Kaempferol as an Alternative Cryosupplement for Bovine Spermatozoa: Cytoprotective and Membrane-Stabilizing Effects.

Kaempferol (KAE) is a natural flavonoid with powerful reactive oxygen species (ROS) scavenging properties and beneficial effects on ex vivo sperm functionality. In this paper, we studied the ability of KAE to prevent or ameliorate structural, functional or oxidative damage to frozen-thawed bovine spermatozoa. The analysis focused on conventional sperm quality characteristics prior to or following thermoresistance tests, namely the oxidative profile of semen alongside sperm capacitation patterns, and the levels of key proteins involved in capacitation signaling. Semen samples obtained from 30 stud bulls were frozen in the presence of 12.5, 25 or 50 µM KAE and compared to native ejaculates (negative control-CtrlN) as well as semen samples cryopreserved in the absence of KAE (positive control-CtrlC). A significant post-thermoresistance test maintenance of the sperm motility (p < 0.001), membrane (p < 0.001) and acrosome integrity (p < 0.001), mitochondrial activity (p < 0.001) and DNA integrity (p < 0.001) was observed following supplementation with all KAE doses in comparison to CtrlC. Experimental groups supplemented with all KAE doses presented a significantly lower proportion of prematurely capacitated spermatozoa (p < 0.001) when compared with CtrlC. A significant decrease in the levels of the superoxide radical was recorded following administration of 12.5 (p < 0.05) and 25 µM KAE (p < 0.01). At the same time, supplementation with 25 µM KAE in the cryopreservation medium led to a significant stabilization of the activity of Mg2+-ATPase (p < 0.05) and Na+/K+-ATPase (p < 0.0001) in comparison to CtrlC. Western blot analysis revealed that supplementation with 25 µM KAE in the cryopreservation medium prevented the loss of the protein kinase A (PKA) and protein kinase C (PKC), which are intricately involved in the process of sperm activation. In conclusion, we may speculate that KAE is particularly efficient in the protection of sperm metabolism during the cryopreservation process through its ability to promote energy synthesis while quenching excessive ROS and to protect enzymes involved in the process of sperm capacitation and hyperactivation. These properties may provide supplementary protection to spermatozoa undergoing the freeze-thaw process.

Characterization of Bitter Taste Receptor-Dependent Autophagy in Oral Epithelial Cells.

Microbial dysbiosis is an important trigger in the development of oral diseases. Oral keratinocytes or gingival epithelial cells (GECs) offer protection against various microbial insults. Recent studies suggest that GECs expressed higher level of bitter taste receptor 14 (T2R14) compared to other taste receptors and toll-like receptors and act as innate immune sentinels. Macroautophagy or autophagy is a cellular conserved process involved in the regulation of host innate immune responses against microbial infection. Here, we describe a robust method for evaluation of T2R14-dependent autophagy flux in GECs. Autophagy flux was detected using Western blot analysis in GECs and further was confirmed using Acridine Orange-dependent flow cytometry analysis.

Eco-friendly green synthesis of N­pyrazole amino chitosan using PEG-400 as an anticancer agent against gastric cancer cells via inhibiting EGFR.

The present study was conducted to develop a green process that provides access to the development of Schiff base derivatives of chitosan with the heterocyclic moiety as a novel class of anti-gastric cancer agent. In the present study, we have synthesized these derivatives by reacting various pyrazoles with chitosan using CAN in PEG400. The compounds were synthesized in 20 min in excellent yield by using CAN at 5% in PEG400 at 80°C in the shortest reaction time of 20 min. The PEG400 could be efficiently recycled for the three consecutive runs. The developed compounds were tested for EGFR-TK inhibition using a Kinase-Glo Plus luminescence kinase assay kit where they exhibited significant activity revealing compound 2d as the most potent analog, while other compounds showed mild to moderate inhibitory activity. MTT assay was conducted to determine the effect of the three most potent EGFR inhibitors (2b, 2c, and 2d) on the proliferation of gastric cancer cells (SGC-7901). The results showed compound 2d as the most potent anticancer agent against SGC7901 cells. The effect of compound 2d was also quantified on the apoptosis and cell phase of SGC7901 cells using flow cytometry assay at various concentrations ranging from 0, 10, 20, and 30 µM. Results suggest that compound 2d showed significant inhibition of SGC-7901 by inducing apoptosis and arresting G0/G1 cell phase. The western blot analysis also revealed that compound 2d significantly inhibited the overexpression of EGFR in SGC-7901 cells. The study successfully demonstrated the development of N­pyrazole amino chitosan as a novel class of agent against gastric cancer via inhibition of EGFR.

Design, synthesis, in silico and biological evaluation of new indole based oxadiazole derivatives targeting estrogen receptor alpha.

A series of new indole-oxadiazole derivatives was designed and synthesized to develop potential anti-breast cancer agents. The compounds exhibited significant inhibitory activity with IC50 values ranging from 1.78 to 19.74 µM against ER-positive human breast cancer (BC) cell lines T-47D and MCF-7. Among them, compounds (5a, 5c, 5e-5h, 5j-5o) displayed superior activity against ER-α dominant (ratio of ER-α/ER-ß is 9/1) T-47D cells compared to the standard drug bazedoxifene (IC50 = 12.78 ± 0.92 µM). Compounds 5c and 5o exhibited remarkable anti-proliferative activity with IC50 values of 3.24 ± 0.46 and 1.72 ± 1.67 µM against T-47D cells, respectively. Further, compound 5o manifested 1589-fold higher ER-α binding affinity (213.4 pM) relative to bazedoxifene (339.2 nM) in a competitive ER-α binding assay, while compound 5c showed a binding affinity of 446.6 nM. The Western blot analysis proved that both compounds influenced the ER-α protein's expression, impeding its subsequent transactivation and signalling pathway within T-47D cells. Additionally, a molecular docking study suggests that compounds 5c and 5o bind in such a fashion that induces conformational changes in the protein, culminating in their antagonistic effect. Also, pharmacokinetic profiles showed that all compounds have drug-like properties. Further, molecular dynamic (MD) simulations and density functional theory (DFT) analysis confirmed the stability, conformational behaviour, reactivity, and biological feasibility of compounds 5c and 5o. In conclusion, based on our findings, compounds 5c and 5o, which exhibit significant ER-α antagonistic activity, can act as potential lead compounds for developing anti-breast cancer agents.

New biflavonoids isolated from Xylia kerrii leaves extract with selective cytotoxicity against MH7A human rheumatoid arthritis synovial fibroblast cells.

Three new biflavonoids (1-3) and two known flavonoids (4, 5) were isolated from Xylia kerrii collected in Thailand. Compounds 1-5 showed selective cytotoxicity against the rheumatoid fibroblast-like synovial MH7A cell line, and these compounds showed weak cytotoxicity against the human lung synovial fibroblast WI-38 VA13 sub 2 RA cell line. Notably, compound 1 was highly selective toward MH7A cells with an IC50 value of 6.9 µM, whereas the IC50 value for WI-38 VA13 sub 2 RA cells was > 100 µM. The western blotting analysis of MH7A cells treated with compound 1 showed increased CDKN2A /p16INK4A and caspase-8 levels.

Synthesis, characterization and irradiation enhances anticancer activity of liposome-loaded iridium(III) complexes.

Herein, we synthesized and characterized two novel iridium (III) complexes: [Ir(bzq)2(PPD)](PF6) (4a, with bzq = deprotonated benzo[h]quinoline and PPD = pteridino[6,7-f][1,10]phenanthroline-11,13-diamine) and [Ir(piq)2(PPD)](PF6) (4b, with piq = deprotonated 1-phenylisoquinoline). The anticancer efficacy of these complexes, 4a and 4b, was investigated using 3-(4,5-dimethylthiazole)-2,5-diphenltetraazolium bromide (MTT). Complex 4a exhibited no cytotoxic activity, while 4b demonstrated moderate efficacy against SGC-7901, A549, and HepG2 cancer cells. To enhance their anticancer potential, we explored two strategies: (I) light irradiation and (II) encapsulation of the complexes in liposomes, resulting in the formation of 4alip and 4blip. Both strategies significantly increased the ability of 4a, 4b to kill cancer cells. The cellular studies indicated that both the free complexes 4a, 4b and their liposomal forms 4alip and 4blip effectively inhibited cell proliferation. The cell cycle arrest analysis uncovered 4alip and 4blip arresting cell growth in the S period. Additionally, we investigated apoptosis and ferroptosis pathways, observing an increase in malondialdehyde (MDA) levels, a reduction of glutathione (GSH), a down-regulation of GPX4 (glutathione peroxidase) expression, and lipid peroxidation. The effects on mitochondrial membrane potential and intracellular Ca2+ concentrations were also examined, revealing that both light-activated and liposomal forms of 4alip and 4blip caused a decline in mitochondrial membrane potential and an enhancement in intracellular Ca2+ levels. In conclusion, these complexes and them encapsulated liposomes induce cell death through apoptosis and ferroptosis.

Validation of a novel western blot assay to monitor patterns and levels of alpha dystroglycan in skeletal muscle of patients with limb girdle muscular dystrophies.

The cell membrane protein, dystroglycan, plays a crucial role in connecting the cytoskeleton of a variety of mammalian cells to the extracellular matrix. The α-subunit of dystroglycan (αDG) is characterized by a high level of glycosylation, including a unique O-mannosyl matriglycan. This specific glycosylation is essential for binding of αDG to extracellular matrix ligands effectively. A subset of muscular dystrophies, called dystroglycanopathies, are associated with aberrant, dysfunctional glycosylation of αDG. This defect prevents myocytes from attaching to the basal membrane, leading to contraction-induced injury. Here, we describe a novel Western blot (WB) assay for determining levels of αDG glycosylation in skeletal muscle tissue. The assay described involves extracting proteins from fine needle tibialis anterior (TA) biopsies and separation using SDS-PAGE followed by WB. Glycosylated and core αDG are then detected in a multiplexed format using fluorescent antibodies. A practical application of this assay is demonstrated with samples from normal donors and patients diagnosed with LGMD2I/R9. Quantitative analysis of the WB, which employed the use of a normal TA derived calibration curve, revealed significantly reduced levels of αDG in patient biopsies relative to unaffected TA. Importantly, the assay was able to distinguish between the L276I homozygous patients and a more severe form of clinical disease observed with other FKRP variants. Data demonstrating the accuracy and reliability of the assay are also presented, which further supports the potential utility of this novel assay to monitor changes in ⍺DG of TA muscle biopsies in the evaluation of potential therapeutics.

An Unusual Case of Serologically Confirmed Post-Partum Lyme Disease Following an Asymptomatic Borrelia burgdorferi Infection Acquired during Pregnancy and Lacking Vertical Transmission in Utero.

In this report, we describe a 23-year-old female who, while pregnant, was exposed to Borrelia burgdorferi but did not develop significant signs or symptoms (joint pain, arthritis) of Lyme disease until shortly after delivering a healthy child at term. Serologic testing confirmed infection with B. burgdorferi. A 3-week course of treatment with doxycycline was completely curative. There was no evidence for congenital or perinatal transmission of this pathogen at any point pre-term or postnatally. The key reasons that could account for this unique clinical scenario are discussed in the context of previously published related reports.

Seroprevalence of Infection by Borrelia Species Responsible for Lyme Disease in the French Alps: Analysis of 27,360 Serology Tests, 2015-2020.

Objectives: Lyme borreliosis incidence is increasing in several areas; moreover, it has recently gained the public's attention. Apart from erythema migrans, Lyme disease diagnosis relies (among others) on serology test; however, the prevalence of positive enzyme-linked immunosorbent assay (ELISA) and western blot (WB) assay has been poorly studied in the general population. We aimed to approach the seroprevalence of infection by Borrelia species responsible for Lyme disease in the French Isere department using city laboratories data. Patients and Methods: We retrieved all serological tests for Borrelia species responsible for Lyme disease performed in the two main networks of city laboratories between 2015 and 2020. All patients with both ELISA and WB IgG were considered seropositive. Results: We analyzed 27,360 tests (ELISA/ELISA+WB). Mean age was 50.9 ± 20.3 years (ranges: 0-101), with 57.1% females. Overall, 11.7% had IgG detected by ELISA, and 4.7% had IgG detected by both ELISA and WB assay. Seropositive status was more frequent in males (7.0% vs. 2.9%, p < 0.001). Seropositivity rate increased with age after a first peak in childhood; men aged 61-70 years had the highest seropositivity rate (10.3%). In addition, seropositivity rate was higher in persons from a rural area. In multivariate analysis, older age, male gender and living in a rural area were independently associated with seropositivity. Seropositivity rate was stable on the 2017-2020 period. Conclusion: The seroprevalence of infection by Borrelia species responsible for Lyme disease is high in Isere; this probably reduces the predictive positive value for Lyme disease of ELISA and WB IgG, suggesting that this serological test should not be performed for nonspecific symptoms.

Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.

Introduction: Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA. Methods: Aiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses. Results and discussion: For 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

Photosensitive Hydrogel with Temperature-Controlled Reversible Nano-Apertures for Single-Cell Protein Analysis.

Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in-gel probing and separation resolution. Here, a highly sensitive temperature-controlled single-cell western blotting (tc-scWB) method is introduced, which is based on a thermo/photo-dualistic-sensitive polyacrylamide hydrogel, namely acrylic acid-functionalized graphene oxide (AFGO) assisted, N-isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high-temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low-temperature for better antibody penetration into the hydrogel. The tc-scWB method enables the separation and profiling of small-molecule-weight proteins with highly crosslinked gel (12% T) in SDS-PAGE. The tc-scWB is demonstrated on three metabolic and ER stress-specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc-scWB is a general toolbox for the analysis of low-abundance small-molecular functional proteins at the single-cell level.

Development and validation of a chemiluminescent western blot assay for glanders (Burkholderia mallei) serodetection.

Glanders, caused by Burkholderia mallei, is a zoonotic disease of equids. Serologic testing for glanders is required by disease-free countries before international movement of equids. The World Organisation for Animal Health Terrestrial Manual recommends the complement fixation test (CFT) for clearance of individual animals for movement, but the CFT is prone to false-positive results. A colorimetric western blot (WB) assay was developed and validated to resolve false-positive CFT results; however, that assay is relatively time-consuming, and the interpretation is subjective. We present here a procedurally similar chemiluminescent WB assay that performs comparably to the validated colorimetric WB assay and offers noticeable benefits of decreased time-to-result and greater ease of interpretation.

Asymptomatic Leishmania infantum infection in dogs and dog owners in an endemic area in southeast France.

The prevalence of asymptomatic leishmaniasis in dogs and their owners in the main endemic areas of France has not been studied to date. The objective of this study was to quantify asymptomatic Leishmania infantum infection in southeast France in healthy people and their dogs using molecular and serological screening techniques. We examined the presence of parasitic DNA using specific PCR targeting kinetoplast DNA (kDNA) and specific antibodies by serology (ELISA for dogs and Western blot for humans) among immunocompetent residents and their dogs in the Alpes-Maritimes. Results from 343 humans and 607 dogs were included. 46.9% (n = 161/343) of humans and 18.3% (n = 111/607) of dogs were PCR positive; 40.2% of humans (n = 138/343) and 9.9% of dogs (n = 60/607) were serology positive. Altogether, 66.2% of humans (n = 227) and 25.7% of dogs (n = 156) had positive serologies and/or positive PCR test results. Short-haired dogs were more frequently infected (71.8%, n = 112) than long-haired dogs (12.2%, n = 19) (p = 0.043). Dogs seemed to be more susceptible to asymptomatic infection according to their breed types (higher infection rates in scenthounds, gun dogs and herding dogs) (p = 0.04). The highest proportion of dogs and human asymptomatic infections was found in the Vence Region, corresponding to 28.2% (n = 20/71) of dogs and 70.5% (n = 31/44) of humans (4.5/100,000 people). In conclusion, the percentage of infections in asymptomatic humans is higher than in asymptomatic dogs in the studied endemic area. It is questionable whether asymptomatic infection in humans constitutes a risk factor for dogs.

Title: Infection asymptomatique à Leishmania infantum chez les chiens et propriétaires de chiens dans une zone endémique du sud-est de la France. Abstract: La prévalence de la leishmaniose asymptomatique chez les chiens et leurs propriétaires dans les principales zones d'endémie françaises n'a pas été étudiée à ce jour. L'objectif de cette étude était de quantifier l'infection asymptomatique à Leishmania infantum dans le sud-est de la France chez des personnes saines et leurs chiens à l'aide de techniques de dépistage moléculaire et sérologique. Nous avons examiné chez des résidents immunocompétents et leurs chiens dans les Alpes-Maritimes la présence d'ADN parasitaire par PCR spécifique ciblant l'ADN du kinétoplaste (ADNk) et d'anticorps spécifiques par sérologie (ELISA pour le chien et Western Blot pour l'homme). Les résultats de 343 humains et 607 chiens ont été inclus; 46,9 % (n = 161/343) des humains et 18,3 % (n = 111/607) des chiens étaient positifs à la PCR et 40,2 % des humains (n = 138/343) et 9,9 % des chiens (n = 60/607) avaient une sérologie positive. Au total, 66,2 % des humains (n = 227) et 25,7 % des chiens (n = 156) avaient des sérologies positives et/ou des résultats de tests PCR positifs. Les chiens à poils courts étaient plus fréquemment infectés (71,8 %, n = 112) que les chiens à poils longs (12,2 %, n = 19) (p = 0,043). Les chiens semblaient plus sensibles à l'infection asymptomatique selon leurs races (taux supérieurs chez les chiens de chasse et chiens de berger) (p = 0,04). La plus forte proportion d'infections asymptomatiques chez les chiens et les humains a été observée dans la Région de Vence, correspondant à 28,2 % (n = 20/71) des chiens et 70,5 % (n = 31/44) des humains (4,5/100 000). personnes). En conclusion, le pourcentage d'infections chez les humains asymptomatiques est plus élevé que chez les chiens asymptomatiques dans la zone d'endémie étudiée. On peut se demander si une infection asymptomatique chez l'homme constitue un facteur de risque pour les chiens.

Investigation of In Vitro Anti-cancer and Apoptotic Potential of Onion-Derived Nanovesicles Against Prostate and Cervical Cancer Cell Lines.

Plant-derived compounds have recently garnered significant interest in the field of medicine due to their rich repertoire of phytochemicals, which holds promise for exploring novel therapies to treat cancer. This study embarks on the first-time investigation of the anti-cancerous effect of onion-derived nanovesicles (ODNVs). ODNVs were isolated employing differential centrifugation followed by ultracentrifugation and subsequent characterization using dynamic light scattering (DLS), field emission scanning electron microscopy (FESEM), and Fourier transform infrared spectroscopy (FTIR). Furthermore, we delineated the anti-cancerous effect of ODNVs on two cancer cell line models HeLa (cervical cancer) and PC-3 (prostate cancer) using MTT assay, DAPI-based DNA damage using immunofluorescence microscopy, colony formation assay, migration assay, cell cycle analysis, and evaluation of apoptosis using flow cytometry and western blotting. The findings revealed dose- and time-dependent anti-proliferative effects of ODNVs on both HeLa and PC3 cell lines, accompanied by selective cytotoxicity against cancer cells. Additional results highlighted that ODNVs prevented colony growth and induced S-phase cell cycle arrest. Apoptosis induction was evaluated through alterations in nuclear morphology and the number of apoptotic cells, which increased significantly after ODNV treatment in both cancer cell lines. Furthermore, annexin V/PI staining evaluation of apoptotic cells by flow cytometry demonstrated that ODNV treatment significantly increased the number of apoptotic cells in both PC-3 and HeLa cells. Finally, Western blot analysis indicated changes in apoptosis-related proteins including bcl-2, bax, and caspase-3, emphasizing that the anti-cancerous effect of ODNVs is attributed to the induction of apoptosis and suggests the  unexplored anti-cancerous potential of ODNVs.

Targeting antibody-mediated complement-independent mechanism in bullous pemphigoid with diacerein.

BACKGROUND: Bullous pemphigoid (BP) is an antibody-mediated blistering disease predominantly affecting the elderly. The pathogenesis involves both complement-dependent and complement-independent mechanisms. The therapeutic potential of targeting complement-independent mechanism has not yet been determined. The mainstay of treatment, corticosteroid, has many side effects, indicating the needs of better treatments. OBJECTIVE: We tempted to establish an in vitro model of BP which resembles complement-independent mechanism and to examine the therapeutic potential of a novel anti-inflammatory agent, diacerein. METHODS: Cultured HaCaT cells were treated with purified antibodies from BP patients, with or without diacerein to measure the cell interface presence of BP180, protein kinase C, and the production of proinflammatory cytokines. An open-label, randomized, phase 2 trial was conducted to compare topical diacerein and clobetasol ointments in patients with mild-to-moderate BP (NCT03286582). RESULTS: The reduced presentation of BP180 at cell interface after treating with BP autoantibodies was noticed in immunofluorescence and western blotting studies. The phenomenon was restored by diacerein. Diacerein also reduced the autoantibody-induced increase of pro-inflammatory cytokines. Reciprocal changes of BP180 and protein kinase C at the cell interface were found after treating with BP autoantibodies. This phenomenon was also reversed by diacerein in a dose-dependent manner. The phase 2 trial showed that topical diacerein reduced the clinical symptoms which were comparable to those of topical clobetasol. CONCLUSION: Diacerein inhibited BP autoantibody-induced reduction of BP180 and production of proinflammatory cytokines in vitro and showed therapeutic potential in patients with BP. It is a novel drug worthy of further investigations.

Establishment of a Novel Anti-Mouse CCR1 Monoclonal Antibody C1Mab-6.

C-C motif chemokine receptor 1 (CCR1/CD191) is a member of G-protein-coupled receptors and is expressed on myeloid cells, such as neutrophils and macrophages. Because the CCR1 signaling promotes tumor expansion in the tumor microenvironment (TME), the modification of TME is an effective strategy for cancer therapy. Although CCR1 is an attractive target for solid tumors and hematological malignancies, therapeutic agents for CCR1 have not been approved. Here, we established a novel anti-mouse CCR1 (mCCR1) monoclonal antibody (mAb), C1Mab-6 (rat IgG2b, kappa), using the Cell-Based Immunization and Screening method. Flow cytometry and Western blot analyses showed that C1Mab-6 recognizes mCCR1 specifically. The dissociation constant of C1Mab-6 for mCCR1-overexpressed Chinese hamster ovary-K1 was determined as 3.9 × 10-9 M, indicating that C1Mab-6 possesses a high affinity to mCCR1. These results suggest that C1Mab-6 could be a useful tool for targeting mCCR1 in preclinical mouse models.

Western blotting: evolution of an old analytical method to a new quantitative tool for biomarker measurements.

Western blotting (WB) is a widely used laboratory technique for detecting specific proteins in biological matrices. Recent advances in antibody production, automation, gel and membrane manufacturing and highly sensitive detection platforms have transformed WB from a labor-intensive and qualitative method into a highly reproducible and quantitative assay suitable for biomarker detection. Despite these significant improvements in the capabilities and efficiency of WB, there remain challenges that hinder its widespread application as a research, diagnostic (in two-tiered assays like Lyme disease testing) and drug development tool. This article describes recent innovations introduced to WB methodology and the remaining challenges that prevent its wider adoption for biomarker measurements throughout the drug development process.

Differential responses to UCP1 ablation in classical brown versus beige fat, despite a parallel increase in sympathetic innervation.

In the cold, the absence of the mitochondrial uncoupling protein 1 (UCP1) results in hyper-recruitment of beige fat, but classical brown fat becomes atrophied. Here we examine possible mechanisms underlying this phenomenon. We confirm that in brown fat from UCP1-knockout (UCP1-KO) mice acclimated to the cold, the levels of mitochondrial respiratory chain proteins were diminished; however, in beige fat, the mitochondria seemed to be unaffected. The macrophages that accumulated massively not only in brown fat but also in beige fat of the UCP1-KO mice acclimated to cold did not express tyrosine hydroxylase, the norepinephrine transporter (NET) and monoamine oxidase-A (MAO-A). Consequently, they could not influence the tissues through the synthesis or degradation of norepinephrine. Unexpectedly, in the cold, both brown and beige adipocytes from UCP1-KO mice acquired an ability to express MAO-A. Adipose tissue norepinephrine was exclusively of sympathetic origin, and sympathetic innervation significantly increased in both tissues of UCP1-KO mice. Importantly, the magnitude of sympathetic innervation and the expression levels of genes induced by adrenergic stimulation were much higher in brown fat. Therefore, we conclude that no qualitative differences in innervation or macrophage character could explain the contrasting reactions of brown versus beige adipose tissues to UCP1-ablation. Instead, these contrasting responses may be explained by quantitative differences in sympathetic innervation: the beige adipose depot from the UCP1-KO mice responded to cold acclimation in a canonical manner and displayed enhanced recruitment, while the atrophy of brown fat lacking UCP1 may be seen as a consequence of supraphysiological adrenergic stimulation in this tissue.

Histological evaluation of monopolar and bipolar radiofrequency microneedling treatment in a porcine model.

BACKGROUND AND OBJECTIVE: Fractional radiofrequency microneedling (FRM) is widely used as an option for skin rejuvenation, however there is a lack of histological evidence for the various energy delivery systems available. The objective was to assess thermal denaturation of tissue and the wound healing response in monopolar mode versus bipolar mode. Histological analysis was performed to demonstrate the efficacy of automatic impedance feedback system in monopolar mode. STUDY DESIGN AND METHODS: In this study, the acute thermal effects caused by monopolar FRM treatment to the dorsal skin of pigs were assessed histologically by hematoxylin & eosin (H&E) staining. Then, one session of either monopolar or bipolar FRM was used to treat one or the other side of the pig using varying power levels and pulse widths. The acute and chronic tissue reactions were assessed using H&E, immunofluorescence, and western blot analysis at 0, 14, 30, and 90 days after treatment. The efficacy of the impedance feedback system was also monitored histologically. RESULTS: High-energy FRM treatment produced tissue loss and necrosis. The power level and pulse duration significantly affected the coagulation amount. Histopathology at 0, 14, 30, and 90 days showed that the skin tissue reaction was more pronounced for bipolar compared to monopolar FRM. Immunofluorescence showed the expression of TGF-ß, Ki67, MMP3, and elastin increased dramatically with both modes, but were higher in the bipolar FRM treated side. The automatic impedance feedback system could effectively adjust the output energy. CONCLUSIONS: We found that bipolar FRM produced greater thermal effects, more collagen coagulation, and more pronounced molecular changes compared with monopolar mode in a porcine animal model.

Novel Pyrimidine-5-Carbonitriles as potential apoptotic and antiproliferative agents by dual inhibition of EGFRWT/T790M and PI3k enzymes; Design, Synthesis, biological Evaluation, and docking studies.

A new series of 6-(4-fluorophenyl)-2-(methylthio) pyrimidine-5-carbonitrile derivatives were designed and synthesized as EGFR/PI3K dual inhibitors, and potential antiproliferative agents. The new 22 compounds were screened by DTP-NCI against all NCI60 cell lines. Almost all compounds showed cytotoxic activity. Compound 7c showed a promising antitumour activity on CNS cancer (SNB-75), and ovarian cancer (OVAR-4) with IC50 < 0.01, and 0.64 µM, respectively. Fortunately, 7c exhibited a better safety profile on normal cells (WI-38) than doxorubicin by 2.2-fold. Compound 7c displayed selective inhibitory activity on EGFRt790m over EGFRWT with IC50 = 0.08, and 0.13 µM, respectively, wherefore it might overcome EGFR-TKIs resistance. In addition to its remarkable inhibitory activity on all PI3K isoforms, specifically PI3K-δ with IC50 = 0.64 µM Compared with LY294002 IC50 = 7.6 µM. Compound 7c arrested the cell cycle of SNB-75 & OVAR-4 at the G0-G1 phase coupled with apoptosis induction. The western blotting analysis approved decreasing the expression level of p-AKT coupled with an increase in Casp3, Casp9, and BAX proteins in the SNB-75 & OVAR-4 after being treated with 7c which may support the suggested mechanism of action of 7c as EGFR/PI3K dual inhibitor. Physicochemical parameters were forecasted using SwissADME online tool. MD showed the interaction of 7c with the crucial amino acids of the active domain of both EGFR/PI3K which may explain its potent inhibitory activities. In vivo study disclosed a significant decrease in tumor weight and the number of nodules in the group of mice treated with 7c compared with the control group.