Clarified cashew apple juice as alternative raw material for biosurfactant production by Bacillus subtilis in a batch bioreactor

Clarified cashew apple juice was evaluated as carbon source for surfactin production by Bacillus subtilis LAMI005 isolated from the tank of chlorination at the Wastewater Treatment Plant on Campus do Pici (WWTP-PICI) in the Federal University of Ceará, Brazil. The highest surfactin concentration using clarified cashew apple juice (CCAJ) supplemented with mineral medium (MM-CCAJ) was 123 mg/L, achieved after 48 h of fermentation. Almost 2-fold less than the amount produced using mineral medium supplemented with 10 g/L of glucose and 8.7 g/L of fructose (MM-GF). However, critical micelle concentration of the biosurfactants produced using MM-CCAJ was 2.5-fold lower than the one produced using MM-GF, which indicates it is a more efficient biosurfactant. Surface tension decreased from 38.50 ± 0.0 to 29.00 ± 0.0 dyne/cm when B. subtilis was grown on MM-CCAJ media (24.68% of reduction on surface tension) and remained constant up to 72 h. Emulsification index was 51.15 and 66.70% using soybean oil and kerosene, respectively. Surfactin produced in MM-CCAJ showed an emulsifying activity of, respectively, 1.75 and 2.3 U when n-hexadecane or soybean oil was tested. However, when mineral medium supplemented with 10 g/L of glucose (MM-G) was used an emulsifying activity of 2.0 and 1.75 U, with n-hexadecane and soybean oil, respectively, was obtained. These results indicate that it is feasible to produce surfactin from CCAJ, a renewable and low-cost carbon source.     

A New Role for TIMP-1 in Modulating Neurite Outgrowth and Morphology of Cortical Neurons

Background

Tissue inhibitor of metalloproteinases-1 (TIMP-1) displays pleiotropic activities, both dependent and independent of its inhibitory activity on matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMP-1 is strongly upregulated in reactive astrocytes and cortical neurons following excitotoxic/inflammatory stimuli, but no information exists on its effects on growth and morphology of cortical neurons.

Principal Findings

We found that 24 h incubation with recombinant TIMP-1 induced a 35% reduction in neurite length and significantly increased growth cones size and the number of F-actin rich microprocesses. TIMP-1 mediated reduction in neurite length affected both dendrites and axons after 48 h treatment. The effects on neurite length and morphology were not elicited by a mutated form of TIMP-1 inactive against MMP-1, -2 and -3, and still inhibitory for MMP-9, but were mimicked by a broad spectrum MMP inhibitor. MMP-9 was poorly expressed in developing cortical neurons, unlike MMP-2 which was present in growth cones and whose selective inhibition caused neurite length reductions similar to those induced by TIMP-1. Moreover, TIMP-1 mediated changes in cytoskeleton reorganisation were not accompanied by modifications in the expression levels of actin, βIII-tubulin, or microtubule assembly regulatory protein MAP2c. Transfection-mediated overexpression of TIMP-1 dramatically reduced neuritic arbour extension in the absence of detectable levels of released extracellular TIMP-1.

Conclusions

Altogether, TIMP-1 emerges as a modulator of neuronal outgrowth and morphology in a paracrine and autrocrine manner through the inhibition, at least in part, of MMP-2 and not MMP-9. These findings may help us understand the role of the MMP/TIMP system in post-lesion pre-scarring conditions.     

Spatial Evaluation and Modeling of Dengue Seroprevalence and Vector Density in Rio de Janeiro,Brazil

Background

Rio de Janeiro, Brazil, experienced a severe dengue fever epidemic in 2008. This was the worst epidemic ever, characterized by a sharp increase in case-fatality rate, mainly among younger individuals. A combination of factors, such as climate, mosquito abundance, buildup of the susceptible population, or viral evolution, could explain the severity of this epidemic. The main objective of this study is to model the spatial patterns of dengue seroprevalence in three neighborhoods with different socioeconomic profiles in Rio de Janeiro. As blood sampling coincided with the peak of dengue transmission, we were also able to identify recent dengue infections and visually relate them to Aedes aegypti spatial distribution abundance. We analyzed individual and spatial factors associated with seroprevalence using Generalized Additive Model (GAM).

Methodology/Principal Findings

Three neighborhoods were investigated: a central urban neighborhood, and two isolated areas characterized as a slum and a suburban area. Weekly mosquito collections started in September 2006 and continued until March 2008. In each study area, 40 adult traps and 40 egg traps were installed in a random sample of premises, and two infestation indexes calculated: mean adult density and mean egg density. Sera from individuals living in the three neighborhoods were collected before the 2008 epidemic (July through November 2007) and during the epidemic (February through April 2008). Sera were tested for DENV-reactive IgM, IgG, Nested RT-PCR, and Real Time RT-PCR. From the before–after epidemics paired data, we described seroprevalence, recent dengue infections (asymptomatic or not), and seroconversion. Recent dengue infection varied from 1.3% to 14.1% among study areas. The highest IgM seropositivity occurred in the slum, where mosquito abundance was the lowest, but household conditions were the best for promoting contact between hosts and vectors. By fitting spatial GAM we found dengue seroprevalence hotspots located at the entrances of the two isolated communities, which are commercial activity areas with high human movement. No association between recent dengue infection and household''s high mosquito abundance was observed in this sample.

Conclusions/Significance

This study contributes to better understanding the dynamics of dengue in Rio de Janeiro by assessing the relationship between dengue seroprevalence, recent dengue infection, and vector density. In conclusion, the variation in spatial seroprevalence patterns inside the neighborhoods, with significantly higher risk patches close to the areas with large human movement, suggests that humans may be responsible for virus inflow to small neighborhoods in Rio de Janeiro. Surveillance guidelines should be further discussed, considering these findings, particularly the spatial patterns for both human and mosquito populations.     

Metabolism of the mesoionic compound (MI‐D) by mouse liver microsome,detection of its metabolite In Vivo,and acute toxicity in mice

The mesoionic derivative 4‐phenyl‐5‐[4‐nitrocinnamoyl]‐1,3,4‐thiadiazolyl‐2‐phenylamine chloride (MI‐D) has antitumoral and anti‐inflammatory effects. In this study, we present aspects of its metabolism and toxicity in mice. MI‐D was metabolized in vitro by liver microsome, generating a main product with a much shorter retention time than MI‐D in high‐performance liquid chromatography (HPLC) analysis but with a spectrum similar to that of the original molecule. Mass spectrometry with electrospray ionization in positive mode analysis of the purified compound by HPLC indicated that the product of metabolism has four additional hydroxyl groups (m/z = 465) compared with MI‐D (m/z = 401). The HPLC analyses of plasma and urine samples from mice treated with MI‐D showed the presence of the metabolite product. The kinetic parameters K_m (19.5 ± 4.5 μM) and V_max [1.5 ± 0.4 units of fluorescence/(100 μg of microsomal protein/mL/s)] were estimated, confirming the metabolism of MI‐D and indicating that the reaction follows Michaelis‐Menten kinetics. Acute toxicity was established on the basis of an estimation of mean lethal dose (LD‐50; 181.2 mg/kg) and histopathological analysis of animals that survived the LD‐50 test. Abdominal adhesions, inflammatory foci, and formation of granulomas were observed. Altogether, the results contribute to the advancement of research in support of MI‐D as a future chemotherapeutic drug. © 2009 Wiley Periodicals, Inc. J Biochem Mol Toxicol 23:394–405, 2009; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20303     

IL2-330G polymorphic allele is associated with decreased risk of Helicobacter pylori infection in adulthood

We evaluated whether polymorphisms in genes coding molecules linked to the innate and adaptive immune response are associated with susceptibility to Helicobacter pylori infection. IL1B-511C → T, IL1B-31 T → C, IL1RN allele 2, IL2-330 T → G, TNFA-307 G → A, TLR2Arg677Trp, TLR2Arg753Gln, TLR4Asp299Gly, and TLR5^392STOP polymorphisms were determined in 541 blood donors. IL2-330 T → G allele carriers had a decreased H. pylori infection risk (OR = 0.63, 95% CI = 0.43–0.93) after adjustment for demographic and environmental factors. Hence, we investigated whether the polymorphism is functional by evaluating IL-2 serum concentration in 150 blood donors and 100 children. IL-2 pro-inflammatory and anti-inflammatory properties were indirectly investigated by determining serum IFN-γ and IL-10/TGF-β levels. The polymorphism was associated with increased mean IL-2 levels in H. pylori-positive adults (2.65 pg/mL vs. 7.78 pg/mL) and children (4.19 pg/mL vs. 8.03 pg/mL). Increased IL-2 was associated with pro-inflammatory activity in adults (IFN-γ = 18.61 pg/mL vs. 25.71 pg/mL), and with anti-inflammatory activity in children (IL-10 = 6.99 vs. 14.17 pg/mL, TGF-β = 45.88 vs. 93.44 pg/mL) (p < 10⁻³ for all). In conclusion, in the context of H. pylori infection, IL2-330 T → G polymorphism is functional and is associated with decreased risk of infection in adults.     

The soluble form of the cancer-associated L1 cell adhesion molecule is a pro-angiogenic factor

A soluble form of the L1 cell adhesion molecule (sL1) is released from various tumor cells and can be found in serum and ascites fluid of uterine and ovarian carcinoma patients. sL1 is a ligand for several Arg-Gly-Asp (RGD)-binding integrins and can be deposited in the extracellular matrix. In this study we describe a novel function of this physiologically relevant form of L1 as a pro-angiogenic factor. We demonstrated that the anti-L1 monoclonal antibody (mAb) chCE7 binds near or to the sixth Ig-like domain of human L1 which contains a single RGD sequence. mAb chCE7 inhibited the RGD-dependent adhesion of ovarian carcinoma cells to sL1 and reversed the sL1-induced proliferation, matrigel invasion and tube formation of bovine aortic endothelial (BAE) cells. A combination of sL1 with vascular endothelial growth factor-A (VEGF-A₁₆₅), which is an important angiogenic inducer in tumors, strongly potentiated VEGF receptor-2 tyrosine phosphorylation in BAE cells. Chick chorioallantoic membrane (CAM) assays revealed the pro-angiogenic potency of sL1 in vivo which could be abolished by chCE7. These results indicate an important role of released L1 in tumor angiogenesis and represent a novel function of antibody chCE7 in tumor therapy.     

Challenge with 17α-ethinylestradiol (EE2) during early development persistently impairs growth,differentiation, and local expression of IGF-I and IGF-II in immune organs of tilapia

The enormous expansion of world-wide aquaculture has led to increasing interest in the regulation of fish immune system. Estrogen has recently been shown to inhibit the endocrine (liver-derived) and autocrine/paracrine local insulin-like growth factor-I system in fish. In order to address the potential actions of estrogen on the IGF system in immune organs, tilapia were fed with 17α-ethinylestradiol (EE2)-enriched food from 10 to 40 days post fertilization (DPF) to induce functional feminization, an approach commonly used in aquaculture. EE2-treated and control fish were sampled at 75 and 165 DPF. The expression levels of ER-α, IGF-I, IGF-II and growth hormone receptor (GH-R) mRNA in spleen and head kidney were determined by real-time PCR and the expressing sites of IGF-I mRNA identified by in situ hybridisation. Ratios of spleen length and weight to body length and weight were determined. At 165 DPF, the length (4.9% vs. 7.6%) and weight (0.084% vs. 0.132%) ratios were significantly lowered in EE2-treated fish and number and size of the melanomacrophage centres were considerably reduced. At 75 DPF, both in spleen and head kidney of EE2-treated fish the expression levels of IGF-I and IGF-II mRNA were markedly diminished. The suppression was more pronounced for IGF-I (spleen: ?12.071-fold; head kidney: ?8.413-fold) than for IGF-II (spleen: ?4.102-fold; head kidney: ?1.342-fold). In agreement, clearly fewer leucocytes and macrophages in head kidney and spleen of EE2-treated fish contained IGF-I mRNA as shown by in situ hybridisation. ER-α mRNA expression in spleen was increased at 75 DPF but unchanged in head kidney. GH-R gene expression showed a mild upregulation at 165 DPF in both tissues. Thus, exposure to EE2 during early development affected distinctly the IGF system in tilapia immune organs. It led to lasting impairment of spleen growth and differentiation that can be attributed to an interaction of EE2 with IGF-I and, less pronouncedly, IGF-II. Especially, the impairment of spleen and melanomacrophage centres might interfere with the antigen presentation capacity of the immune system and, thus, alter susceptibility to infection.     

Leishmania (Viannia) braziliensis transfectants overexpressing the miniexon gene lose virulence in vivo

The miniexon gene has a central role in the processing of polycistronic pre-mRNA of kinetoplastids. It is added to the 5' extremity of each mRNA, supplying the 5'-capped structure to the molecule. Previous studies in Leishmania (Leishmania) major showed that the overexpression of the miniexon array attenuates the virulence of the parasite in in vivo assays. The results presented here extend those findings to Viannia subgenus. Leishmania (Viannia) braziliensis was transfected with a cosmid harboring a tandem array of one hundred miniexon gene copies and then characterized by Northern blot analysis. The overexpression of the exogenous gene was confirmed and its effect on the virulence of L. (V.) braziliensis was investigated in hamsters. In BALB/c mice we could not detect parasites during the course of 15 weeks of infection. In addition, hamsters infected with transfectants overexpressing the miniexon gene exhibited only a minor footpad swelling of late onset and failed to develop progressive lesion, these attenuated parasites could be recovered from the inoculation site 1 year after infection. The persistence of parasites in the host indicates that a stable line overexpressing the miniexon may be tested as live vaccine against leishmaniasis.