Search

Understanding the responses of olive trees to drought stress is crucial for improving cultivation and developing drought-tolerant varieties. Water transport and storage within the plant is a key factor in drought-tolerance strategies. Water management can be based on a variety of factors such as stomatal control, osmoprotectant molecules, proteins and wood properties. The aim of the study was to evaluate the water management strategy under drought stress from an anatomical and biochemical point of view in three young Italian olive cultivars (Giarraffa, Leccino and Maurino) previously distinguished for their physiological and metabolomic responses. For each cultivar, 15 individuals in pots were exposed or not to 28 days of water withholding. Every 7 days, the content of sugars (including mannitol), proline, aquaporins, osmotins, and dehydrins, in leaves and stems, as well as the chemical and anatomical characteristics of the wood of the three cultivars, were analyzed. 'Giarraffa' reduced glucose levels and increased mannitol production, while 'Leccino' accumulated more proline. Both 'Leccino' and 'Maurino' increased sucrose and aquaporin levels, possibly due to their ability to remove embolisms. 'Maurino' and 'Leccino' accumulated more dehydrins and osmotins. While neither genotype nor stress affected wood chemistry, 'Maurino' had a higher vessel-to-xylem area ratio and a larger hydraulic diameter, which allows it to maintain a high transpiration rate but may make it more susceptible to cavitation. The results emphasized the need for an integrated approach, highlighting the importance of the relative timing and sequence of each parameter analyzed, allowing, overall, to define a "strategy" rather than a "response" to drought of each cultivar. [ABSTRACT FROM AUTHOR]

Melanin naturally exists in organisms and is synthetized by tyrosinase (TYR); however, its over-production may lead to aberrant pigmentation and skin conditions. Loquat (Eriobotrya japonica (Thunb.) Lindl.) flowers contain a variety of bioactive compounds, while studies on their suppressive capabilities against melanin synthesis are limited. Loquat flower isolate product (LFP) was obtained by ethanol extraction and resin purification, and its inhibitory efficiency against TYR activity was investigated by enzyme kinetics and multiple spectroscopy analyses. In addition, the impact of LFP on melanin synthesis-related proteins' expression in mouse melanoma B16 cells was analyzed using Western blotting. HPLC-MS/MS analysis indicated that LFP was composed of 137 compounds, of which 12 compounds, including flavonoids (quercetin, isorhamnoin, p-coumaric acid, etc.) and cinnamic acid and its derivatives, as well as benzene and its derivatives, might have TYR inhibitory activities. LFP inhibited TYR activity in a concentration-dependent manner with its IC50 value being 2.8 mg/mL. The inhibition was an anti-competitive one through altering the enzyme's conformation rather than chelating copper ions at the active center. LFP reduced the expression of TYR, tyrosinase-related protein (TRP) 1, and TRP2 in melanoma B16 cells, hence inhibiting the synthesis of melanin. The research suggested that LFP had the potential to reduce the risks of hyperpigmentation caused by tyrosinase and provided a foundation for the utilization of loquat flower as a natural resource in the development of beauty and aging-related functional products. [ABSTRACT FROM AUTHOR]

Delivering nucleic acid therapeutics across cell membranes is a significant challenge. Cell-penetrating peptides (CPPs) containing arginine (R), tryptophan (W), and histidine (H) show promise for siRNA delivery. To improve siRNA delivery and silence a model STAT3 gene, we hypothesized that oleyl acylation to CPPs, specifically (WRH)n, would enhance STAT3 silencing efficiency in breast and ovarian cancer cells. Using Fmoc/tBu solid-phase peptide chemistry, we synthesized, purified, and characterized the oleyl-conjugated (WRH)n (n = 1–4) peptides. The peptide/siRNA complexes were non-cytotoxic at N/P 40 (~20 μM) against MDA-MB-231, MCF-7, SK-OV-3, and HEK-293 cells after 72 h incubation. All peptide/siRNA complexes showed serum stability at N/P ≥ 40. The synthesized conjugates, with a diameter of <100 nm, formed nano-complexes with siRNA and exhibited a stable range of zeta potential values (13–18 mV at N/P = 40). Confocal microscopy and flow cytometry analysis provided qualitative and quantitative evidence of a successful cellular internalization of siRNA. The peptides oleyl-(WRH)3 and oleyl-(WRH)4 showed ~60% and ~75% cellular uptake of siRNA, respectively, in both MDA-MB-231 and SK-OV-3 cells. Western blot analysis of oleyl-(WRH)4 demonstrated effective silencing of the STAT-3 gene, with ~75% silencing in MDA-MB-231 cells and ~45% in SK-OV-3 cells. [ABSTRACT FROM AUTHOR]

In the pathological process of Alzheimer's disease, neuronal cell death is closely related to the accumulation of reactive oxygen species. Our previous studies have found that oxidative stress can activate microtubule affinity-regulating kinases, resulting in elevated phosphorylation levels of tau protein specifically at the Ser262 residue in N1E-115 cells that have been subjected to exposure to hydrogen peroxide. This process may be one of the pathogenic mechanisms of Alzheimer's disease. Vitamin E is a fat-soluble, naturally occurring antioxidant that plays a crucial role in biological systems. This study aimed to examine the probable processes that contribute to the inhibiting effect on the abnormal phosphorylation of tau protein and the neuroprotective activity of a particular type of vitamin E, α-tocotrienol. The experimental analysis revealed that α-tocotrienol showed significant neuroprotective effects in the N1E-115 cell line. Our data further suggest that one of the mechanisms underlying the neuroprotective effects of α-tocotrienol may be through the inhibition of microtubule affinity-regulated kinase activation, which significantly reduces the oxidative stress-induced aberrant elevation of p-Tau (Ser262) levels. These results indicate that α-tocotrienol may represent an intriguing strategy for treating or preventing Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The utility of serum glial fibrillary acidic protein (GFAP) in acute ischemic stroke (AIS) has been extensively studied in recent years. Here, we aimed to assess its potential role as a cargo protein of extracellular vesicles (EVs) secreted by astrocytes (ADEVs) in response to brain ischemia. Plasma samples from eighteen AIS patients at 24 h (D1), 7 days (D7), and one month (M1) post-symptoms onset, and nine age, sex, and cardiovascular risk factor-matched healthy controls were obtained to isolate EVs using the Exoquick ULTRA EV kit. Subsets of presumed ADEVs were identified further by the expression of the glutamate aspartate transporter (GLAST) as a specific marker of astrocytes with the Basic Exo-Flow Capture kit. Western blotting has tested the presence of GFAP in ADEV cargo. Post-stroke ADEV GFAP levels were elevated at D1 and D7 but not M1 compared to controls (p = 0.007, p = 0.019, and p = 0.344, respectively). Significant differences were highlighted in ADEV GFAP content at the three time points studied (n = 12, p = 0.027) and between D1 and M1 (z = 2.65, p = 0.023). A positive correlation was observed between the modified Rankin Scale (mRS) at D7 and ADEV GFAP at D1 (r = 0.58, p = 0.010) and D7 (r = 0.57, p = 0.013), respectively. ADEV GFAP may dynamically reflect changes during the first month post-ischemia. Profiling ADEVs from peripheral blood could provide a new way to assess the central nervous system pathology. [ABSTRACT FROM AUTHOR]

Hong Kong oysters (Crassostrea hongkongensis) are an important marine bivalve with nutritional and commercial value. The expanded off-bottom farming scale in recent years makes the oysters more susceptible to exposure to abiotic stresses, such as salinity stress, an important environmental factor that has been proven to have significant effects on oyster growth and development. However, the molecular mechanism is still unclear. Cyclin E is an important protein in the process of cell cycle regulation that is indispensable for propelling G1/S phase transition in a dose-dependent manner. In order to investigate whether the salinity stress affects cyclin E expression in oysters, the cDNA sequence of C. hongkongensis cyclin E (Ch-CCNE) was isolated from a gill cDNA library, and the 2.8 kbp length cDNA fragment contained a complete open reading frame (ORF) encoding 440 amino acid residues. Ch-CCNE mRNA was highly expressed in the gonad and low in the adductor mussel, mantle, gill, labial palp, and digestive gland. The recombinant CCNE protein was expressed and purified in a pET32a(+)-CCNE/Escherichia coli BL21(DE3) system via IPTG induction and was used for generating mice anti-Ch-CCNE antiserums. Western blot analysis showed that the CCNE protein in the gill was maintained at low expression levels under either hypo- (5 ppt) or hyper- (35 ppt) salinity, and could be produced at high levels under appropriate salinity during a 10-day exposure period. The immuno-localization indicated that the Ch-CCNE protein was distributed in the nucleus. These results suggested that either hypo- or hyper-salinity stress could inhibit the CCNE expression of Hong Kong oysters and their negative impact on cell division and proliferation. [ABSTRACT FROM AUTHOR]

One of the emerging trends in modern analytical and bioanalytical chemistry involves the substitution of enzyme labels (such as horseradish peroxidase) with nanozymes (nanoparticles possessing enzyme-like catalytic activity). Since enzymes and nanozymes typically operate through different catalytic mechanisms, it is expected that optimal reaction conditions will also differ. The optimization of substrates for nanozymes usually focuses on determining the ideal pH and temperature. However, in some cases, even this step is overlooked, and commercial substrate formulations designed for enzymes are utilized. This paper demonstrates that not only the pH but also the composition of the substrate buffer, including the buffer species and additives, significantly impact the analytical signal generated by nanozymes. The presence of enhancers such as imidazole in commercial substrates diminishes the catalytic activity of nanozymes, which is demonstrated herein through the use of 3,3′-diaminobenzidine (DAB) and Prussian Blue as a model chromogenic substrate and nanozyme. Conversely, a simple modification to the substrate buffer greatly enhances the performance of nanozymes. Specifically, in this paper, it is demonstrated that buffers such as citrate, MES, HEPES, and TRIS, containing 1.5–2 M NaCl or NH4Cl, substantially increase DAB oxidation by Prussian Blue and yield a higher signal compared to commercial DAB formulations. The central message of this paper is that the optimization of substrate composition should be an integral step in the development of nanozyme-based assays. Herein, a step-by-step optimization of the DAB substrate composition for Prussian Blue nanozymes is presented. The optimized substrate outperforms commercial formulations in terms of efficiency. The effectiveness of the optimized DAB substrate is affirmed through its application in several commonly used immunostaining techniques, including tissue staining, Western blotting assays of immunoglobulins, and dot blot assays of antibodies against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Detection of erythropoietin (Epo) was difficult until a method was developed by the World Anti-Doping Agency (WADA). WADA recommended the Western blot technique using isoelectric focusing (IEF)-PAGE to show that natural Epo and injected erythropoiesis-stimulating agents (ESAs) appear in different pH areas. Next, they used sodium N-lauroylsarcosinate (SAR)-PAGE for better differentiation of pegylated proteins, such as epoetin β pegol. Although WADA has recommended the use of pre-purification of samples, we developed a simple Western blotting method without pre-purification of samples. Instead of pre-purification, we used deglycosylation of samples before SDS-PAGE. The double detection of glycosylated and deglycosylated Epo bands increases the reliability of the detection of Epo protein. All of the endogenous Epo and exogenous ESAs shift to 22 kDa, except for Peg-bound epoetin β pegol. All endogenous Epo and exogenous ESAs were detected as 22 kDa deglycosylated Epo by liquid chromatography/mass spectrum (LC/MS) analysis. The most important factor for the detection of Epo is the selection of the antibody against Epo. WADA recommended clone AE7A5, and we used sc-9620. Both antibodies are useful for the detection of Epo protein by Western blotting. [ABSTRACT FROM AUTHOR]

Aucubin is an iridoid glycoside that displays various pharmacological actions including antioxidant activity. However, there are few reports available on the neuroprotective effects of aucubin against ischemic brain injury. Thus, the aim of this study was to investigate whether aucubin protected against damage to hippocampal function induced by forebrain ischemia-reperfusion injury (fIRI) in gerbils, and to examine whether aucubin produced neuroprotection in the hippocampus against fIRI and to explore its mechanisms by histopathology, immunohistochemistry, and Western analysis. Gerbils were given intraperitoneal injections of aucubin at doses of 1, 5, and 10 mg/kg, respectively, once a day for seven days before fIRI. As assessed by the passive avoidance test, short-term memory function following fIRI significantly declined, whereas the decline in short-term memory function due to fIRI was ameliorated by pretreatment with 10 mg/kg, but not 1 or 5 mg/kg, of aucubin. Most of the pyramidal cells (principal cells) of the hippocampus died in the Cornu Ammonis 1 (CA1) area four days after fIRI. Treatment with 10 mg/kg, but not 1 or 5 mg/kg, of aucubin protected the pyramidal cells from IRI. The treatment with 10 mg/kg of aucubin significantly reduced IRI-induced superoxide anion production, oxidative DNA damage, and lipid peroxidation in the CA1 pyramidal cells. In addition, the aucubin treatment significantly increased the expressions of superoxide dismutases (SOD1 and SOD2) in the pyramidal cells before and after fIRI. Furthermore, the aucubin treatment significantly enhanced the protein expression levels of neurotrophic factors, such as brain-derived neurotrophic factor and insulin-like growth factor-I, in the hippocampal CA1 area before and after IRI. Collectively, in this experiment, pretreatment with aucubin protected CA1 pyramidal cells from forebrain IRI by attenuating oxidative stress and increasing neurotrophic factors. Thus, pretreatment with aucubin can be a promising candidate for preventing brain IRI. [ABSTRACT FROM AUTHOR]

Previous studies demonstrated that enzymatic hydrolysis enhances wheat bran (WB) biological properties. This study evaluated the immunostimulatory effect of a WB hydrolysate (HYD) and a mousse enriched with HYD (MH) before and after in vitro digestion on murine and human macrophages. The antiproliferative activity of the harvested macrophage supernatant on colorectal cancer (CRC) cells was also analyzed. MH showed significantly higher content than control mousse (M) in soluble poly- and oligosaccharides (OLSC), as well as total soluble phenolic compounds (TSPC). Although in vitro gastrointestinal digestion slightly reduced the TSPC bioaccessibility of MH, ferulic acid (FA) levels remained stable. HYD showed the highest antioxidant activity followed by MH, which demonstrated a greater antioxidant activity before and after digestion as compared with M. RAW264.7 and THP-1 cells released the highest amounts of pro-inflammatory cytokines after being treated with 0.5 mg/mL of digested WB samples. Treatment with digested HYD-stimulated RAW264.7 supernatant for 96 h showed the most anticancer effect, and spent medium reduced cancer cell colonies more than direct WB sample treatments. Although a lack of inner mitochondrial membrane potential alteration was found, increased Bax:Bcl-2 ratio and caspase-3 expression suggested activation of the mitochondrial apoptotic pathway when CRC cells were treated with macrophage supernatants. Intracellular reactive oxygen species (ROS) were positively correlated with the cell viability in CRC cells exposed to RAW264.7 supernatants (r = 0.78, p < 0.05) but was not correlated in CRC cells treated with THP-1 conditioned media. Supernatant from WB-stimulated THP-1 cells may be able to stimulate ROS production in HT-29 cells, leading to a decrease of viable cells in a time-dependent manner. Therefore, our present study revealed a novel anti-tumour mechanism of HYD through the stimulation of cytokine production in macrophages and the indirect inhibition of cell proliferation, colony formation, and activation of pro-apoptotic proteins expression in CRC cells. [ABSTRACT FROM AUTHOR]

Traditional Western blotting is one of the most used analytical techniques in biological research. However, it can be time-consuming and suffer from a lack of reproducibility. Consequently, devices with different degrees of automation have been developed. These include semi-automated techniques and fully automated devices that replicate all stages downstream of the sample preparation, including sample size separation, immunoblotting, imaging, and analysis. We directly compared traditional Western blotting with two different automated systems, iBind™ Flex, which is a semi-automated system designed to perform the immunoblotting, and JESS Simple Western™, a fully automated and capillary-based system performing all steps downstream of sample preparation and loading, including imaging and image analysis. We found that a fully automated system can save time and importantly offer valuable sensitivity. This is particularly beneficial for limited sample amounts. The downside of automation is the cost of devices and reagents. Nevertheless, automation can be a good option to increase output and facilitate sensitive protein analyses. [ABSTRACT FROM AUTHOR]

Amyloidosis refers to a clinically heterogeneous group of disorders characterized by the extracellular deposition of amyloid proteins in various tissues of the body. To date, 42 different amyloid proteins that originate from normal precursor proteins and are associated with distinct clinical forms of amyloidosis have been described. Identification of the amyloid type is essential in clinical practice, since prognosis and treatment regimens both vary according to the particular amyloid disease. However, typing of amyloid protein is often challenging, especially in the two most common forms of amyloidosis, i.e., the immunoglobulin light chain amyloidosis and transthyretin amyloidosis. Diagnostic methodology is based on tissue examinations as well as on noninvasive techniques including serological and imaging studies. Tissue examinations vary depending on the tissue preparation mode, i.e., whether it is fresh-frozen or fixed, and they can be carried out by ample methodologies including immunohistochemistry, immunofluorescence, immunoelectron microscopy, Western blotting, and proteomic analysis. In this review, we summarize current methodological approaches used for the diagnosis of amyloidosis and discusses their utility, advantages, and limitations. Special attention is paid to the simplicity of the procedures and their availability in clinical diagnostic laboratories. Finally, we describe new methods recently developed by our team to overcome limitations existing in the standard assays used in common practice. [ABSTRACT FROM AUTHOR]

Estrogens have important roles in endometrial cancer (EC) and exert biological effects through the classical estrogen receptors (ERs) ERα and ERβ, and the G-protein–coupled ER, GPER. So far, the co-expression of these three types of ERs has not been studied in EC. We investigated ERα, ERβ, GPER mRNA and protein levels, and their intracellular protein distributions in EC tissue and in adjacent control endometrial tissue. Compared to control endometrial tissue, immunoreactivity for ERα in EC tissue was weaker for nuclei with minor, but unchanged, cytoplasmic staining; mRNA and protein levels showed decreased patterns for ERα in EC tissue. For ERβ, across both tissue types, the immunoreactivity was unchanged for nuclei and cytoplasm, although EC tissues again showed lower mRNA and protein levels compared to adjacent control endometrial tissue. The immunoreactivity of GPER as well as mRNA levels of GPER were unchanged across cancer and control endometrial tissues, while protein levels were lower in EC tissue. Statistically significant correlations of estrogen receptor α (ESR1) versus estrogen receptor β (ESR2) and GPER variant 3,4 versus ESR1 and ESR2 was seen at the mRNA level. At the protein level studied with Western blotting, there was significant correlation of ERα versus GPER, and ERβ versus GPER. While in clinical practice the expression of ERα is routinely tested in EC tissue, ERβ and GPER need to be further studied to examine their potential as prognostic markers, provided that specific and validated antibodies are available. [ABSTRACT FROM AUTHOR]

The ameliorative effect of medicarpin (MC) was investigated by animal behavioral experiments such as Morris water maze (MWM), Y-maze, and passive avoidance test (PAT), using scopolamine-induced cognitively impaired mice. The scopolamine (5 mg/kg), donepezil (5 mg/kg), and MC (5 and 15 mg/kg) were administered by intraperitoneal injection at a volume of 0.3 mL. In the MWM, the escape latency times of MC-treated groups were significantly decreased compared with the scopolamine-treated negative control, and times spent in the platform zone of MC-treated groups were increased dose-dependently. In the Y-maze, the zone alternations of the MC-treated group were increased to the level of the donepezil-treated positive control. In the PAT, the crossing times of MC-treated groups were significantly higher than those of the negative control with dose-dependency. On the other hand, the monoamine oxidase (MAO)-A, MAO-B, and acetylcholinesterase (AChE) activities, relating to cognitive functions, in hippocampus treated with MC were decreased. In addition, the AChE activity in SH-SY5Y cells was significantly decreased. In Western blots, phosphorylated cyclic adenosine monophosphate (cAMP) response element-binding protein (p-CREB), brain-derived neurotrophic factor (BDNF), phosphorylated protein kinase B (p-Akt), and dopamine D2 receptor (D2R) levels in the hippocampus were higher than those of the negative control. In addition, p-CREB, BDNF, p-Akt, and D2R levels in SH-SY5Y cells treated with MC were significantly increased. These results showed that MC ameliorated a cognitive function along with increased BDNF and D2R expressions, and they suggested that MC could be used for the treatment of neurological disorders such as Alzheimer's disease and Parkinson's disease. [ABSTRACT FROM AUTHOR]

The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spawned an ongoing demand for new research reagents and interventions. Herein we describe a panel of monoclonal antibodies raised against SARS-CoV-2. One antibody showed excellent utility for immunohistochemistry, clearly staining infected cells in formalin-fixed and paraffin embedded lungs and brains of mice infected with the original and the omicron variants of SARS-CoV-2. We demonstrate the reactivity to multiple variants of concern using ELISAs and describe the use of the antibodies in indirect immunofluorescence assays, Western blots, and rapid antigen tests. Finally, we illustrate the ability of two antibodies to reduce significantly viral tissue titers in K18-hACE2 transgenic mice infected with the original and an omicron isolate of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

In vivo biotinylation using wild-type and mutants of biotin ligases is now widely applied for the study of cellular proteomes. The commercial availability of kits for the highly efficient purification of biotinylated proteins and their excellent compatibility with LC-MS/MS protocols are the main reasons for the choice of biotin ligases. Since they are all enzymes, however, just a very low expression in cells is required for experiments. Therefore, it can be difficult to perform the quantifications of these enzymes in various samples. Traditional methods, such as western blotting, are not always fit for the detection of the expression levels. Therefore, real-time qRT-PCR, a technology that is more sensitive, was used in this study to quantify the expression of BirA fusions. Using this method, we detected high expression levels of BirA fusions in models of interactions of pluripotency transcription factors to carry out their relative quantification. We also found the absence of the competing endogenous proteins SOX2 and OCT4, as well as no cross-reactivity between BAP/BirA and the endogenous biotinylation system in HEK293T cells. Thus, these data indicated that the high level of biotinylation is due to the in vivo interaction of BAP-X and BirA-Y (X,Y = SOX2, OCT4) in the cell rather than their random collision, a big difference in the expression level of BirA fusions across samples or endogenous biotinylation. [ABSTRACT FROM AUTHOR]

Absence seizures are hyperexcitations within the cortico-thalamocortical (CTC) network, however the underlying causative mechanisms at the cellular and molecular level are still being elucidated and appear to be multifactorial. Dysfunctional feed-forward inhibition (FFI) is implicated as one cause of absence seizures. Previously, we reported altered excitation onto parvalbumin-positive (PV+) interneurons in the CTC network of the stargazer mouse model of absence epilepsy. In addition, downstream changes in GABAergic neurotransmission have also been identified in this model. Our current study assessed whether dysfunctional FFI affects GABAA receptor (GABAAR) subunit expression in the stargazer primary somatosensory cortex (SoCx). Global tissue expression of GABAAR subunits α1, α3, α4, α5, β2, β3, γ2 and δ were assessed using Western blotting (WB), while biochemically isolated subcellular fractions were assessed for the α and δ subunits. We found significant reductions in tissue and synaptic expression of GABAAR α1, 18% and 12.2%, respectively. However, immunogold-cytochemistry electron microscopy (ICC-EM), conducted to assess GABAAR α1 specifically at synapses between PV+ interneurons and their targets, showed no significant difference. These data demonstrate a loss of phasic GABAAR α1, indicating altered GABAergic inhibition which, coupled with dysfunctional FFI, could be one mechanism contributing to the generation or maintenance of absence seizures. [ABSTRACT FROM AUTHOR]

Simple Summary: Human papillomaviruses (HPV) are one of the deadliest viruses causing cancer resulting in over 300,000 deaths annually in the world. HPV16 is the most oncogenic form of HPV and is responsible for over half of all HPV-driven tumors. The viral E7 gene encodes a powerful oncoprotein that can cause infected cells to have uncontrolled growth. A previous study showed that HPV16 isolates with amino acid alterations in the E7 gene were nearly always found in women with HPV infection but no cancer. To study the role of these variants, we expressed them in cultured cells. Most variants produced lower levels of protein, and selected variants had reduced activity in allowing cells to grow in assays that measure tumor cell invasiveness. The data indicate that inhibition of E7 could be a practical approach to treating HPV-driven cancers. The human papillomavirus (HPV) type 16 E7 oncogene is critical to carcinogenesis and highly conserved. Previous studies identified a preponderance of non-synonymous E7 variants amongst HPV16-positive cancer-free controls compared to those with cervical cancer. To investigate the function of E7 variants, we constructed full-length HPV16 E7 genes and tested variants at positions H9R, D21N, N29S, E33K, T56I, D62N, S63F, S63P, T64M, E80K, D81N, P92L, and P92S (found only in controls); D14E, N29H cervical intraepithelial neoplasia (CIN2), and P6L, H51N, R77S (CIN3). We determined the steady-state level of cytoplasmic and nuclear HPV16 E7 protein. All variants from controls showed a reduced level of E7 protein, with 7/13 variants having lower protein levels. In contrast, 2/3 variants from the CIN3 precancer group had near-wild type E7 levels. We assayed the activity of representative variants in stably transfected NIH3T3 cells. The H9R, E33K, P92L, and P92S variants found in control subjects had lower transforming activity than D14E and N29H variants (CIN2), and the R77S (CIN3) had activity only slightly reduced from wild-type E7. In addition, R77S and WT E7 caused increased migration of NIH3T3 cells in a wound-healing assay compared with H9R, E33K, P92L, and P92S (controls) and D14E (CIN2). These data provide evidence that the E7 variants found in HPV16-positive cancer-free women are partially defective for transformation and cell migration, further demonstrating the importance of fully active E7 in cancer development. [ABSTRACT FROM AUTHOR]

Wheat allergies are potentially life-threatening because of the high risk of anaphylaxis. Wheats belong to four genotypes represented in thousands of lines and varieties. Monitoring changes to wheat allergens is critical to prevent inadvertent ntroduction of hyper-allergenic varieties via breeding. However, validated methods for this purpose are unavailable at present. As a proof-of-concept study, we tested the hypothesis that salt-soluble wheat allergens in our mouse model will be identical to those reported for humans. Groups of Balb/cJ mice were rendered allergic to durum wheat salt-soluble protein extract (SSPE). Using blood from allergic mice, a mini hyper-IgE plasma bank was created and used in optimizing an IgE Western blotting (IEWB) to identify IgE binding allergens. The LC-MS/MS was used to sequence the allergenic bands. An ancient Aegilops tauschii wheat was grown in our greenhouse and extracted SSPE. Using the optimized IEWB method followed by sequencing, the cross-reacting allergens in A. tauschii wheat were identified. Database analysis showed all but 2 of the durum wheat allergens and all A. tauschii wheat allergens identified in this model had been reported as human allergens. Thus, this model may be used to identify and monitor potential changes to salt-soluble wheat allergens caused by breeding. [ABSTRACT FROM AUTHOR]

Simple Summary: The quality of rapeseed (Brassica napus) oil depends mainly on the degree of hydrocarbon chain saturation of fatty acids. In previous studies, seeds of the B. napus near-isogenic lines were used as raw materials by iTRAQ analysis at 20–35 days after pollination, and three differential proteins related to oleic acid metabolisms were found to be related to acetylation modification. Thus, we analyzed lysine acetylation using the same raw materials. The function of the corresponding genes of fatty acid metabolisms-related differential proteins was verified by identifying the overexpression of BnACP363K, BnACP363R, and BnACP363 in Arabidopsis thaliana. The results show that the acetylation modification of BnaACP3 may have a selective effect on oleic acid and slow down the conversion of oleic acid to linoleic acid. This is the first report of oleic acid synthesis regulation by acetylation. In this study, lysine acetylation analysis was conducted using two Brassica napus near-isogenic lines, HOCR and LOCR, containing high and low oleic acid contents, respectively, to explore this relationship. Proteins showing differences in quantitative information between the B. napus lines were identified in lysine acetylation analysis, and KEGG pathways were analyzed, yielding 45 enriched proteins, most of which are involved in carbon fixation in photosynthetic organisms, photosynthesis, ascorbate and aldarate metabolism, and glycolysis. Potential key genes related to fatty acid metabolisms were determined. To further explore the effect of acetylation modification on fatty acid metabolisms, the acyl-ACP3 related gene BnaACP363K was cloned, and a base mutation at No.63 was changed via overlapping primer PCR method. This study is the first to demonstrate that acetylation modification can regulate oleic acid metabolisms, which provides a promising approach for the study of the molecular mechanism of oleic acid in rapeseed. [ABSTRACT FROM AUTHOR]

Recent approvals of siRNA-based products motivated the scientific community to explore siRNA as a treatment option for several intractable ailments, especially cancer. The success of approved siRNA therapy requires a suitable and safer drug delivery agent. Herein, we report a series of oleyl conjugated histidine–arginine peptides as a promising nonviral siRNA delivery tool. The conjugated peptides were found to bind with the siRNA at N/P ratio ≥ 2 and demonstrated complete protection for the siRNA from early enzymatic degradation at N/P ratio ≥ 20. Oleyl-conjugated peptide -siRNA complexes were found to be noncytotoxic in breast cancer cells (MCF-7 and MDA-MB-231) and normal breast epithelial cells (MCF 10A) at N/P ratio of ~40. The oleyl-R3-(HR)4 and oleyl-R4-(HR)4 showed ~80-fold increased cellular uptake in MDA-MB-231 cells at N/P 40. Moreover, the conjugated peptides-siRNA complexes form nanocomplexes (~115 nm in size) and have an appropriate surface charge to interact with the cell membrane and cause cellular internalization. Furthermore, this study provides a proof-of-concept that oleyl-R5-(HR)4 can efficiently silence STAT-3 gene (~80% inhibition) in MDA-MB-231 cells with similar effectiveness to Lipofectamine. Further exploration of this approach holds a great promise in discovering a successful in vivo siRNA delivery agent with a favorable pharmacokinetic profile. [ABSTRACT FROM AUTHOR]

α-Synuclein (α-syn) is a protein involved in neuronal degeneration. However, the family of synucleins has recently been demonstrated to be involved in the mechanisms of oncogenesis by selectively accelerating cellular processes leading to cancer. Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal human cancers, with a specifically high neurotropism. The molecular bases of this biological behavior are currently poorly understood. Here, α-synuclein was analyzed concerning the protein expression in PDAC and the potential association with PDAC neurotropism. Tumor (PDAC) and extra-tumor (extra-PDAC) samples from 20 patients affected by PDAC following pancreatic resections were collected at the General Surgery Unit, University of Pisa. All patients were affected by moderately or poorly differentiated PDAC. The amount of α-syn was compared between tumor and extra-tumor specimen (sampled from non-affected neighboring pancreatic areas) by using in situ immuno-staining with peroxidase anti-α-syn immunohistochemistry, α-syn detection by using Western blotting, and electron microscopy by using α-syn-conjugated immuno-gold particles. All the methods consistently indicate that each PDAC sample possesses a higher amount of α-syn compared with extra-PDAC tissue. Moreover, the expression of α-syn was much higher in those PDAC samples from tumors with perineural infiltration compared with tumors without perineural infiltration. [ABSTRACT FROM AUTHOR]

Leishmaniasis is a heterogeneous group of neglected tropical diseases with various clinical syndromes, which is caused by obligate intracellular protozoa of the genus Leishmania and transmitted by the bite of a female phlebotomine sandfly. Humans and several animal species are considered as reservoirs of the disease. Among other animal species, dogs are the most important reservoirs in a domestic environment, maintaining the endemic focus of the parasite. The behavior of the disease progression and the clinical symptoms of the disease in the infected dog is mainly associated with depressed cellular immunity and strong humoral response. This study aimed to assess the role of Western blotting in the analysis of the idiotype expression of the two main immunoglobulins (IgG1 and IgG2) in dogs that are naturally infected with Leishmania infantum (L. infantum) and treated with N-methyl meglumine antimoniate. Interestingly, for the first time, our study identified several L. infantum antigen polypeptides (14, 31, 33, 49, 64, 66, 99, and 169 kDa) that more frequently stimulate an immune reaction in recovered dogs after treatment, whereas in the non-recovered group of dogs, four antigen polypeptides of L. infantum with molecular weights of 31, 49, 66, and 115 kDa with unfavorable prognosis were identified. Clearly, these interesting findings confirm the strong association between the detected immunodominant bands and the successful recovery in treated dogs that can be used for differentiating the treated dogs from the untreated dogs, as well as the markers of a favorable or unfavorable prognosis and, as a consequence, the prediction of the clinical outcome of the disease. Likewise, these data could be helpful in the implementation of novel vaccines from the detected antigens. [ABSTRACT FROM AUTHOR]

Ca2+ regulation in equine muscle is important for horse performance, yet little is known about this species-specific regulation. We reported recently that horse encode unique gene and protein sequences for the sarcoplasmic reticulum (SR) Ca2+-transporting ATPase (SERCA) and the regulatory subunit sarcolipin (SLN). Here we quantified gene transcription and protein expression of SERCA and its inhibitory peptides in horse gluteus, as compared to commonly-studied rabbit skeletal muscle. RNA sequencing and protein immunoblotting determined that horse gluteus expresses the ATP2A1 gene (SERCA1) as the predominant SR Ca2+-ATPase isoform and the SLN gene as the most-abundant SERCA inhibitory peptide, as also found in rabbit skeletal muscle. Equine muscle expresses an insignificant level of phospholamban (PLN), another key SERCA inhibitory peptide expressed commonly in a variety of mammalian striated muscles. Surprisingly in horse, the RNA transcript ratio of SLN-to-ATP2A1 is an order of magnitude higher than in rabbit, while the corresponding protein expression ratio is an order of magnitude lower than in rabbit. Thus, SLN is not efficiently translated or maintained as a stable protein in horse muscle, suggesting a non-coding role for supra-abundant SLN mRNA. We propose that the lack of SLN and PLN inhibition of SERCA activity in equine muscle is an evolutionary adaptation that potentiates Ca2+ cycling and muscle contractility in a prey species domestically selected for speed. [ABSTRACT FROM AUTHOR]

Mycoplasma bovis is associated with several clinical syndromes of cattle. Currently, limited information is available on the sensitivity (Se) and specificity (Sp) of serological assays used for the detection of M. bovis-specific antibodies. Consequently, it is difficult to critically evaluate the outcomes of studies that use these assays. Therefore, the current study used bovine sera sourced from M. bovis exposure studies from three countries to estimate the Se and Sp of two commercial M. bovis enzyme-linked immunosorbent assays (ELISA), BIO K302 and BIO K260, and Western blotting. Western blotting had the highest Se estimate of 74% (95% confidence interval (CI): 16-98%), compared to the BIO K302: 47% (95% CI: 10-87%) and BIO K260: 28% (95% CI: 1-92%). However, for Sp, the BIO K302: 96% (95% CI: 87-99%) and the BIO K260: 100% (95% CI: 93-100%) out-performed Western blotting: 88% (95% CI: 56-98%). Western blotting was the best assay for detecting seroconversion, correctly identifying 61% (95% CI: 29-86%) of exposed animals compared to 35% for BIO K302 (95% CI: 21-54%) and 8% for BIO K260 (95% CI: 0-87%). While none of the methods assessed had high Se and Sp, the availability of these estimates will aid in the interpretation of studies that use these assays. The results of this study highlight the difficulties encountered when using serology to detect exposure to M. bovis in cattle. [ABSTRACT FROM AUTHOR]

Reference proteins and biomarkers are important for the quantitative evaluation of protein abundance. Chlamydomonas reinhardtii was grown under five stress conditions (dark, cold, heat, salt, and glucose supplementation), and the OD750 and total protein contents were evaluated on days 0, 1, 2, 4, and 6 of culture. Antibodies for 20 candidate proteins were generated, and the protein expression patterns were examined by western blotting. Reference protein(s) for each treatment were identified by calculating the Pearson's correlation coefficient (PCC) between target protein abundance and total protein content. Histone H3, beta tubulin 1 (TUB-1), ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RBCL), and mitochondrial F1F0 ATP synthase subunit 6 (ATPs-6) were the top reference proteins, because they were expressed stably under multiple stress conditions. The average relative-fold change (ARF) value of each protein was calculated to identify biomarkers. Heat shock protein 90B (HSP90B), flagellar associated protein (FAP127) and ATP synthase CF0 A subunit (ATPs-A) were suitable biomarkers for multiple treatments, while receptor of activated protein kinase C1 (RCK1), biotin carboxylase (BCR1), mitochondrial phosphate carrier protein (MPC1), and rubisco large subunit N-methyltransferase (RMT1) were suitable biomarkers for the dark, cold, heat, and glucose treatments, respectively [ABSTRACT FROM AUTHOR]

Previously, we described prostate cancer (PCa) detection (83% sensitivity; 67% specificity) in seminal plasma by CE-MS/MS. Moreover, advanced disease was distinguished from organ-confined tumors with 80% sensitivity and 82% specificity. The discovered biomarkers were naturally occurring fragments of larger seminal proteins, predominantly semenogelin 1 and 2, representing endpoints of the ejaculate liquefaction. Here we identified proteases putatively involved in PCa specific protein cleavage, and examined gene expression and tissue protein levels, jointly with cell localization in normal prostate (nP), benign prostate hyperplasia (BPH), seminal vesicles and PCa using qPCR, Western blotting and confocal laser scanning microscopy. We found differential gene expression of chymase (CMA1), matrix metalloproteinases (MMP3, MMP7), and upregulation of MMP14 and tissue inhibitors (TIMP1 and TIMP2) in BPH. In contrast tissue protein levels of MMP14 were downregulated in PCa. MMP3/TIMP1 and MMP7/TIMP1 ratios were decreased in BPH. In seminal vesicles, we found low-level expression of most proteases and, interestingly, we also detected TIMP1 and low levels of TIMP2. We conclude that MMP3 and MMP7 activity is different in PCa compared to BPH due to fine regulation by their inhibitor TIMP1. Our findings support the concept of seminal plasma biomarkers as non-invasive tool for PCa detection and risk stratification. [ABSTRACT FROM AUTHOR]

Elevated blood concentrations of homocysteine have been well established as a risk factor for cardiovascular diseases and neuropsychiatric diseases, yet the etiologic relationship of homocysteine to these disorders remains poorly understood. Protein N-homocysteinylation has been hypothesized as a contributing factor; however, it has not been examined globally owing to the lack of suitable detection methods. We recently developed a selective chemical method to label N-homocysteinylated proteins with a biotin-aldehyde tag followed by Western blotting analysis, which was further optimized in this study. We then investigated the variation of protein N-homocysteinylation in plasma from rats on a vitamin B12 deficient diet. Elevated "total homocysteine" concentrations were determined in rats with a vitamin B12 deficient diet. Correspondingly, overall levels of plasma protein N-homocysteinylation displayed an increased trend, and furthermore, more pronounced and statistically significant changes (e.g., 1.8-fold, p-value: 0.03) were observed for some individual protein bands. Our results suggest that, as expected, a general metabolic correlation exists between "total homocysteine" and N-homocysteinylation, although other factors are involved in homocysteine/homocysteine thiolactone metabolism, such as the transsulfuration of homocysteine by cystathionine β-synthase or the hydrolysis of homocysteine thiolactone by paraoxonase 1 (PON1), may play more significant or direct roles in determining the level of N-homocysteinylation. [ABSTRACT FROM AUTHOR]

Maternal protein components change markedly during mammalian oogenesis. Many of these proteins have yet to be characterized and verified. In this study, a proteomics approach was used to evaluate changes in proteins during oogenesis in the Swamp Buffalo (Bubalus bubalis). Proteins from 500 immature oocytes and 500 in vitro matured oocytes were subjected to two-dimensional electrophoresis, and more than 400 spots were detected. Image analysis indicated that 17 proteins were differentially expressed between the two groups. Eight proteins were identified by mass spectrometry. In mature oocytes, three proteins were down-regulated: major vault protein (MVP), N-acetyllactosaminide β-1,6-N-acetylglucosaminyl-transferase (GCNT-2), and gem-associated protein (GEMIN)8, whereas five other proteins, heat shock protein (HSP)60, Ras-responsive element-binding protein 1 (RREB-1), heat shock cognate 71 kDa protein (HSC71), hemoglobin subunit α (HBA), and BMP-2-inducible protein kinase (BMP-2K), were up-regulated. The expression profiles of HSP60 and GEMIN8 were further verified by Western blotting. The changes in HSP60 protein expression demonstrate the increasing need for mitochondrial protein importation to facilitate macromolecular assembly during oocyte maturation. The down-regulation of GEMIN8 production implies that RNA splicing is impaired in mature oocytes. [ABSTRACT FROM AUTHOR]

Since 2007, resonant coupling wireless power transfer (WPT) technology has been attracting attention and has been widely researched for practical use. Moreover, dosimetric evaluation has also been discussed to evaluate the potential health risks of the electromagnetic field from this WPT technology based on the International Commission on Non-Ionizing Radiation Protection (ICNIRP) guidelines. However, there has not been much experimental evaluation of the potential health risks of this WPT technology. In this study, to evaluate whether magnetic resonant coupling WPT induces cellular stress, we focused on heat shock proteins (Hsps) and determined the expression level of Hsps 27, 70 and 90 in WI38VA13 subcloned 2RA human fibroblast cells using a western blotting method. The expression level of Hsps under conditions of magnetic resonant coupling WPT for 24 h was not significantly different compared with control cells, although the expression level of Hsps for cells exposed to heat stress conditions was significantly increased. These results suggested that exposure to magnetic resonant coupling WPT did not cause detectable cell stress. [ABSTRACT FROM AUTHOR]

Toll-like receptors (TLRs) not only form an important part of the innate immune system but also serve to activate the adaptive immune system in response to cancer. Real-time PCR; immunohistochemical stain and Western blotting analyses were performed to clarify molecular alterations in colorectal cancer (CRC) patients. We identified Toll-like receptor 1 (TLR1), TLR2, TLR4 and TLR8 gene expression levels and downstream gene, i.e., interleukin-6 (IL-6), IL-8, interferon-α (IFN-α) and myeloid differentiation primary-response protein-88 (MyD88), expression levels in CRC patients and in cancer cell lines. CRC tissues have higher TLR1, TLR2, TLR4, TLR8, IL-6 and IL-8 gene expression levels than do the normal colon mucosa (p < 0.05). TLR2 expression varied in different cell types (mucosa and lymphocytes). There was no difference in the MyD88 and IFN-α gene expression levels between cancerous and normal colon mucosa. CRC patients had higher levels of IL-6 (p = 0.002) and IL-8 (p = 0.038) expression than healthy volunteers did; and higher IL-6 and IL-8 expression was also found to signify a higher risk of recurrence. CL075 (3M002) treatments can reduce the production of IL-8 in different cancer cell lines. The signaling pathway of TLRs in cancer tissue is different from that in normal cells; and is MyD88-independent. Higher expression levels of TLR1, TLR2, TLR 4 and TLR 8 mRNA were related to upregulation inflammatory cytokines IL-6 and IL-8 gene expression in tissue and to the upregulation of IL-6 in blood. The concentration of IL-6 in serum can be used as an indicator of the possibility of CRC recurrence. Treatment with 3M002 can reduce IL-6 production in vitro and may prevent CRC recurrence. Our findings provide evidence that TLR1, TLR2, TLR4 and TLR8 gene expression induce downstream IL-6 and IL-8 gene expression; detection of these expression levels can serve as a CRC marker. [ABSTRACT FROM AUTHOR]

The global coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spawned an ongoing demand for new research reagents and interventions. Herein we describe a panel of monoclonal antibodies raised against SARS-CoV-2. One antibody showed excellent utility for immunohistochemistry, clearly staining infected cells in formalin-fixed and paraffin embedded lungs and brains of mice infected with the original and the omicron variants of SARS-CoV-2. We demonstrate the reactivity to multiple variants of concern using ELISAs and describe the use of the antibodies in indirect immunofluorescence assays, Western blots, and rapid antigen tests. Finally, we illustrate the ability of two antibodies to reduce significantly viral tissue titers in K18-hACE2 transgenic mice infected with the original and an omicron isolate of SARS-CoV-2.

Previously we have shown that 3,6-dihydroxyflavone (3,6-DHF) is a potent agonist of the human peroxisome proliferator-activated receptor (hPPAR) with cytotoxic effects on human cervical cancer cells. To date, the mechanisms by which 3,6-DHF exerts its antitumor effects on cervical cells have not been clearly defined. Here, we demonstrated that 3,6-DHF exhibits a novel antitumor activity against HeLa cells with IC50 values of 25 μM and 9.8 μM after 24 h and 48 h, respectively. We also showed that the anticancer effects of 3,6-DHF are mediated via the toll-like receptor (TLR) 4/CD14, p38 mitogen-activated protein kinase (MAPK), Jun-N terminal kinase (JNK), extracellular-signaling regulated kinase (ERK), and cyclooxygenase (COX)-2 pathways in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. We found that 3,6-DHF showed a similar IC50 (113 nM) value to that of the JNK inhibitor, SP600125 (IC50 = 118 nM) in a JNK1 kinase assay. Binding studies revealed that 3,6-DHF had a strong binding affinity to JNK1 (1.996 × 105 M-1) and that the 6-OH and the carbonyl oxygen of the C ring of 3,6-DHF participated in hydrogen bonding interactions with the carbonyl oxygen and the amide proton of Met111, respectively. Therefore, 3,6-DHF may be a candidate inhibitor of JNKs, with potent anticancer effects. [ABSTRACT FROM AUTHOR]

This study investigated the effects of cathepsin L on proteolysis of beef myofibrillar proteins in vivo and in vitro. Results indicated that cathepsin L affected the degradation of desmin and troponin-T during postmortem aging, and the extent of degradation increased from 1 d to 14 d postmortem. No detectable degradation of titin, nebulin, and α-actinin in the presence of cathepsin L inhibitor was observed during postmortem aging. In vitro, cathepsin L affected the degradation of titin, nebulin, and troponin-T, and the extent of degradation increased with increasing incubation time. Nevertheless, cathepsin L did not cause the degradation of α-actinin and desmin, regardless of incubation temperature. The different results between in vitro and in vivo experiments might mainly depend on different treatment temperatures. Overall, these results indicated that cathepsin L participated in the degradation of myofibrillar proteins and meat tenderization. [ABSTRACT FROM AUTHOR]

Sweet pepper is susceptible to changes in the environmental conditions, especially temperatures below 15 °C. In this work, two sets of pepper fruits (Capsicum annuum L.) which underwent distinct temperature profiles in planta were investigated. Accordingly, two harvesting times corresponding to each set were established: Harvest 1, whose fruits developed and ripened at 14.9 °C as average temperature; and Harvest 2, with average temperature of 12.4 °C. The oxidative metabolism was analyzed in all fruits. Although total ascorbate content did not vary between Harvests, a shift from the reduced to the oxidized form (dehydroascorbate), accompanied by a higher ascorbate peroxidase activity, was observed in Harvest 2 with respect to Harvest 1. Moreover, a decrease of the ascorbate-generating enzymatic system, the γ-galactono-lactone dehydrogenase, was found at Harvest 2. The activity values of the NADP-dependent dehydrogenases analyzed seem to indicate that a lower NADPH synthesis may occur in fruits which underwent lower temperature conditions. In spite of the important changes observed in the oxidative metabolism in fruits subjected to lower temperature, no oxidative stress appears to occur, as indicated by the lipid peroxidation and protein oxidation profiles. Thus, the antioxidative systems of pepper fruits seem to be involved in the response against temperature changes. [ABSTRACT FROM AUTHOR]

The Mustang, Musculoskeletal Temporally Activated Novel-1 Gene (MUSTN1) plays an important role in regulating musculoskeletal development in mammals. We evaluated the developmental and tissue-specific regulation of MUSTN1 mRNA and protein abundance in Erlang Mountainous (EM) chickens. Results indicated that MUSTN1 mRNA/protein was expressed in most tissues with especially high expression in heart and skeletal muscle. The MUSTN1 protein localized to the nucleus in myocardium and skeletal muscle fibers. There were significant differences in mRNA and protein abundance among tissues, ages and between males and females. In conclusion, MUSTN1 was expressed the greatest in skeletal muscle where it localized to the nucleus. Thus, in chickens MUSTN1 may play a vital role in muscle development. [ABSTRACT FROM AUTHOR]

In this study, the anticancer activity of chamaejasmine was characterized in the human breast cancer cell line, MDA-MB-231. Cell viability and cell cycle distribution were determined by MTT assay and flow cytometry, respectively. Western blotting was performed to determine changes in levels of various proteins. Results showed that treatment with chamaejasmine (4-16 μM) inhibited cell proliferation, which correlated with G2/M phase arrest and apoptosis in MDA-MB-231 cells. Chamaejasmine treatment of MDA-MB-231 cells resulted in induction of WAF1/p21 and KIP1/p27, decrease in cyclins A and cyclins B1. Cyclin-dependent kinase (cdk) 2 and cdc2 was also decreased after chamaejasmine treatment. Moreover, inhibition of nuclear translocation, phosphorylation of NF-κB, activation of IKKα and IKKβ, inhibition of phosphorylation and degradation of IκBα were also detected in this work. Our findings suggested that chamaejasmine could be explored as a preventive and perhaps as a chemotherapeutic agent in the management of breast cancer. [ABSTRACT FROM AUTHOR]

Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons). Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4) using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes. [ABSTRACT FROM AUTHOR]

The aggregation of proteins into insoluble amyloid fibrils coincides with the onset of numerous diseases. An array of techniques is available to study the different stages of the amyloid aggregation process. Recently, emphasis has been placed upon the analysis of oligomeric amyloid species, which have been hypothesized to play a key role in disease progression. This paper reviews techniques utilized to study aggregation of the amyloid-β protein (Aβ) associated with Alzheimer's disease. In particular, the review focuses on techniques that provide information about the size or quantity of oligomeric Aβ species formed during the early stages of aggregation, including native-PAGE, SDS-PAGE, Western blotting, capillary electrophoresis, mass spectrometry, fluorescence correlation spectroscopy, light scattering, size exclusion chromatography, centrifugation, enzyme-linked immunosorbent assay, and dot blotting. [ABSTRACT FROM AUTHOR]

Endometrial cancer (EC) is the most frequent gynaecologic cancer in postmenopausal women. We used 2D-DIGE and mass spectrometry to identify candidate biomarkers in endometrial cancer, analysing the serum protein contents of 10 patients versus 10 control subjects. Using gel-based proteomics, we identified 24 candidate biomarkers, considering only spots with a fold change in volume percentage ≥ 1.5 or intensity change ≤ 0.6, which were significantly different between cases and controls (p < 0.05). We used Western blotting analysis both in the serum and tissue of 43 patients for data validation. Among the identified proteins, we selected Suprabasin (SBSN), an oncogene previously associated with poor prognosis in different cancers. SBSN principal isoforms were subjected to Western blotting analysis in serum and surgery-excised tissue: both isoforms were downregulated in the tissue. However, in serum, isoform 1 was upregulated, while isoform 2 was downregulated. Data-mining on the TCGA and GTEx projects, using the GEPIA2.0 interface, indicated a diminished SBSN expression in the Uterine Corpus Endometrial Cancer (UCEC) database compared to normal tissue, confirming proteomic results. These results suggest that SBSN, specifically isoform 2, in tissue or serum, could be a potential novel biomarker in endometrial cancer. [ABSTRACT FROM AUTHOR]

Anemia is a major complication of chronic renal failure. To treat this anemia, prolylhydroxylase domain enzyme (PHD) inhibitors as well as erythropoiesis-stimulating agents (ESAs) have been used. Although PHD inhibitors rapidly stimulate erythropoietin (Epo) production, the precise sites of Epo production following the administration of these drugs have not been identified. We developed a novel method for the detection of the Epo protein that employs deglycosylation-coupled Western blotting. With protein deglycosylation, tissue Epo contents can be quantified over an extremely wide range. Using this method, we examined the effects of the PHD inhibitor, Roxadustat (ROX), and severe hypoxia on Epo production in various tissues in rats. We observed that ROX increased Epo mRNA expression in both the kidneys and liver. However, Epo protein was detected in the kidneys but not in the liver. Epo protein was also detected in the salivary glands, spleen, epididymis and ovaries. However, both PHD inhibitors (ROX) and severe hypoxia increased the Epo protein abundance only in the kidneys. These data show that, while Epo is produced in many tissues, PHD inhibitors as well as severe hypoxia regulate Epo production only in the kidneys. [ABSTRACT FROM AUTHOR]

Resveratrol, a phenolic compound possessing vital biological activities such as anti-cancer, is present abundantly in grape skin, a waste produced during the processing of grape juice. The objectives of this study were to prepare resveratrol-gold nanoparticles and a resveratrol nanoemulsion from grape skin and study their inhibition effects on pancreatic cancer cells BxPC-3. The spherical-shaped citrate gold nanoparticles (GNPs) and resveratrol-gold nanoparticles (R-GNPs) were, respectively, prepared with a surface plasmon resonance peak at 528 and 538 nm, mean particle size of 20.8 and 11.9 nm, and zeta-potential at −32.7 and −66.7 mV, by controlling an appropriate concentration of citrate/resveratrol and gold chloride as well as stirring time and temperature. The resveratrol nanoemulsion, composed of soybean oil, Tween 80, and sucrose fatty acid ester in glycerol and water, possessed a high storage stability with a mean particle size of 14.1 nm, zeta-potential of −49.7 mV, and encapsulation efficiency of 95.5%. An antiproliferation study revealed that both R-GNPs and resveratrol nanoemulsion could effectively inhibit the growth of pancreatic cancer cells BxPC-3, with the latter showing a higher inhibition effect. Western blot analysis implied that both can down-regulate expressions of cyclin A, cyclin B, CDK1, and CDK2 and up-regulate expressions of p53 and p21, accompanied by enhancing cytochrome C expression, decreasing BcL-2 expression, increasing Bax expression, and leading to the elevation of caspase-8, caspase-9, and caspase-3 activities for cell apoptosis execution. Future research is needed to study the inhibition of pancreatic tumors in vivo by R-GNPs and resveratrol nanoemulsions. [ABSTRACT FROM AUTHOR]

Actively involved in tumor maintenance, cAMP-dependent protein kinase A (PKA) has been proposed as a putative biomarker in cancer. Recently, an active PKA form has been identified in human sera and PKA autoantibodies have been detected in cancer patients. However, their serum functions, as well as diagnostic significance, remain largely unknown. Although several PKA detection assays have been developed, none refer to a laboratory diagnostic procedure. Among these, ELISA and Western blotting (WB) assays have been employed in PKA detection. Since, to the best of our knowledge, there are no data showing its presence in human urine samples, herein, we explore the possibility of PKA's existence in this biological specimen. Interestingly, among the 30 screened urines by quantitative sandwich ELISA, we recognized detectable PKA levels in 5 different samples, and of those two exhibited a considerable high concentration. To corroborate these results, we also evaluated PKA's presence in both positive and negative ELISA urines by WB. Remarkably, immunoblotting analysis confirmed PKA's existence in certain, but not in all, human urine specimens. Despite being quite preliminary, these findings firstly identify PKA in urine samples and provide evidence for its potential clinic usage as a diagnostic analyte in laboratory medicine. [ABSTRACT FROM AUTHOR]

The kidney is a main site of erythropoietin production in the body. We developed a new method for the detection of Epo protein by deglycosylation-coupled Western blotting. Detection of deglycosylated Epo enables the examination of small changes in Epo production. Using this method, we investigated the effects of angiotensin II (ATII) on Epo production in the kidney. ATII stimulated the plasma Epo concentration; Epo, HIF2α, and PHD2 mRNA expression in nephron segments in the renal cortex and outer medulla; and Epo protein expression in the renal cortex. In situ hybridization and immunohistochemistry revealed that ATII stimulates Epo mRNA and protein expression not only in proximal tubules but also in collecting ducts, especially in intercalated cells. These data support the regulation of Epo production in the kidney by the renin–angiotensin–aldosterone system (RAS). [ABSTRACT FROM AUTHOR]

Bioactive plant compounds and extracts are of special interest for the development of pharmaceuticals. Here, we describe the screening of more than 1100 aqueous plant extracts and synthetic reference compounds for their ability to stimulate or inhibit insulin secretion. To quantify insulin secretion in living MIN6 β cells, an insulin–Gaussia luciferase (Ins-GLuc) biosensor was used. Positive hits included extracts from Quillaja saponaria, Anagallis arvensis, Sapindus mukorossi, Gleditsia sinensis and Albizia julibrissin, which were identified as insulin secretion stimulators, whereas extracts of Acacia catechu, Myrtus communis, Actaea spicata L., Vaccinium vitis-idaea and Calendula officinalis were found to exhibit insulin secretion inhibitory properties. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) were used to characterize several bioactive compounds in the selected plant extracts, and these bioactives were retested for their insulin-modulating properties. Overall, we identified several plant extracts and some of their bioactive compounds that may be used to manipulate pancreatic insulin secretion. [ABSTRACT FROM AUTHOR]