Bioprocess parameters and oxygen transfer characteristics in beta-lactamase production by Bacillus species

After screening potential beta-lactamase producers in a medium containing penicillin G, an inducible (Bacillus subtilis NRS 1125) and a constitutive (Bacillus licheniformis 749/C ATCC 25972) beta-lactamase producer were selected. As the highest enzyme activity was obtained with B. licheniformis 749/C, the effects of the concentration of carbon sources, i.e., glucose, fructose, sucrose, citric acid, and glycerol, and nitrogen sources, i.e., (NH(4))(2)HPO(4), NH(4)Cl, yeast extract, casamino acids and peptone, pH, and temperature on beta-lactamase production were investigated with B. licheniformis 749/C in laboratory scale bioreactors. Among the investigated media, the highest volumetric activity was obtained as 270 U cm(-)(3) in the medium containing 10.0 kg m(-)(3) glucose, 1.18 kg m(-)(3) (NH(4))(2)HPO(4), 8.0 kg m(-)(3) yeast extract, and the salt solution at 32 degrees C and pH(0) = 6.0. By using the designed medium, fermentation and oxygen transfer characteristics of the bioprocess were investigated at V = 3.0 dm(3) bioreactor systems with a V(R) = 1.65 dm(3) working volume at Q(O)/V(R) = 0.5 vvm and N = 500 min(-1). At the beginning of the process the Damk?hler number was <1, indicating that the process was at biochemical reaction limited condition; at t = 2-5 h both mass-transfer and biochemical reaction resistances were effective; and at t = 6-10 h (Da >1) the bioprocess was at mass transfer limited condition. Overall oxygen transfer coefficients (K(L)a) varied between 0.01 and 0.03 s(-)(1), enhancement factor (K(L)a/K(L)a(O)) varied between 1.2 and 2.3, and volumetric oxygen uptake rate varied between 0.001 and 0.003 mol m(-)(3) s(-)(1) throughout the bioprocess. The specific oxygen uptake and the specific substrate consumption rates were the highest at t = 2 h and then decreased with the cultivation. The maximum yield of cells on substrate and the maximum yield of cells on oxygen values were obtained, respectively, as Y(X/S) = 0.34 and Y(X/O) = 1.40, at t = 5 h, whereas the highest yield of substrate on oxygen was obtained as Y(S/O) = 6.94 at t = 3.5 h. The rate of oxygen consumption for maintenance and the rate of substrate consumption for maintenance values were found, respectively, as m(O) = 0.13 kg kg(-)(1) h(-)(1) and m(S) = 3.02 kg kg(-)(1) h(-)(1).     

Molecular cloning of mouse collectin liver 1

Collectins are members of the superfamily of vertebrate C-type lectins that contain a collagen-like region, and are involved in first-line host defense. We earlier cloned and characterized a new kind of collectin, collectin liver 1 (CL-L1). In this study, we isolated the mouse homologue of CL-L1 encoding 277 amino acid residues; its deduced protein sequence was 88% identical with human CL-L1. Mouse CL-L1 mRNA was expressed mainly in the liver and stomach, but was found also in muscles, testes, intestines, and embryos. In mouse embryos, the level of CL-L1 mRNA gradually increased with embryonic age. In 16-day-old mouse embryos, CL-L1 mRNA was expressed in the liver, amnion, and visceral yolk sac. The mouse CL-L1 gene, Cll1 was found on chromosome 15 in a region syntenic with human chromosome 8q. CL-L1 was a highly conserved protein in mammals, birds, and fish.     

Interleukin (IL)-4/IL-9 and exogenous IL-16 induce IL-16 production by BEAS-2B cells, a bronchial epithelial cell line

Previous studies have suggested that bronchial epithelial cells may perpetuate airway inflammation. We have reported that the bronchial epithelial cell line BEAS-2B can produce interleukin (IL)-16, a potent chemoattractant for CD4+ T cells. IL-16 is thought to regulate airway inflammation in asthmatics. Recent studies showed that IL-4 induces inflammatory cytokines in bronchial epithelial cells and that IL-9 is a candidate gene for development of asthma. The present study demonstrated that BEAS-2B cells produced specifically IL-16 by synergistic effects of IL-4 + IL-16, or IL-9 + IL-16, and that the synthesized IL-16 induced migration of CD4+ T cells. This study is a first report indicating that IL-16 production may be maintained by an autocrine machinery by epithelial cell-derived IL-16 with IL-4 and IL-9 in asthma.     

Phage display derived therapeutic antibodies have enriched aliphatic content: Insights for developability issues

Phage display is one of the most widely used technology for antibody discovery and engineering. Number of therapeutic antibodies derived from phage display increases rapidly due to its ease of use and ability to control antibody sequence information. Although there are numerous antibody candidates as promising therapeutics, most of them fail at later stages of development due to undesired biophysical properties. Antibody candidates with poor properties should be prevented or improved in early development phases to minimize enormous loss of time and resources. In this study, we showed that phage display derived therapeutic antibodies show higher self-interaction and polyspecificity compared to non-phage display derived ones. To identify molecular determinants behind this, physicochemical properties of CDR regions of 137 therapeutic antibodies were analyzed. We found multiple significant differences in both heavy and light chain CDR regions. Most profoundly, aliphatic content of HCDR3, HCDR2, and LCDR3 regions were enriched in phage display derived antibodies compared to non-phage display derived ones. Physicochemical determinants documented here seem to play important roles in polyspecific and aggregation-prone natures of antibodies which should be avoided in early development phases.     

Plasmodium vivax Malaria in Pregnant Women in the Brazilian Amazon and the Risk Factors Associated with Prematurity and Low Birth Weight: A Descriptive Study

Introduction

Plasmodium vivax is the most prevalent malaria species in the American region. Brazil accounts for the higher number of the malaria cases reported in pregnant women in the Americas. This study aims to describe the characteristics of pregnant women with malaria in an endemic area of the Brazilian Amazon and the risk factors associated with prematurity and low birth weight (LBW).

Methods/Principal Findings

Between December 2005 and March 2008, 503 pregnant women with malaria that attended a tertiary health centre were enrolled and followed up until delivery and reported a total of 1016 malaria episodes. More than half of study women (54%) were between 20–29 years old, and almost a third were adolescents. The prevalence of anaemia at enrolment was 59%. Most women (286/503) reported more than one malaria episode and most malaria episodes (84.5%, 846/1001) were due to P. vivax infection. Among women with only P. vivax malaria, the risk of preterm birth and low birth weight decreased in multigravidae (OR, 0.36 [95% CI, 0.16–0.82]; p = 0.015 and OR 0.24 [95% CI, 0.10–0.58]; p = 0.001, respectively). The risk of preterm birth decreased with higher maternal age (OR 0.43 [95% CI, 0.19–0.95]; p = 0.037) and among those women who reported higher antenatal care (ANC) attendance (OR, 0.32 [95% CI, 0.15–0.70]; p = 0.005).

Conclusion

This study shows that P. vivax is the prevailing species among pregnant women with malaria in the region and shows that vivax clinical malaria may represent harmful consequences for the health of the mother and their offsprings particularly on specific groups such as adolescents, primigravidae and those women with lower ANC attendance.     

Enhanced G2/M Arrest,Caspase Related Apoptosis and Reduced E-Cadherin Dependent Intercellular Adhesion by Trabectedin in Prostate Cancer Stem Cells

Trabectedin (Yondelis, ET-743) is a marine-derived tetrahydroisoquinoline alkaloid. It is originally derived from the Caribbean marine tunicate Ecteinascidia turbinata and currently produced synthetically. Trabectedin is active against a variety of tumor cell lines growing in culture. The present study focused on the effect of trabectedin in cell proliferation, cell cycle progression, apoptosis and spheroid formation in prostate cancer stem cells (CSCs). Cluster of differentiation (CD) 133^+high/CD44^+high prostate CSCs were isolated from the DU145 and PC-3 human prostate cancer cell line through flow cytometry. We studied the growth-inhibitory effects of trabectedin and its molecular mechanisms on human prostate CSCs and non-CSCs. DU-145 and PC-3 CSCs were treated with 0.1, 1, 10 and 100 nM trabectedin for 24, 48 and 72 h and the growth inhibition rates were examined using the sphere-forming assay. Annexin-V assay and immunofluorescence analyses were performed for the detection of the cell death. Concentration-dependent effects of trabectedin on the cell cycle were also evaluated. The cells were exposed to the different doses of trabectedin for 24, 48 and 72 h to evaluate the effect of trabectedin on the number and diameter of spheroids. According to the results, trabectedin induced cytotoxicity and apoptosis at the IC₅₀ dose, resulting in a significant increase expression of caspase-3, caspase-8, caspase-9, p53 and decrease expression of bcl-2 in dose-dependent manner. Cell cycle analyses revealed that trabectedin induces dose-dependent G2/M-phase cell cycle arrest, particularly at high-dose treatments. Three-dimensional culture studies showed that trabectedin reduced the number and diameter of spheroids of DU145 and PC3 CSCs. Furthermore, we have found that trabectedin disrupted cell-cell interactions via E-cadherin in prostasphere of DU-145 and PC-3 CSCs. Our results showed that trabectedin inhibits cellular proliferation and accelerates apoptotic events in prostate CSCs; and may be a potential effective therapeutic agent against prostate cancer.     

Competition for Antigen between Th1 and Th2 Responses Determines the Timing of the Immune Response Switch during Mycobaterium avium Subspecies paratuberulosis Infection in Ruminants

Johne''s disease (JD), a persistent and slow progressing infection of ruminants such as cows and sheep, is caused by slow replicating bacilli Mycobacterium avium subspecies paratuberculosis (MAP) infecting macrophages in the gut. Infected animals initially mount a cell-mediated CD4 T cell response against MAP which is characterized by the production of interferon (Th1 response). Over time, Th1 response diminishes in most animals and antibody response to MAP antigens becomes dominant (Th2 response). The switch from Th1 to Th2 response occurs concomitantly with disease progression and shedding of the bacteria in feces. Mechanisms controlling this Th1/Th2 switch remain poorly understood. Because Th1 and Th2 responses are known to cross-inhibit each other, it is unclear why initially strong Th1 response is lost over time. Using a novel mathematical model of the immune response to MAP infection we show that the ability of extracellular bacteria to persist outside of macrophages naturally leads to switch of the cellular response to antibody production. Several additional mechanisms may also contribute to the timing of the Th1/Th2 switch including the rate of proliferation of Th1/Th2 responses at the site of infection, efficiency at which immune responses cross-inhibit each other, and the rate at which Th1 response becomes exhausted over time. Our basic model reasonably well explains four different kinetic patterns of the Th1/Th2 responses in MAP-infected sheep by variability in the initial bacterial dose and the efficiency of the MAP-specific T cell responses. Taken together, our novel mathematical model identifies factors of bacterial and host origin that drive kinetics of the immune response to MAP and provides the basis for testing the impact of vaccination or early treatment on the duration of infection.     

Preparation and characterization of novel albumin-sericin nanoparticles as siRNA delivery vehicle for laryngeal cancer treatment

Abstract

Small interfering RNA (siRNA)-based gene silencing strategy has high potential on suppressing specific molecular targets, involved in cancer progression. However, the lack of an effective nanocarrier system that safely delivers siRNA to its target still limits the clinical applications of siRNA. This study aimed to develop albumin-sericin nanoparticles (Alb-Ser NPs) as a novel siRNA delivery system for laryngeal cancer treatment. Nanoparticle formulations composed of albumin and sericin at different ratios (1:1, 2:1, 1:2 w/w) were synthesized by desolvation method. The nanoparticles were modified with poly-L-lysine (PLL) for siRNA binding and decorated with hyaluronic acid (HA) to target laryngeal cancer cell line, Hep-2. HA/PLL/Alb-Ser NPs were individually loaded with siRNAs for casein kinase 2 (CK2), Absent, Small, or Homeotic-Like (ASH2L), and Cyclin D1 genes, which are overexpressed in Hep-2 cells. Downregulation of genes was confirmed by real-time PCR (RT-PCR). Size, morphological, and thermogravimetric characterizations revealed that Alb-Ser NPs having 2:1 (w/w) ratio are the most optimized formulation. Between 36.8 and 61.3% of siRNA entrapment efficiencies were achieved. HA/PLL-siRNA/Alb-Ser (2:1) NPs-mediated gene silencing resulted in a significant inhibition of cell growth and induction of apoptosis in cells. Our findings showed that HA/PLL/Alb-Ser (2:1) NPs were promising as a siRNA carrier.     

Sonic hedgehog signaling is associated with resistance to zoledronic acid in CD133high/CD44high prostate cancer stem cells

Molecular Biology Reports - Cancer stem cells (CSCs) are a unique population that has been linked to drug resistance and metastasis and recurrence of prostate cancer. The sonic hedgehog (SHH)...     

Slow oocyte freezing and thawing in couples with no sperm or an insufficient number of sperm on the day of <Emphasis Type="Italic">in vitro</Emphasis>fertilization

Background  

The clinical results of in vitro fertilization of slowly frozen-thawed oocytes are known to be significantly worse than those obtained by fresh oocytes. Little is known about the factors affecting the clinical outcome of frozen-thawed oocytes. The aim of this retrospective study was to explore the role of oocyte cryopreservation in the group of patients with no available sperm on the day of in vitro fertilization. Additionally, the effects of the female serum FSH level and sperm quality on the clinical outcome of frozen-thawed oocytes were evaluated.