Hasil untuk "Biochemistry"

Bartosz Orzechowski, Jan Miciński, Katarzyna Ząbek et al.

With the aim to investigate the immunomodulatory potential of selenitetriglycerides (SeTG), a new lipophilic Se (IV) compound, 30 sheep (15 sheep/treatment) were used in a completely random design to receive the SeGT supplement as follows: (1) no SeGT supplement (Control) and (2) daily dosage of 2 mL of SeGT (equivalent to 1 mg Se/kg BW) during the first 7 days of the evaluation, which lasted 28 d. Individually, blood samples were collected on days 0, 14, and 28 to measure and assess parameters of innate cellular and humoral immunity, including respiratory burst activity (RBA) and potential killing activity (PKA) of monocytes and granulocytes, proliferative response of lymphocytes stimulated with ConA (Concovalin A) and LPS (lipopolisaccharidde), lysozyme activity, ceruloplasmin activity, and gamma globulin levels. From the 14th day, supplemental SeTG saw significant increases (<i>p</i> ≤ 0.001) in RBA and PKA parameters, as well as enhanced proliferative responses of lymphocytes compared with controls. Both innate humoral immunity (elevated lysozyme activity) and adaptive humoral immunity (increased gamma globulin levels) were positively influenced (<i>p</i> ≤ 0.01), whereas ceruloplasmin activity remained unchanged. Under the conditions in which the current experiment was carried out, SeGT showed good promise to modulate immunity in a short period (28 d). Further research should explore experiments with a greater number of animals over long-term periods of evaluation under production system conditions.

Lourdes Hontecillas-Prieto, Daniel J. García-Domínguez, Carlos Jiménez-Cortegana et al.

Abstract Background Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin's lymphoma worldwide and is characterized by its heterogeneity. Although first-line therapy improves survival outcomes for DLBCL patients, approximately one third will relapse, often with a poor prognosis. Among the factors influencing prognosis and response to treatment in cancer patients, including those with lymphoma, overweight and obesity have emerged as significant considerations. However, the role of excess weight in DLBCL remains controversial, with studies reporting both negative and positive effects on cancer outcomes. In this translational substudy of the R2-GDP-GOTEL trial, we have evaluated the impact of excess weight as a predictor of treatment response and survival in patients with relapsed/refractory (R/R) DLBCL, and examining its relationship with immune cell dynamics. Methods Of the 79 patients who received the R2-GDP scheme in the phase II trial, weight and height parameters were obtained in 75 patients before starting treatment. Blood samples were analyzed by flow cytometry. Statistical analyses were performed to determine the prognostic value of overweight and obesity at baseline in R/R DLBCL patients. Results Our results indicate that overweight (including obese) patients exhibit longer survival compared to patients of ideal weight. This group also demonstrated a reduction of regulatory T cells with supposedly protumor activity and an increase of Natural Killer (NK)-like T cells with supposedly antitumor activity. Additionally, we have found that excess weight correlates with better treatment response, associated with elevated levels of vitamin D and CD8 + NK cells. Conclusions Our findings suggest that excess weight does not exacerbate the progression of DLBCL. Instead, it appears to confer a survival advantage and improve treatment response, with the immune system playing a possible pivotal role in mediating these effects. Trial registration EudraCT, ID:2014–001620-29.

Bakul Akter, Silvia Aishee, Abdullah Hridoy et al.

Etoricoxib (ETC), a selective cyclooxygenase enzyme (COX-2) inhibitor, is widely utilized to manage pain and inflammation. Nevertheless, its therapeutic efficacy is limited by poor aqueous solubility, low bioavailability, and significant cardiovascular risks, including increased blood pressure, thrombosis, and the potential for myocardial infarction. This study aimed to address these limitations through structural modifications of etoricoxib. A total of 21 derivatives were designed by introducing various functioning sets at the R3, R2, and R1 sites of ETC. Quantum chemical calculations were performed to assess alterations in physicochemical properties, such as HOMO–LUMO energy gaps, electrostatic potential, enthalpy, and dipole moments. Notably, most of the derivatives showed improved binding affinities, particularly ETC9 and ETC19, demonstrating the highest binding interactions in molecular docking studies (-10.1 and -10.8 kcal/mol, respectively). Furthermore, molecular dynamics (MD) simulations accomplished by exploiting the YASARA dynamics software program with the AMBER14 energy field throughout 100 ns revealed that the ETC9 and ETC19 derivatives exhibited enhanced stability and flexibility profiles compared to the parent drug, ETC. ADMET and PASS predictions confirmed the drug-like properties of most derivatives, particularly ETC19 and ETC9, which also showed improved absorption, better blood-brain barrier penetration, and reduced toxicity. These outcomes underscore the prospect of the de novo-designed etoricoxib analogues as safer and more effective alternatives, effectively addressing the pharmacological limitations and safety concerns associated with the parent drug.

Yinghua Zhu, Hongde Li, Hongbo Hu et al.

Sai Shashank Chavali, Steven Z. Chou, Wenxiang Cao et al.

Abstract Arp2/3 complex nucleates branched actin filaments for cell and organelle movements. Here we report a 2.7 Å resolution cryo-EM structure of the mature branch junction formed by S. pombe Arp2/3 complex that provides details about interactions with both mother and daughter filaments. We determine a second structure at 3.2 Å resolution with the phosphate analog BeFx bound with ADP to Arp3 and ATP bound to Arp2. In this ADP-BeFx transition state the outer domain of Arp3 is rotated 2° toward the mother filament compared with the ADP state and makes slightly broader contacts with actin in both the mother and daughter filaments. Thus, dissociation of Pi from the ADP-Pi transition state reduces the interactions of Arp2/3 complex with the actin filaments and may contribute to the lower mechanical stability of mature branch junctions with ADP bound to the Arps. Our structures also reveal that the mother filament in contact with Arp2/3 complex is slightly bent and twisted, consistent with the preference of Arp2/3 complex binding curved actin filaments. The small degree of twisting constrains models of actin filament mechanics.

Sarah Frank, Elisa Gabassi, Stephan Käseberg et al.

Molecular and cellular phenotyping of human brain organoids carrying various MID1 mutations reveals an unexpected role of the composition of the MID1 isoform pool during early patterning processes. The X-linked form of Opitz BBB/G syndrome (OS) is a monogenic disorder in which symptoms are established early during embryonic development. OS is caused by pathogenic variants in the X-linked gene MID1 . Disease-associated variants are distributed across the entire gene locus, except for the N-terminal really interesting new gene (RING) domain that encompasses the E3 ubiquitin ligase activity. By using genome-edited human induced pluripotent stem cell lines, we here show that absence of isoforms containing the RING domain of MID1 causes severe patterning defects in human brain organoids. We observed a prominent neurogenic deficit with a reduction in neural tissue and a concomitant increase in choroid plexus-like structures. Transcriptome analyses revealed a deregulation of patterning pathways very early on, even preceding neural induction. Notably, the observed phenotypes starkly contrast with those observed in MID1 full-knockout organoids, indicating the presence of a distinct mechanism that underlies the patterning defects. The severity and early onset of these phenotypes could potentially account for the absence of patients carrying pathogenic variants in exon 1 of the MID1 gene coding for the N-terminal RING domain.

Justus Kaufmann, Maximilian Haist, Ivan-Maximiliano Kur et al.

The growth pattern of oropharyngeal squamous cell carcinomas (OPSCC) varies from compact tumor cell aggregates to diffusely infiltrating tumor cell-clusters. The influence of the growth pattern on local tumor control and survival has been studied mainly for surgically treated oral cavity carcinomas on a visual basis. In this study, we used multiplex immunofluorescence staining (mIF) to examine the antigens pan-cytokeratin, p16INK4a, Ki67, CD271, PD-L1, and CD8 in pretherapeutic biopsies from 86 OPSCC. We introduce Tumor-stroma contact ratio (TSC), a novel parameter, to quantify the relationship between tumor cells in contact with the stromal surface and the total number of epithelial tumor cells. mIF tumor cores were analyzed at the single-cell level, and tumor-stromal contact area was quantified using the R package ''Spatstat''. TSC was correlated with the visually assessed invasion pattern by two independent investigators. Furthermore, TSC was analyzed in relation to clinical parameters and patient survival data to evaluate its potential prognostic significance.Higher TSC correlated with poor response to (chemo-)radiotherapy (r = 0.3, p < 0.01), and shorter overall (OS) and progression-free (PFS) survival (median OS: 13 vs 136 months, p < 0.0001; median PFS: 5 vs 85 months, p < 0.0001). Visual categorization of growth pattern according to established criteria of tumor aggressiveness showed interobserver variability increasing with more nuanced categories (2 categories: k = 0.7, 95 %-CI: 0.55 - 0.85; 4 categories k = 0.48, 95 %-CI: 0.35 - 0.61).In conclusion, TSC is an objective and reproducible computer-based parameter to quantify tumor-stroma contact area. We demonstrate its relevance for the response of oropharyngeal carcinomas to primary (chemo-)radiotherapy.

Deny Jennes, Soumya Ramankutty, S. Anoop et al.

Platelet rich plasma (PRP) therapy is an integral part of regenerative medicine as the platelets possess a good healing capacity owing to the presence of a wide variety of growth factors in the platelet granules found in the cytoplasm of the platelet. Autologous PRP was prepared from the blood of the patient itself, without any preservatives. Storage of PRP was one of the main hurdles of the treatment modality. During storage, the platelet counts may get reduced, undergo activation or get contaminated with bacteria as no preservatives are used in the preparation of autologous PRP. Cytological changes and microbial quality of the PRP during storage at 4oC and -20oC for seven days were analysed in this study. Reduction in platelet count and the chance of microbial contamination were less when autologous PRP was stored at -20°C compared to 4°C.

Stefanny F. Amaro, Cristiane C. Maciel, Jéssica S. Rodrigues et al.

This research aims to study flexible sensors based on a poly(butylene adipate-co-terephthalate) (PBAT) biodegradable polymer and graphite. Sensors were modified through the layer-by-layer (LbL) technique to improve their electrochemical behavior for paraquat (PQ) detection. Nanostructured films were obtained by alternating layers of anionic and cationic materials, carbon nanotubes (CNTs), and polypyrrole (PPY), respectively. The devices, with and without modification, were characterized by contact angle, scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FTIR). Electrochemical characterization was labeled via cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). PQ molecules were detected using the differential pulse voltammetry (DPV) technique in a concentration range of 0.1 to 2.1 µM. The sensor detection limit (LOD) was obtained using the analytical curve, with it being equal to 0.073 µM. The LbL film gPBAT(PPY/CNT)_n sensor showed good stability, reproducibility, and repeatability, with recovery values ranging from 99.4% to 109.3% for PQ when the analyzed samples were contaminated with tap water. The produced electrodes have the advantage of being flexible, disposable, reproducible, and of low manufacturing cost, which makes them attractive for portable environmental analysis.

Buket Akcan, Asim Örem, Yahya Altinkaynak et al.

Background and ObjectiveDue to the frequency of meal ingestion, individuals spend the majority of the day, ~18 h, in a status of post-prandial (PP) stress. Remnant-like lipoprotein particles (RLPs) are predominant in PP phase playing an important role in the development of atherosclerosis. Endothelial progenitor cells (EPCs) have been suggested to play a role in vessel wall homeostasis and in reducing atherosclerosis. However, there is no information about peripheral blood EPCs number following PP stress. We investigated the association between circulating EPCs levels and extent of PP lipemia in healthy subjects following a high-fat meal.Materials and MethodsA total of 84 healthy subjects (42 men, 42 women) aged 17–55 years were included in the study. PP lipemic response of subjects was determined by Oral Fat-Loading Test (OFLT). All the subjects were classified on the basis of their plasma TG levels after PP lipemic stressors in categories 1 (low), 2 (moderate), and 3 (high). Circulating EPCs numbers were measured by the flow cytometry method.ResultsThere was a significant difference in terms of lipid parameters between men and women: high-density lipoprotein cholesterol (HDL-C) was significantly lower in men than in women (p &lt; 0.001). Total cholesterol (TC) (p = 0.004), low-density lipoprotein cholesterol (LDL-C) (p &lt; 0.001), triglyceride (TG) (p &lt; 0.001), and TG-AUC (p &lt; 0.001) were significantly higher in men than in women. There was no significant difference between the genders in terms of CD34+KDR+ and CD34+KDR+CD133+cell number and MMP-9 levels. Vascular endothelial growth factor (VEGF) levels were significantly higher in men than women (p = 0.004). TC, LDL-C, and TG were significantly higher in the 3rd category than 1st and 2nd categories (p &lt; 0.001) in women. Age, body mass index (BMI), fat rate, TG, TC, and LDL-C were significantly higher in the 3rd category than 1st category (p &lt; 0.001, p = 0.002, p = 0.002, p = 0.01, p = 0.007, p = 0.004; respectively), in men. Circulating numbers of EPCs in men were significantly higher in the PP hyperlipidemia group than in the low TG levels category, independently from age (p &lt; 0.05). Circulating EPC levels showed a positive correlation with OFLT response in men (r = 0.414, p &lt; 0.05). Also, OFLT response showed a strong positive correlation with fasting TG levels (r = 0.930, p &lt; 0.001). EPC levels in categories of women were not different.ConclusionIncreased EPCs levels in subjects with different PP hyperlipidemia may be associated with a response to endothelial injury, related to increased atherogenic remnant particles at the PP phase.

Lukas Becker, Jasleen Singh Badwal, Fabian Brandl et al.

Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.

Soleyman Bafadam, Maryam Mahmoudabady, Saeed Niazmand et al.

Introduction: Inadequate control of diabetes mellitus (DM) leads to considerable cardiovascular implications like diabetic cardiomyopathy (DCM). Cardiomyocyte apoptosis is one of the main mechanisms of DCM pathogenesis associated with hyperglycemia, oxidative stress, inflammation, hyperlipidemia and several other factors. Trigonella foenum-graecum (Fenugreek) has been long used as a traditional medicine and has many therapeutic effects, including anti-diabetic, anti-hyperlipidemia, anti-inflammatory and anti-oxidant properties. The current study aimed to investigate cardioprotective effects of fenugreek seed on diabetic rats. Methods: Diabetes was induced in forty-two male rats by injection of streptozotocin (STZ) (60 mg/ kg). Diabetic animals were treated with three different doses of fenugreek seed extract (50, 100 and 200 mg/kg) or metformin (300 mg/kg) for six weeks by gavage. Nondiabetic rats served as controls. Glucose, cholesterol, and triglycerides levels were measured in the blood samples, and oxidative stress markers as well as gene expression of ICAM1, Bax and Bcl2 were assessed in the cardiac tissues of the experimental groups. Results: Diabetic rats exhibited increased serum glucose, cholesterol and triglycerides levels, elevated markers of oxidative stress thiobarbituric acid–reacting substances (TBARS) levels , total thiol groups (SH), catalase (CAT) and superoxide dismutase (SOD) activity, and enhanced apoptosis cell death (ratio of Bax/Bcl2). Fenugreek seed extract considerably improved metabolism abnormalities, attenuated oxidative stress and diminished apoptosis index. Conclusion: Our study suggests that fenugreek seed may protect the cardiac structure in STZ-induced diabetic rats by attenuating oxidative stress and apoptosis.

Nicole Nasby-Lucas, Heidi Dewar, Oscar Sosa-Nishizaki et al.

Abstract Background Most information on shortfin makos (Isurus oxyrinchus) in the eastern North Pacific (ENP) currently comes from fisheries data and short-term tracking studies. Although range has been inferred from catch and conventional tag data, little is known about the migration patterns and behavior in the ENP. This long-term electronic tagging study was designed to examine in detail the movement patterns and behavior of mako sharks in the ENP. Results In this study, a total of 105 mako sharks (104–280 cm fork length) were successfully tagged in the California Current between 2002 and 2014 with Argos satellite tags, including 93 satellite-linked radio-transmitting (SLRT) tags and 71 pop-up satellite archival tags (PSATs). This included 29 males that are in the size range of maturity, but only one mature female. Mean track durations from SLRT data were 337 days (max 1025), and PSAT tags were 136 days (max 272). Estimated minimum distance traveled in 1 year ranged from 6945 to 18,800 km/year. Habitats utilized included the entire California Current, the Sea of Cortez and offshore in the areas of the North Pacific Subtropical Gyre, North Pacific Transition Zone and North Equatorial Current. Seasonal movements within the California Current coincided with periods of higher primary productivity and chlorophyll a, and sea surface temperatures (SSTs) between 15 and 25 °C. SST ranged from 11 to 31 °C throughout the range, indicating a broad thermal tolerance. Conclusions Some of the key findings include the discovery of a high degree of variability between individuals in their vertical and horizontal movements, a strong influence of body size and season on mako shark movements, and the repetitive use of certain areas by individuals. These results expand our understanding of the distribution of mako sharks in the ENP. Although mako sharks are thought to comprise a single stock throughout the North Pacific, horizontal distribution of tagged mako sharks in this study was limited to the ENP demonstrating some spatial substructure. This study provides important data that can be used to identify fishery and gear vulnerabilities and inform management.

Jinling Jia, Jingyu Wang, Junlei Zhang et al.

Background/Aims: Chondrocyte apoptosis is largely responsible for cartilage degeneration in osteoarthritis (OA). MicroRNAs (miRNAs) play an important role in chondrogenesis and cartilage remodeling. This study explored the effect of miR-125b on inflammatory injury in chondrogenic cells. Methods: LPS was used to simulate inflammatory injury in murine chondrogenic ATDC5 cell lines. Targeting effect of miR-125b on MIP-1α 3’UTR was assessed by dual luciferase activity assay. Regulatory effect of miR-125b on MIP-1α expression and the potential regulatory mechanism on inflammatory injury were assessed by Western blot. Results: miR-125b expression was decreased in LPS-induced ATDC5 cells and overexpression of miR-125b inhibited LPS-induced cell viability decline, the rise of apoptosis and inflammatory factors’ productions. MIP-1α expression was negatively related to miR-125b, and miR-125b directly targeted with 3’UTR of MIP-1α. Knockdown of miR-125b promoted LPS-induced inflammatory response via upregulation of MIP-1α. miR-125b expression in LPS-induced ATDC5 cells was negatively related with activations of NF-κB and JNK signaling pathways. Overexpression of miR-125b inhibited LPS-induced inflammation injury via suppressing MIP-1α expression and inhibiting activations of NF-κB and JNK signaling pathways. Conclusion: miR-125b could play an important role in inflammatory injury of chondrogenic cells and miR-125b affected inflammatory injury of ATDC5 cells via regulating expression of MIP-1α and regulating NF-κB and JNK signaling pathways.

VAIBHAV KUMAR, KISHWAR ALI, AMRESH KUMAR et al.

AP2/ERF a plant specific transcription factor plays a crucial role in the expression and regulation of abiotic stress related genes. In this study, we have characterized a rice transcription factor named Os-AP2/ERF-N22. It contains single AP2 domain which spans from 5th to 70th amino acid residue in the protein which is 243 amino acid residue long. The partial sequence of the gene encoding Os-AP2/ERF transcription factor was amplified and cloned into pET29a bacterial expression vector. The histidine-tagged truncated Os-AP2/ERF-N22 protein was expressed in BL21(DE3) strain of E. coli after induction with 0.5mM IPTG. A time course induction study was used to optimize the protein expression and the recombinant Os-AP2/ERF-N22 was purified using Ni-NTA agarose column chromatography. The 26.74 kDa recombinant Os-AP2/ERF-N22 was visualized using SDS-PAGE analysis and its expression was further confirmed by western blot analysis using anti-his primary antibody and alkaline phosphatase conjugated secondary antibody. Antibody against the purified truncated AP2/ERF-N22 was custom synthesized and it was used to quantitate the expression of Os-AP2/ERF-N22 transcription factor in rice at the protein level.

Madhan R. Tirumalai, Fathi Karouia, Quyen Tran et al.

Evolution: Bacteria gain advantageous mutations under simulated microgravity Bacteria grown for an extended period of time under simulated microgravity adopt growth advantages. George Fox and colleagues from the University of Houston, Texas, USA, cultured Escherichia coli bacteria for 1000 generations in a high aspect rotating vessel to simulate the low fluid shear microgravity environment encountered during spaceflight. They then performed growth competition assays and found that the 1000-generation adapted bacteria outcompeted control bacteria grown without simulated microgravity. Genomic sequencing of the adapted bacteria revealed 16 mutations, five of which altered protein sequences. These DNA changes likely explain the growth advantage of the bacteria grown for multiple generations in simulated microgravity. Similar adaptations during prolonged space missions could result in nastier pathogens that might threaten the health of astronauts. Fortunately, the microbes did not appear to acquire antibiotic resistance over the 1000 generation in the modeled microgravity culture.

Sebastiano Calandra, Patrizia Tarugi, Helen E. Speedy et al.

This review integrates historical biochemical and modern genetic findings that underpin our understanding of the low-density lipoprotein (LDL) dyslipidemias that bear on human disease. These range from life-threatening conditions of infancy through severe coronary heart disease of young adulthood, to indolent disorders of middle- and old-age. We particularly focus on the biological aspects of those gene mutations and variants that impact on sterol absorption and hepatobiliary excretion via specific membrane transporter systems (NPC1L1, ABCG5/8); the incorporation of dietary sterols (MTP) and of de novo synthesized lipids (HMGCR, TRIB1) into apoB-containing lipoproteins (APOB) and their release into the circulation (ANGPTL3, SARA2, SORT1); and receptor-mediated uptake of LDL and of intestinal and hepatic-derived lipoprotein remnants (LDLR, APOB, APOE, LDLRAP1, PCSK9, IDOL). The insights gained from integrating the wealth of genetic data with biological processes have important implications for the classification of clinical and presymptomatic diagnoses of traditional LDL dyslipidemias, sitosterolemia, and newly emerging phenotypes, as well as their management through both nutritional and pharmaceutical means.

C.B. Cowey