Chimeric Human Papillomavirus-16 Virus-like Particles Presenting HIV-1 P18I10 Peptide: Expression, Purification, Bio-Physical Properties and Immunogenicity in BALB/c Mice.

Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG (p = 0.6409) was observed as Gardasil anti-HPV vaccine-immunized mice. Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1.

Evaluation of the Antidiabetic Potential of Extracts of Urtica dioica, Apium graveolens, and Zingiber officinale in Mice, Zebrafish, and Pancreatic ß-Cell.

Medicinal plants are commonly used in the treatment of diabetes, particularly as they contain flavonoids and phenolic compounds. The present study aims to investigate the activities of a polyherbal formulation made from Urtica dioica, Apium graveolens, and Zingiber officinale (UAZ) against streptozotocin-nicotinamide ((STZ-NA)-induced type 2 diabetes in CD1 mice, glucose-induced type 2 diabetes (T2DM) in zebrafish, and high glucose-induced damage in RINm5F pancreatic ß-cells. In fasting mice, plasma glucose, glycosylated hemoglobin (HbA1C), lipid hydroperoxides (LOOH), thiobarbituric acid reactive substances (TBARS), and lipid profiles were significantly increased, whereas insulin, enzymatic antioxidants, and carbohydrate metabolic enzymes were altered significantly in diabetic mice. Zebrafish had similar glucose levels, liver enzymes, and lipid profiles compared to mice. The study investigated the effects of the extract in enhancing cell viability, insulin secretion, and reducing lipid peroxidation and intracellular reactive oxygen species (ROS) levels in RINm5F cells damaged by high glucose. All the above biochemical parameters were enhanced in both mice and zebrafish treated; the combined extract UAZ normalized all the biochemical parameters. The medicinal plant extracts, used either separately or in combination, ameliorated the adverse effect of glucose on cell viability and functionality of beta-RINm5F cells.

Early initiation of hydroxyurea (hydroxycarbamide) using individualised, pharmacokinetics-guided dosing can produce sustained and nearly pancellular expression of fetal haemoglobin in children with sickle cell anaemia.

Hydroxyurea (hydroxycarbamide) is an effective treatment for sickle cell anaemia (SCA), but clinical responses depend primarily upon the degree of fetal haemoglobin (HbF) induction and the heterogeneity of HbF expression across erythrocytes. The number and characteristics of HbF-containing cells (F-cells) are not assessed by traditional HbF measurements. Conventional hydroxyurea dosing (e.g. fixed doses or low starting doses with stepwise escalation) produces a moderate heterocellular HbF induction, but haemolysis and clinical complications continue. Robust, pancellular HbF induction is needed to minimise or fully inhibit polymerisation of sickle haemoglobin. We treated children with hydroxyurea using an individualised, pharmacokinetics-guided regimen starting at predicted maximum tolerated dose (MTD). We observed sustained HbF induction (mean >30%) for up to 6 years, which was not dependent on genetic determinants of HbF expression. Nearly 70% of patients had ≥80% F-cells (near-pancellular), and almost half had ≥90% F-cells (pancellular). The mean HbF/F-cell content was ~12 pg. Earlier age of initiation and better medication adherence were associated with high F-cell responses. In summary, early initiation of hydroxyurea using pharmacokinetics-guided starting doses at predicted MTD can achieve sustained near-pancellular or pancellular HbF expression and should be considered an achievable goal for children with SCA treated with hydroxyurea at optimal doses. Clinical trial registration number: NCT02286154 (clinicaltrials.gov).

Automated Oxygen Gradient Ektacytometry: A Novel Biomarker in Sickle Cell Anemia.

Sickle cell anemia (SCA) is a hereditary hemoglobinopathy with a variable phenotype. There is no single biomarker that adequately predicts disease severity and can be used to monitor treatment response in patients in clinical trials and clinical care. The use of clinical outcomes, such as vaso-occlusive crises (VOC), requires long and expensive studies, sometimes with inconclusive results. To address these limitations, there are several biomarkers under study to improve the ability to predict complications and assess treatment response in both clinical and research settings. Oxygen gradient ektacytometry, also called as oxygenscan, is an assay that measures the effects of deoxygenation and reoxygenation on red blood cell (RBC) deformability and is gaining popularity in SCA research, because it captures the dynamic sickling capacity of a patient's RBCs as they are subjected to an oxygen gradient under steady shear stress. We describe here the oxygenscan methodology and evaluate the correlation between oxygenscan parameters and more well-known biomarkers of SCA such as fetal hemoglobin (HbF), F-cells, and dense red blood cells (DRBCs). Our data indicate that the oxygenscan curve is affected by all these parameters and the result incorporates the effects of %HbF, %F-cells, RBC hydration, and RBC membrane deformability.

Curcumin inhibits alloxan-induced pancreatic islet cell damage via antioxidation and antiapoptosis.

The present study elucidates the possible protective effects of curcumin on ß-cells damaged by oxidative stress and its significance in controlling diabetes mellitus in in vitro experiments. Pancreatic islet (RIN-m5F) cells were treated with 25 mmol/L alloxan (AXN) to induce cell damage and the protective effects of curcumin were observed. The results showed that curcumin significantly promoted the cellular activity of AXN-treated RIN-m5F cells, decreased the ratio of apoptosis, downregulated the level of malondialdehyde, upregulated the levels of superoxide dismutase and reactive oxygen species, increased the expression of Bcl-2, cleaved caspase-3, and cleaved PARP1, and decreased the expression of Bax in AXN-treated cells. These results suggest that curcumin inhibits AXN-induced damage in RIN-m5F cells via antioxidative and antiapoptotic mechanisms.

Antidiabetic Activity of Gold Nanoparticles Synthesized Using Wedelolactone in RIN-5F Cell Line.

We synthesized the gold nanoparticles (AuNPs) using wedelolactone (WDL) and characterized them using UV-visible spectroscopy, fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopic (SEM), transmission electron microscopic (TEM), energy dispersive X-ray diffraction, and atomic force microscopic (AFM) studies. The electronic spectrum exhibited an absorption peak at 535 nm. The FT-IR results proved that WDL was stabilized on the surface of AuNPs by acting as a capping or reducing agent. The crystalline structure was affirmed by XRD pattern and the spherical shape of WDL-AuNPs was evidenced by SEM, TEM, and AFM. The synthesized WDL-AuNPS were evaluated for anti-diabetic activity in pancreatic RIN-5F cell lines. In vitro results showed that WDL-AuNPs did not only improve the insulin secretion affected by di-(2-ethylhexyl) phthalate (DEHP), but also the cell viability in RIN5F cells. WDL-AuNPs treatment modulates the pro-apoptotic proteins and anti-apoptotic proteins expression to prevent the cells undergoing apoptosis in DEHP-exposed RIN-5F cells. The exposure of DEHP causes an increase in ROS production and lipid peroxidation levels. The free radical scavenging and antioxidant properties of WDL-AuNPs increase the deleterious effect caused by DEHP. On the other side, WDL-AuNPs increase mRNA expressions of insulin-signaling proteins in RIN-5F cells. This study concludes that WDL-AuNPs can be successfully used to regulate the expression of Bcl-2 family proteins, reduce lipid peroxidation, and to improve the secretion of antioxidants and insulin through the GLUT2 pathway in RIN-5F cell lines.

Wide range of F cell levels in healthy Thai adults: Influence of Swiss-type hereditary persistence of foetal haemoglobin & ß-haemoglobinopathy.

Background & objectives: Swiss-type hereditary persistence of foetal haemoglobin (HPFH) has been shown to be responsible for the wide range of F cell levels in healthy Thai adults. However, a survey for F cells in healthy Thai adults has not been performed. This study was conducted to determine the F cell distribution in adult Thai blood donors and to assess the possible involvement of ß-thalassaemia and haemoglobin E (HbE) carriers in increased HbF levels. Methods: Thai blood donors (n=375, 205 males and 170 females) were included in the study. Blood samples were collected for measuring haemoglobin (Hb) concentration and haematocrit (Hct) and F cell levels. Hb and Hct levels were determined by automated blood counter, while F cells were quantified by flow cytometric analysis of F cells stained by fluorescein isothiocyanate-conjugated anti γ-globin monoclonal antibody. Finally, F cell levels were compared between blood samples having mean corpuscular volume (MCV ) <80 fl and ≥80 fl as well as between ß-haemoglobinopathies (HbE and ß-thalassaemia carriers) and normal adults. Results: F cell levels varied markedly spanning 0.80-39.2 per cent with a positively skewed distribution. Thirty two per cent of these individuals had F cell levels more than the 4.5 per cent cut-off point. F cell levels in females were significantly higher than those in males (P<0.05). F cell levels in individuals having MCV <80 fl were significantly higher than those having MCV ≥80 fl (P<0.05). ß-haemoglobinopathy (HbE and ß-thalassaemia carriers) had significantly higher F cell levels than normal individuals (P<0.05). Interpretation & conclusions: The present results showed that besides Swiss-type HPFH, the ß-haemoglobinopathy was expected to be involved in increased F cell levels in adult Thais. Thus, influence of ß-haemoglobinopathy must be considered in interpreting F cell levels in area endemic of this globin disorder.

microRNA-29a functions as a tumor suppressor in nasopharyngeal carcinoma 5-8F cells through targeting VEGF.

OBJECTIVES: microRNA-29 (miR-29) family miRNAs have been mentioned as tumor suppressive genes in several human cancers. The purpose of this study was to investigate the function of miR-29a in nasopharyngeal carcinoma (NPC) cells. MATERIALS AND METHODS: Human NPC cell line 5-8F was transfected with mimic, inhibitor or scrambled controls specific for miR-29a. Subsequently, cell viability, migration, apoptosis and expression changes of VEGF were assessed by trypan blue staining, MTT assay, transwell assay, flow cytometry, Western blot and RT-qPCR. TargetScan online database was used to predict the targets of miR-29a, and luciferase reporter assay was carried out for testing the targeting relationship between VEGF and miR-29a. Western blot analysis was performed to determine the expression changes of core proteins in PI3K/AKT and JAK/STAT pathways. RESULTS: Overexpression of miR-29a suppressed 5-8F cells viability and relative migration, but increased apoptotic cell rate. Consistently, Bcl-2 was downregulated, Bax was upregulated, and caspase-3 and -9 were cleaved by miR-29a overexpression. VEGF was a target gene of miR-29a. Besides, VEGF silence exerted similar effects like miR-29a, as the viability and migration were repressed and apoptosis was induced. Finally, we found that PI3K/AKT and JAK/STAT pathways were deactivated by miR-29a or VEGF silence. CONCLUSION: These findings highlighted the tumor suppressive effects of miR-29a on NPC cells, as its overexpression inhibited 5-8F cells viability, migration, and induced apoptosis. miR-29a exerted tumor suppressive functions might be via targeting VEGF and deactivating PI3K/AKT and JAK/STAT pathways.

Variability of the f-cell ratio after treatment of traumatic hemorrhagic shock.

PURPOSE: Measuring total blood volume (TBV) in critically ill patients, using isotope techniques to determine red cell volume (RBCV) and plasma volume (PV) is laborious. Recently, PV measurement using a single bolus dose of tracers has been validated, thus, allowing TBV calculation using large venous hematocrit (LVHCT). However, this technique relies on using a correlation factor, the f-cell ratio, to adjust for differences in LVHCT and total body hematocrit (TBHCT). The normal f-cell ratio is 0.9 but has never been studied in patients recovering from hemorrhagic shock (HS). This study assesses the f-cell ratio at different phases after HS to determine accuracy in calculating TBV. METHODS: 114 injured patients requiring immediate operation for HS were studied. All patients had measurements of PV and RBCV via isotope dilution enabling measurements of TBHCT. Correlation of LVHCT and TBHCT were used to calculate the f-cell ratio in the fluid sequestration (n = 81) and in the fluid mobilization period (n = 108). RESULTS: The f-cell ratio (mean ±â€¯SD) averaged 0.89 ±â€¯0.15 and 0.90 ±â€¯0.01 in the first and second halves of the fluid sequestration period versus 0.90 ±â€¯0.2 and 0.80 ±â€¯0.07 in the first and second 48 h of the fluid mobilization period. The f-cell ratio was significantly lower (p=<0.001) in the mobilization period. CONCLUSIONS: These data show for the first time that using PV and LVHCT to calculate TBV after HS is unreliable. The mechanisms causing this variation in f-cell ratio is unknown but likely related to capillary/interstitial dynamics and needs further scientific study.

MafB Is Critical for Glucagon Production and Secretion in Mouse Pancreatic α Cells In Vivo.

The MafB transcription factor is expressed in pancreatic α and ß cells during development but becomes exclusive to α cells in adult rodents. Mafb-null (Mafb-/- ) mice were reported to have reduced α- and ß-cell numbers throughout embryonic development. To further analyze the postnatal function of MafB in the pancreas, we generated endocrine cell-specific (MafbΔEndo ) and tamoxifen-dependent (MafbΔTAM ) Mafb knockout mice. MafbΔEndo mice exhibited reduced populations of insulin-positive (insulin+) and glucagon+ cells at postnatal day 0, but the insulin+ cell population recovered by 8 weeks of age. In contrast, the Arx+ glucagon+ cell fraction and glucagon expression remained decreased even in adulthood. MafbΔTAM mice, with Mafb deleted after pancreas maturation, also demonstrated diminished glucagon+ cells and glucagon content without affecting ß cells. A decreased Arx+ glucagon+ cell population in MafbΔEndo mice was compensated for by an increased Arx+ pancreatic polypeptide+ cell population. Furthermore, gene expression analyses from both MafbΔEndo and MafbΔTAM islets revealed that MafB is a key regulator of glucagon expression in α cells. Finally, both mutants failed to respond to arginine, likely due to impaired arginine transporter gene expression and glucagon production ability. Taken together, our findings reveal that MafB is critical for the functional maintenance of mouse α cells in vivo, including glucagon production and secretion, as well as in development.

Histomorphologic characterization of the hepatopancreas of freshwater prawn Macrobrachium rosenbergii (De Man, 1879) / Caracterização histomorfológica do hepatopâncreas do camarão-de-água-doce Macrobrachium rosenbergii (De Man, 1879)

This study aimed to describe the Macrobrachium rosenbergii hepatopancreas histomorphology. The hepatopancreas is constituted by a set of blind end tubules, divided into proximal, middle, and distal regions, with the epithelium formed by E, F, B, R, and M cells differently of other species. Measurements of the length and width of the tubules were 419.64+69.09µm and 117.42+16.99µm, respectively. The percentage of each cell type per region was: proximal region (40%B, 20%F, 6.7%M, 33.3%R), middle region (45.4%B, 18.2%F, 9.1%M, and 27.3%R) and distal region (36.4%E, 27.2%B, 18.2%F, 9.1%M, 9.1%R). Cell B that stores glycogen and lipids, is the most commonly found cell in proximal and middle regions. In the distal region, the E, responsible for the mitosis, is the most prominent. M, responsible by nutrient storage, is numerically constant among the portions differently in the Macrobrachium amazonicum. The study for the first time also suggests that in addition to digestive enzymes, the F cell produces protective mucus. The present study generated for the first time a morphometric profile of M. rosenbergii hepatopancreas, demonstrating differences from other species, and can be an important tool for new studies in nutrition, reproduction, and production with the species.(AU)

O objetivo do presente estudo foi descrever a histomorfologia do hepatopâncreas do camarão-de-água-doce Macrobrachium rosenbergii. Observou-se que ele é constituído por um conjunto de túbulos de fundo cego, sendo cada túbulo dividido em regiões proximal, média e distal, com o epitélio formado por cinco tipos de células (E, F, B, R, M), diferentemente de outras espécies. As medidas de comprimento e largura dos túbulos foram de 419,64+69,09µm e 117,42+16,99µm, respectivamente. A porcentagem de cada tipo celular por região foi: região proximal (40%B, 20%F, 6,7%M, 33,3%R), região média (45,4%B, 18,2%F, 9,1%M e 27,3%R) e região distal (36,4%E, 27,2%B, 18,2%F, 9,1%M, 9,1%R). Assim, a B, que armazena glicogênio e lipídeos, é a célula mais encontrada nas regiões proximal e média. Na região distal, a célula E, responsável pela mitose, é a mais encontrada. A célula M, responsável pelo acúmulo de nutrientes, tem um número constante de células nas porções do túbulo, diferentemente do Macrobrachium amazonicum. O estudo também sugere, pela primeira vez, que a célula F produz, além de enzimas digestivas, um muco protetor para o túbulo hepatopancreático. O presente estudo foi o primeiro a gerar um perfil morfométrico do hepatopâncreas do M. rosenbergii e demonstrou diferenças em relação a outras espécies, bem como serviu de importante ferramenta para novos estudos que abranjam a produção, a nutrição e a reprodução para a espécie.(AU)

Expression, purification and characterization of the human MTA2-RBBP7 complex.

The repressive Nucleosome Remodeling and histone Deacetylation (NuRD) complex remodels the chromatin structure by coupling ATP-dependent remodeling activity with histone deacetylase function and plays important roles in regulating gene transcription, DNA damage repair and chromatin assembly. The complex is composed of six subunits: Metastasis Associated proteins MTA1/2/3 initially recruit histone chaperones RBBP4/7 followed by the histone deacetylases HDAC1/2 forming a core complex. Further association of the CpG-binding protein MBD2/3, p66α/ß and the ATP-dependent helicase CDH3/4 constitutes the NuRD complex. Recent structural studies on truncated human proteins or orthologous have revealed that the stoichiometry of the MTA1-RBBP4 complex is 2:4. This study reports expression and purification of the intact human MTA2-RBBP7 complex using HEK293F cells as expression system. In analogy with findings on the Drosophila NuRD complex, we find that also the human MTA-RBBP can be isolated in vitro. Taken together with previous findings this suggests, that MTA-RBBP is a stable complex, with a central role in the initial assembly of the human NuRD complex. Refined 3D volumes of the complex generated from negative stain electron microscopy (EM) data reveals an elongated architecture that is capable of hinge like motion around the center of the particle.