAT. This dissertation provides fundamental insights into bovine BA metabolism during the transition from pregnancy to lactation and characterizes condition-dependent and lactation-induced changes. The transition from gestation to lactation is characterized by metabolic, physiological and hormonal changes. In early lactation, the energy requirement for milk synthesis exceeds the energy areadue to feed intake, which can lead to the cow having a negative energy balance (NEB). Energy reserves are mobilized from fat and muscle tissue and metabolized in the liver and mammary gland, among other places. Excessive mobilization of body fat, which occurs especially in overconditioned dairy cows, can have a negative effect on liver function and is associated with an increased incidence of metabolic diseases such as ketosis or fatty liver. In humans, liver dysfunction has been shown to have an effect on bile acid metabolism. Bile acids (BA) are formed in the liver, conjugated with the amino acids glycine or taurine and stored in the gallbladder. To support the digestion of dietary fats and fat-soluble vitamins, they are released into the intestine and modified by the intestinal microbiome, absorbed via the intestinal mucosa and transported back to the liver via the portal vein. BAs can also act as signaling molecules, receptor-mediated signaling cascades and influence other metabolic processes, including lipid and glucose metabolism. The aim of this thesis was to investigate the influence of lactation-induced and condition-dependent lipolysis on BA metabolism in dairy cows. Cows with feeding-induced (15-7 weeks before calving), high body condition (HBCS, N=19) and normal body condition (NBCS, N=19) in the period around calving (-7, 1, 3 and 12 weeks relative to calving) were examined. Using a targeted metabolomics approach, 20 BAs were measured in the liver, subcutaneous adipose tissue (scAT) and blood serum. In addition, the mRNA of BA-associated enzymes, transporters and receptors in the liver and scAT was investigated. The HBCS animals had a large number of BAs in low concentrations and showed an increased hepatic mRNA expression of enzymes of an alternative, usually less relevant synthesis pathway than the NBCS animals. The results suggest that the increased lipolysis affects bovine BA metabolism. With the onset of lactation, an increase in BA could be observed in both groups, which was accompanied by an increased expression of the key enzyme CYP7A1 and is probably an adaptation to the increasing energy demand due to milk synthesis. Although BA has already been detected in some peripheral tissues in dairy cows, their origin and function are largely unexplored. Our investigations show that BA is present in bovine scAT, whereby complete de novo synthesis can probably be excluded (mRNA data). The detection of mRNA expression of specific transporters and receptors in scAT suggests that BA affect signaling cascades in scAT. The present dissertation provides fundamental insights into the BA metabolism of dairy cows in the transition period between pregnancy and lactation and characterizes condition-dependent changes.
adipose tissue, BCS, bile acids, body condition, dairy cow, liver, transition period
Exposure to Ultraviolet B (UVB) radiation can trigger a diverse array of biological responses that have the potential to contribute to the onset of skin cancer. Natural compounds, such as tea polyphenols, have been shown to protect against UVB-induced damage by modulating oxidative stress, inflammatory response, and cell proliferation. The chemopreventive and anti-inflammatory properties of South African rooibos (Aspalathus linearis) and honeybush (Cyclopia spp.) herbal teas have been shown to mainly target the early stages of cancer development through mechanisms that involve intracellular interleukin-1α (IL-1α) inhibition. Thus, the aim was to investigate the preventive effects of unfermented rooibos and honeybush aqueous extracts against UVB-induced oxidative stress and inflammation in HaCaTs. Honeybush was found to reduce the accumulation of UVB-induced IL-1α while maintaining cell viability and without affecting apoptosis. Furthermore, only honeybush extract was able to decrease the secretion of interleukin-6 (IL-6) caused by UVB exposure. Honeybush and rooibos extracts significantly decreased the secretion of UVB-induced interleukin-8 (IL-8). Except for rooibos extract at a concentration of 0.2 mg/mL, both extracts restored the expression of antioxidant genes to levels observed prior to UVB exposure. The anti-inflammatory effects of these herbal tea extracts are likely attributed to the antioxidant properties of their polyphenolic constituents, which modulate the oxidative stress-induced pathways governing inflammatory responses.
Honeybush, inflammation, Keratinocytes, Oxidative stress, Rooibos, UVB
Automation in sample preparation improves accuracy, productivity, and precision in bioanalysis. Moreover, it reduces resource consumption for repetitive procedures. Automated sample analysis allows uninterrupted handling of large volumes of biological samples originating from preclinical and clinical studies. Automation significantly helps in management of complex testing methods where generation of large volumes of data is required for process monitoring. Compared to traditional sample preparation processes, automated procedures reduce associated expenses and manual error, facilitate laboratory transfers, enhance data quality, and better protect the health of analysts. Automated sample preparation techniques based on robotics potentially increase the throughput of bioanalytical laboratories. Robotic liquid handler, an automated sample preparation system built on a robotic technique ensures optimal laboratory output while saving expensive solvents, manpower, and time. Nowadays, most of the traditional extraction processes are being automated using several formats of online techniques. This review covered most of the automated sample preparation techniques reported till date, which accelerated and simplified the sample preparation procedure for bioanalytical sample analysis. This article critically analyzed different developmental aspects of automated sample preparation techniques based on robotics as well as conventional sample preparation methods that are accelerated using automated technologies.
Automation in Sample Preparation, Bioanalysis
Protein kinase C-iota (PKC-ι) is an oncogene overexpressed in many cancer cells including prostate, breast, ovarian, melanoma, and glioma cells. Previous in vitro studies have shown that 5-amino-1-((1R,2S,3R,4R)-2-3-dihydroxy-4-(hydroxymethyl)cyclopentyl)-1H-imidazole-4-carboxamide (ICA-1S), a PKC-ι-specific inhibitor, has low toxicity in both acute and sub-acute mouse model toxicological testing and is an effective therapeutic against several cancer cell lines showing significant reductions in tumor growth when treating athymic nude mice with xenografted carcinoma cell lines. To further assess ICA-1S as a possible therapeutic agent, chronic mouse model toxicological testing was performed in vivo to provide inferences concerning the long-term effects and possible health hazards from repeated exposure over a substantial part of the animal’s lifespan. Subjects survived well after 30, 60, and 90 days of doses ranging from 50 mg/kg to 100 mg/kg. Heart, liver, kidney, and brain tissues were then analyzed for accumulations of ICA-1S including the measured assessment of aspartate transaminase (AST), alkaline phosphatase (ALK-P), gamma-glutamyl transferase (GGT), troponin, and C-reactive protein (CRP) serum levels to assess organ function. Predictive in vitro/in silico methods were used to predict compound-induced direct hepatocyte toxicity or renal proximal tubular cell (PTC) toxicity in humans based on the high-content imaging (HCI) of compound-treated cells in combination with phenotypic profiling. In conclusion, ICA-1S shows low toxicity in both acute and chronic toxicology studies, and shows promise as a potential therapeutic.
acute and chronic toxicity, PKC-ι, specific inhibition
Bile acids (BA) play a crucial role not only in lipid digestion but also in the regulation of overall energy homeostasis, including glucose and lipid metabolism. The aim of this study was to investigate BA profiles and mRNA expression of BA-related genes in the liver of high versus normal body condition in dairy cows. We hypothesized that body condition and the transition from gestation to lactation affect hepatic BA concentrations as well as the mRNA abundance of BA-related receptors, regulatory enzymes, and transporters. Therefore, we analyzed BA in the liver as well as the mRNA abundance of BA-related synthesizing enzymes, transporters, and receptors in the liver during the transition period in cows with different body conditions around calving. In a previously established animal model, 38 German Holstein cows were divided into groups with high body condition score (HBCS; n = 19) or normal body condition score (NBCS; n = 19) based on BCS and backfat thickness (BFT). Cows were fed diets aimed at achieving the targeted differences in BCS and BFT (NBCS: BCS <3.5, BFT <1.2 cm; HBCS: BCS >3.75, BFT >1.4 cm) until they were dried off at wk 7 before parturition. Both groups were fed identical diets during the dry period and subsequent lactation. Liver biopsies were taken at wk −7, 1, 3, and 12 relative to parturition. For BA measurement, a targeted metabolomics approach with liquid chromatography electrospray ionization MS/MS was used to analyze BA in the liver. The mRNA abundance of targeted genes related to BA synthesizing enzymes, transporters, and receptors in the liver was analyzed using microfluidic quantitative PCR. In total, we could detect 14 BA in the liver: 6 primary and 8 secondary BA, with glycocholic acid (GCA) being the most abundant one. The increase of glycine-conjugated BA after parturition, in parallel to increasing serum glycine concentrations may originate from an enhanced mobilization of muscle protein to meet the high nutritional requirements in early lactating cows. Higher DMI in NBCS cows compared with HBCS cows was associated with higher liver BA concentrations such as GCA, deoxycholic acid, and cholic acid. The mRNA abundance of BA-related enzymes measured herein suggests the dominance of the alternative signaling pathway in the liver of HBCS cows. Overall, BA profiles and BA metabolism in the liver depend on both, the body condition and lactation-induced effects in periparturient dairy cows.
bile acids, body condition, liver, periparturient period
Abnormal trinucleotide repeat expansions alter protein conformation causing malfunction and contribute to a significant number of incurable human diseases. Scarce structural insights available on disease-related homorepeat expansions hinder the design of effective therapeutics. Here, we present the dynamic structure of human PHOX2B C-terminal fragment, which contains the longest polyalanine segment known in mammals. The major α-helical conformation of the polyalanine tract is solely extended by polyalanine expansions in PHOX2B, which are responsible for most congenital central hypoventilation syndrome cases. However, polyalanine expansions in PHOX2B additionally promote nascent homorepeat conformations that trigger length-dependent phase transitions into solid condensates that capture wild-type PHOX2B. Remarkably, HSP70 and HSP90 chaperones specifically seize PHOX2B alternative conformations preventing phase transitions. The precise observation of emerging polymorphs in expanded PHOX2B postulates unbalanced phase transitions as distinct pathophysiological mechanisms in homorepeat expansion diseases, paving the way towards the search of therapeutics modulating biomolecular condensates in central hypoventilation syndrome.