Microbial production host selection for converting second-generation feedstocks into bioproducts

Background  

Increasingly lignocellulosic biomass hydrolysates are used as the feedstock for industrial fermentations. These biomass hydrolysates are complex mixtures of different fermentable sugars, but also inhibitors and salts that affect the performance of the microbial production host. The performance of six industrially relevant microorganisms, i.e. two bacteria (Escherichia coli and Corynebacterium glutamicum), two yeasts (Saccharomyces cerevisiae and Pichia stipitis) and two fungi (Aspergillus niger and Trichoderma reesei) were compared for their (i) ability to utilize monosaccharides present in lignocellulosic hydrolysates, (ii) resistance against inhibitors present in lignocellulosic hydrolysates, (iii) their ability to utilize and grow on different feedstock hydrolysates (corn stover, wheat straw, sugar cane bagasse and willow wood). The feedstock hydrolysates were generated in two manners: (i) thermal pretreatment under mild acid conditions followed by enzymatic hydrolysis and (ii) a non-enzymatic method in which the lignocellulosic biomass is pretreated and hydrolyzed by concentrated sulfuric acid. Moreover, the ability of the selected hosts to utilize waste glycerol from the biodiesel industry was evaluated.     

Medical bioremediation of age-related diseases

Catabolic insufficiency in humans leads to the gradual accumulation of a number of pathogenic compounds associated with age-related diseases, including atherosclerosis, Alzheimer's disease, and macular degeneration. Removal of these compounds is a widely researched therapeutic option, but the use of antibodies and endogenous human enzymes has failed to produce effective treatments, and may pose risks to cellular homeostasis. Another alternative is "medical bioremediation," the use of microbial enzymes to augment missing catabolic functions. The microbial genetic diversity in most natural environments provides a resource that can be mined for enzymes capable of degrading just about any energy-rich organic compound. This review discusses targets for biodegradation, the identification of candidate microbial enzymes, and enzyme-delivery methods.     

Prevalence of mood dysfunction in epilepsy patients in Croatia

Fifty consecutive and consenting epilepsy patients from the Zagreb Epilepsy Center were examined for the presence of depressive symptoms using the Beck Depression Inventory (BDI). This questionnaire has been previously validated for use in the Croatian population. Mean age of the patients was 30.8 +/- 13.5 years, 60.4% were females. Majority of them were employed (72.9%) and single (62.5%), and 35.4% had a university degree. Most of them had complex partial seizures (n=40, 80%), and 6 (12%) were diagnosed with idiopathic generalized epilepsy. Assessment with the BDI showed that 33.3% of patients had recent depressive symptoms: 6.3% had mild depressive symptoms, 8.4% moderate and 18.6% severe depressive symptoms. Three patients (6.4%) attempted suicide in the past, two of them had current suicidal ideation, and all of them were severely depressed. This is the first and preliminary study assessing mood dysfunction in epilepsy patients in Croatia. Increased prevalence of depression in epilepsy patients suggests specific approach and need for early treatment.     

Role of sarcolemmal ATP-sensitive potassium channel in oxidative stress-induced apoptosis: mitochondrial connection

From time of their discovery, sarcolemmal ATP-sensitive K+ (sarcK ATP) channels were thought to have an important protective role in the heart during stress whereby channel opening protects the heart from stress-induced Ca2+ overload and resulting damage. In contrast, some recent studies indicate that sarcK ATP channel closing can lead to cardiac protection. Also, the role of the sarcK ATP channel in apoptotic cell death is unclear. In the present study, the effects of channel inhibition on apoptosis and the specific interaction between the sarcK ATP channel and mitochondria were investigated. Apoptotic cell death of cultured HL-1 and neonatal cardiomyocytes following exposure to oxidative stress was significantly increased in the presence of sarcK ATP channel inhibitor HMR-1098 as evidenced by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling and caspase-3,7 assays. This was paralleled by an increased release of cytochrome c from mitochondria to cytosol, suggesting activation of the mitochondrial death pathway. sarcK ATP channel inhibition during stress had no effect on Bcl-2, Bad, and phospho-Bad, indicating that the increase in apoptosis cannot be attributed to these modulators of the apoptotic pathway. However, monitoring of mitochondrial Ca2+ with rhod-2 fluorescent indicator revealed that mitochondrial Ca2+ accumulation during stress is potentiated in the presence of HMR-1098. In conclusion, this study provides novel evidence that opening of sarcK ATP channels, through a specific Ca2+-related interaction with mitochondria, plays an important role in preventing cardiomyocyte apoptosis and mitochondrial damage during stress.     

Immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type 1

Herpes simplex viruses (HSV) have developed several immunoevasive strategies. Here we demonstrate a novel mechanism by which HSV type 1 may interfere with the immune response through infection of immature dendritic cells (DC) and selective downmodulation of costimulatory molecules. In our study we show productive infection of immature monocyte-derived DC, which closely resemble sessile Langerhans cells, by sequential expression of immediate-early, early, and late viral proteins and of glycoprotein D mRNA, as well as production of infectious virus of moderate titers. Infection was cytopathic, with the progressive loss of 20 to 45% of cells from 24 to 48 h after infection, with no more than 80% of DC found to be infected. These results are in contrast to those of previous findings of nonpermissive or abortive infection of monocytes and mature monocyte-derived DC. Infection of immature DC also led to selective and asynchronous downregulation of CD1a, CD40, CD54 (ICAM-1) (12 h postinfection), CD80 (24 h postinfection), and CD86 (48 h postinfection) but not of CD11c or major histocompatibility complex class I and II molecules when compared to DC exposed to UV-inactivated virus. Thus, we propose that productive infection of epidermal Langerhans cells in vivo may lead to delayed activation of T cells, allowing more time for replication of HSV type 1 in epidermal cells.     

Reasons for extraction of permanent teeth in urban and rural populations of Croatia

The survey aimed to determine the reasons for extraction of permanent teeth by general dental practitioners in urban and rural population of the Senj region, Adriatic coast, Croatia. During a two-year period (1998-9), a total of 2006 teeth were extracted in both regions, in patients aged 15+. The causes were defined as follows: (1) decay or root without a crown (radix relicta), (2) periodontal disease, (3) endodontic or periapical diseases and (4) other reasons--orthodontics/prosthodontics and dental trauma. The statistical Chi-square-test was used to determine the significant difference between the populations and the sexes. Dental caries was the most frequent cause for extraction (over 50%), followed by endodontic and periapical diseases (23%) as the result of untreated caries and at the end periodontal disease (21%). Urban population more often lose teeth due to periodontal disease (22.75%) than rural (18.93%, p < 0.05). Similarly, this is more frequent in the urban male population (25.61%) than the female urban population (20%, p < 0.05). In rural areas, people more often lost teeth as a result of endodontic and periapical disease (25.85%) than in the urban locations (19.07%, p < 0.01) and this is more frequent in women from rural areas (28.37%) than the rural men (22.44%, p < 0.05). Periodontal disease was not the main cause of tooth loss in either the rural or the urban population. Dental caries and its sequel remain the most important challenge for the dental service. It also reveals the inadequacy of dental services. Education of both the population and the general dental practitioners must be conducted in order to improve oral hygiene and to insist on conservative rather than extraction therapy.     

The effect of baby friendly hospital initiative and postnatal support on breastfeeding rates--Croatian experience

The effects of implementation WHO/UNICEF Breastfeeding Hospital Initiative (BFHI) and community postnatal support on breastfeeding rates were examined during and after the breastfeeding promotion campaign in one county of Croatia. Comparison with a control group indicated increase of breastfeeding prevalence in a period of BFHI implementation (1994-1998) - 68% vs. 87% at infant age 1 mo., 30% vs. 54% at 3 mo., 11.5% vs. 28% at 6 mo., and 2% vs. 3.5% at infant age 11-12 mo. (chi-square test, p < 0.05). More considerable increase has been noticed in period 1999-2000 which is characterized by breastfeeding support groups activity: 68% vs. 87% at infant age 1 mo., 30% vs. 66% at 3 mo., 11.5% vs. 49% at 6 mo., and 2% vs. 23% at infant age 11-12 mo. (chi-square test, p < 0.05). Our conclusion is that activities aiming to promote breastfeeding in maternity hospitals have had limited success. They have resulted in satisfactory increase of breastfeeding prevalence in early infant's period, but for far-reaching effect postnatal support is also required.     

Mechanism of thrombin-induced vasodilation in human coronary arterioles

Thrombin (Thromb), activated as part of the clotting cascade, dilates conduit arteries through an endothelial pertussis toxin (PTX)-sensitive G-protein receptor and releases nitric oxide (NO). Thromb also acts on downstream microvessels. Therefore, we examined whether Thromb dilates human coronary arterioles (HCA). HCA from right atrial appendages were constricted by 30-50% with endothelin-1. Dilation to Thromb (10(-4)-1 U/ml) was assessed before and after inhibitors with videomicroscopy. There was no tachyphylaxis to Thromb dilation (maximum dilation = 87.0%, ED(50) = 1.49 x 10(-2)). Dilation to Thromb was abolished with either hirudin or denudation but was not affected by PTX. Neither N(omega)-nitro-l-arginine methyl ester (n = 7), indomethacin (n = 9), (1)H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (n = 6), tetraethylammonium chloride (n = 5), nor iberiotoxin (n = 4) reduced dilation to Thromb. However, KCl (maximum dilation = 89 +/- 5 vs. 20 +/- 10%; P < 0.05; n = 7), tetrabutylammonium chloride (maximum dilation = 79 +/- 7 vs. 21 +/- 4%; P < 0.05; n = 5), and charybdotoxin (maximum dilation = 89 +/- 4 vs. 10 +/- 2%; P < 0.05; n = 4) attenuated dilation to Thromb. In contrast to animal models, Thromb-induced dilation in human arterioles is independent of G(i)-protein activation and NO release. However, Thromb dilation is endothelium dependent, is maintained on consecutive applications, and involves activation of K(+) channels. We speculate that an endothelium-derived hyperpolarizing factor contributes to Thromb-induced dilation in HCA.     

Herpes simplex virus infection of human dendritic cells induces apoptosis and allows cross-presentation via uninfected dendritic cells

HSV efficiently infects dendritic cells (DCs) in their immature state and induces down-regulation of costimulatory and adhesion molecules. As in mice, HSV infection of human DCs also leads to their rapid and progressive apoptosis, and we show that both early and late viral proteins contribute to its induction. Because topical HSV infection is confined to the epidermis, Langerhans cells are expected to be the major APCs in draining lymph nodes. However, recent observations in murine models show T cell activation to be mediated by nonepidermal DC subsets, suggesting cross-presentation of viral Ag. In this study we provide an explanation for this phenomenon, demonstrating that HSV-infected apoptotic DCs are readily phagocytosed by uninfected bystander DCs, which, in turn, stimulate virus-specific CD8+ T cell clones.