Molecular phylogenetic reconstruction improves the taxonomic understanding of Indian Dipcadi (Asparagaceae) and reveals a new species from the bank of Hiranyakeshi River, Maharashtra, India.

Dipcadi (Scilloideae: Asparagaceae) is a genus of bulbous monocots with approximately 40 species, of which 13 occur in India. Species delimitation within the genus has been troublesome hindering a comprehensive phylogenetic analysis. The most recent phylogeny of the subfamily Ornithogaloideae included six species of Dipcadi only from Africa. Here, we reconstructed the phylogeny of Ornithogaloideae including 23 accessions comprising 13 recognized taxa (11 species and two varieties) of Indian Dipcadi. The phylogenetic analyses were based on nucleotide sequences of three plastid regions (rbcL, matK and trnL-F spacer) and one nuclear region (ITS). Pseudogaltonia clavata exhibited sister relationship to Dipcadi. Our combined nuclear + plastid dataset analyses revealed a monophyletic Dipcadi with five clades, Clade I-V. Clade I, II and III included mainly Indian species whereas Clade V included mostly African species. Clade IV comprised D. serotinum. Clade I included nine taxa including our newly described species, D. mukaianum. The new species was phylogenetically placed with D. erythraeum, D. saxorum and D. ursulae. Morphologically, the species resembled D. montanum and D. ursulae but differed in characters such as tepal cohesion, number of ovules per locule and foul-smelling flowers. Clade II and III included 11 and six taxa, respectively. D. erythraeum which has a native range from Egypt to western India was found in Clades I and V. The widespread Dipcadi species, viz. D. erythraeum and D. serotinum showed polyphyly however, the monophyly of Dipcadi is established. Our studies suggest that additional molecular markers (plastid as well as nuclear) should be tested for their taxonomy utility. Further work on the historical biogeography of Dipcadi on the subfamily Ornithogaloideae with more genetic data will yield insights how aridification of the landscape would have shaped the evolution of the geographical clades.

Post-infarction ventricular septal defect: percutaneous or surgical management in the UK national registry.

AIMS: Post-infarction ventricular septal defect (PIVSD) is a mechanical complication of acute myocardial infarction (AMI) with a poor prognosis. Surgical repair is the mainstay of treatment, although percutaneous closure is increasingly undertaken. METHODS AND RESUTS: Patients treated with surgical or percutaneous repair of PIVSD (2010-2021) were identified at 16 UK centres. Case note review was undertaken. The primary outcome was long-term mortality. Patient groups were allocated based upon initial management (percutaneous or surgical). Three-hundred sixty-two patients received 416 procedures (131 percutaneous, 231 surgery). 16.1% of percutaneous patients subsequently had surgery. 7.8% of surgical patients subsequently had percutaneous treatment. Times from AMI to treatment were similar [percutaneous 9 (6-14) vs. surgical 9 (4-22) days, P = 0.18]. Surgical patients were more likely to have cardiogenic shock (62.8% vs. 51.9%, P = 0.044). Percutaneous patients were substantially older [72 (64-77) vs. 67 (61-73) years, P < 0.001] and more likely to be discussed in a heart team setting. There was no difference in long-term mortality between patients (61.1% vs. 53.7%, P = 0.17). In-hospital mortality was lower in the surgical group (55.0% vs. 44.2%, P = 0.048) with no difference in mortality after hospital discharge (P = 0.65). Cardiogenic shock [adjusted hazard ratio (aHR) 1.97 (95% confidence interval 1.37-2.84), P < 0.001), percutaneous approach [aHR 1.44 (1.01-2.05), P = 0.042], and number of vessels with coronary artery disease [aHR 1.22 (1.01-1.47), P = 0.043] were independently associated with long-term mortality. CONCLUSION: Surgical and percutaneous repair are viable options for management of PIVSD. There was no difference in post-discharge long-term mortality between patients, although in-hospital mortality was lower for surgery.

Acute aortic regurgitation due to fibrous strand rupture.

A 60-year-old male presented with sudden onset chest pain and pulmonary edema. The investigation confirmed torrential aortic regurgitation of a bicuspid valve. At surgery, a ruptured fibrous strand was identified which had been supporting the left-right cusp commissure with loss of attachment to the aortic wall. This case demonstrates that fibrous strands may be present as a supporting structure of the aortic valve, and rupture can be a rare cause of torrential aortic regurgitation, similar in pathogenesis to how it may be associated with acute severe mitral regurgitation and chordae tendineae rupture.

Cost-effectiveness of Denosumab for the Treatment of Postmenopausal Osteoporosis in Malaysia.

From the perspective of Malaysian health care providers, denosumab was cost-effective in the treatment of postmenopausal osteoporosis, with an optimal outcome starting at age 60 years. Our results provide important insights into the value for money of anti-osteoporotic agents that can serve as a reference for other countries with comparable epidemiological data. INTRODUCTION: The study aimed to compare the cost-effectiveness of denosumab with alendronate and no treatment in the management of postmenopausal osteoporosis among the Malaysian population. METHODS: A well-validated Markov model was used to estimate the cost-effectiveness of denosumab in a hypothetical cohort of postmenopausal osteoporotic women between 50 and 80 years old who had no history of fractures. A 10-year time horizon from the perspective of Malaysian health care providers was used in this analysis. The model parameters, including transition probabilities and costs, were based on Malaysian sources. Treatment efficacy data were obtained from a network meta-analysis. The study outcomes were presented as incremental cost per quality-adjusted life-year (QALY) gained. Sensitivity analyses were performed to ensure the robustness of the results. A cost-effectiveness threshold was set at MYR 21,438 (USD 5175) per QALY. RESULTS: Denosumab was found to be a cost-effective option for postmenopausal osteoporotic women aged 60 and older. The incremental cost-effectiveness ratios (ICERs) for denosumab versus alendronate ranged from MYR 16,955 (USD 4093) per QALY at age 60 to MYR 4380 (USD 1057) per QALY at age 80. The cost-effectiveness of denosumab improved monotonically with increasing age. Denosumab was 72.8-92.7% likely to be cost-effective at the cost-effectiveness threshold. Sensitivity analyses demonstrated that the results were robust across all parameter variations, with the annual cost of denosumab being the most sensitive. CONCLUSIONS: From the perspective of the Malaysian health care provider, denosumab appears to be a cost-effective treatment choice for postmenopausal osteoporotic women over 60 years of age.

Cardiac tamponade from peripherally-inserted central venous catheters in neonates: Three case reports.

Pericardial effusion (PCE) and cardiac tamponade (CT) are rare but life-threatening complications of percutaneouslyinserted central catheter (PICC) use in neonates. There is often a lack in index of suspicion in a neonate with sudden deterioration, resulting in high mortality. We describe a series of three cases of PICC-related PCE/CT in low birth weight infants whose timely diagnosis with echocardiography and pericardiocentesis led to successful resuscitation and survival. We suggest that echocardiographic skills to determine tip position and pericardiocentesis be taught in neonatal resuscitation programs to ensure good outcomes of this otherwise fatal complication.

The association between the reflux symptoms index and nasal symptoms among patients with non-allergic rhinitis.

BACKGROUND: Laryngopharyngeal reflux symptoms assessed with the reflux symptom index can overlap with non-allergic rhinitis symptoms. This study aims to explore the association between the reflux symptom index and nasal symptoms in non-allergic rhinitis patients. METHODS: A cross-sectional study was conducted on consecutive adults with non-allergic rhinitis. The reflux symptom index (score of more than 13 = laryngopharyngeal reflux) and nasal symptoms (categorised as mild (total score of 0-3), moderate (4-7) or severe (8-12)) were assessed. RESULTS: The study included 227 participants (aged 58.64 ± 12.39 years, 59.5 per cent female). The reflux symptom index scores increased with total nasal symptom scores (mild vs moderate vs severe, 8.61 ± 6.27 vs 12.94 ± 7.4 vs 16.40 ± 8.10; p < 0.01). Logistic regression indicated that laryngopharyngeal reflux is more likely in patients with severe nose block (odds ratio 5.47 (95 per cent confidence interval = 2.16-13.87); p < 0.01). CONCLUSION: Laryngopharyngeal reflux symptoms are associated with nasal symptom severity, and nasal symptoms should be primarily treated. Those with predominant nose block and laryngopharyngeal reflux symptoms are more likely to have laryngopharyngeal reflux.

Inhibitory Effect of Imiquimod-Induced Psoriasis-Like Skin Inflammation in Mice by Histamine H4 Receptor Agonist 4-Methylhistamine.

Psoriasis is a chronic inflammatory immune-mediated autoimmune skin disorder. The histamine H4 receptor (H4R) agonist 4-methylhistamine (4-MH) plays an important role in immunomodulation of inflammatory responses associated with allergic inflammatory diseases. In this study, we investigated the effects of H4R agonist 4-MH on the development of imiquimod (IMQ)-induced psoriasis-like skin inflammation in mice and explored the immunoregulatory mechanism involved. The total clinical severity scores were significantly ameliorated by treatment with 4-MH (20 mg/kg) and 4-MH (40 mg/kg). Histological analysis of the skin revealed that 4-MH (20 mg/kg) and 4-MH (40 mg/kg) significantly attenuated the psoriatic phenotypes, including epidermal hyperplasis, hyperkeratosis and lymphocytes infiltration. Treatment with 4-MH (20 mg/kg) and 4-MH (40 mg/kg) led to reductions in the levels of Th1 cytokines (TNF-α, IFN-α, and IL-27) in the serum and dorsal skin, whereas Th17 cytokines levels (IL-17A and IL-23) did not change in response to treatment with 4-MH (20 mg/kg) and 4-MH (40 mg/kg). Furthermore, the number of CD4(+) CD25(+) FoxP3(+) regulatory T (Treg) cells was significantly increased by treatment with 4-MH (40 mg/kg). Taken together, these results imply that H4R agonist 4-MH might be an effective immunomodulatory approach for treatment of patients with psoriasis and the effects may be related to inhibited epidermal alteration, selectively reduced Th1 pro-inflammatory cytokines, and recruited CD4(+) CD25(+) FoxP3(+) Treg cells.

Management of neonatal jaundice in primary care.

The Clinical Practice Guidelines on Management of Neonatal Jaundice 2003 was updated by a multidisciplinary development group and approved by the Ministry of Health Malaysia in 2014. A systematic review of 13 clinical questions was conducted using evidence retrieved mainly from Medline and Cochrane databases. Critical appraisal was done using the Critical Appraisal Skills Programme checklist. Recommendations were formulated based on the accepted 103 evidences and tailored to local setting as stated below. Neonatal jaundice (NNJ) is a common condition seen in primary care. Multiple risk factors contribute to severe NNJ, which if untreated can lead to adverse neurological outcomes. Visual assessment, transcutaneous bilirubinometer (TcB) and total serum bilirubin (TSB) are the methods used for the detection of NNJ. Phototherapy remains the mainstay of the treatment. Babies with severe NNJ should be followed-up to detect and manage sequelae. Strategies to prevent severe NNJ include health education, identification of risk factors, proper assessment and early referral.

Tetrafluoroethane (R134a) hydrate formation within variable volume reactor accompanied by evaporation and condensation.

Vast size hydrate formation reactors with fast conversion rate are required for the economic implementation of seawater desalination utilizing gas hydrate technology. The commercial target production rate is order of thousand tons of potable water per day per train. Various heat and mass transfer enhancement schemes including agitation, spraying, and bubbling have been examined to maximize the production capacities in scaled up design of hydrate formation reactors. The present experimental study focused on acquiring basic knowledge needed to design variable volume reactors to produce tetrafluoroethane hydrate slurry. Test vessel was composed of main cavity with fixed volume of 140 ml and auxiliary cavity with variable volume of 0 ∼ 64 ml. Temperatures at multiple locations within vessel and pressure were monitored while visual access was made through front window. Alternating evaporation and condensation induced by cyclic volume change provided agitation due to density differences among water and vapor, liquid and hydrate R134a as well as extended interface area, which improved hydrate formation kinetics coupled with latent heat release and absorption. Influences of coolant temperature, piston stroke/speed, and volume change period on hydrate formation kinetics were investigated. Suggestions of reactor design improvement for future experimental study are also made.

Effects of Dietary Fermented Chlorella vulgaris (CBT(®)) on Growth Performance, Relative Organ Weights, Cecal Microflora, Tibia Bone Characteristics, and Meat Qualities in Pekin Ducks.

Fermented Chlorella vulgaris was examined for its effects on growth performance, cecal microflora, tibia bone strength, and meat qualities in commercial Pekin ducks. A total of three hundred, day-old male Pekin ducks were divided into three groups with five replicates (n = 20 ducklings per replicate) and offered diets supplemented with commercial fermented C. vulgaris (CBT(®)) at the level of 0, 1,000 or 2,000 mg/kg, respectively for 6 wks. The final body weight was linearly (p = 0.001) increased as the addition of fermented C. vulgaris into diets increased. Similarly, dietary C. vulgaris linearly increased body weight gain (p = 0.001) and feed intake (p = 0.001) especially at the later days of the feeding trial. However, there was no C. vulgaris effect on feed efficiency. Relative weights of liver were significantly lowered by dietary fermented C. vulgaris (linear effect at p = 0.044). Dietary fermented C. vulgaris did not affect total microbes, lactic acid bacteria, and coliforms in cecal contents. Finally, meat quality parameters such as meat color (i.e., yellowness), shear force, pH, or water holding capacity were altered by adding fermented C. vulgaris into the diet. In our knowledge, this is the first report to show that dietary fermented C. vulgaris enhanced meat qualities of duck meats. In conclusion, our study indicates that dietary fermented C. vulgaris exerted benefits on productivity and can be employed as a novel, nutrition-based strategy to produce value-added duck meats.

Carcass Characteristics and Meat Quality of Korean Native Ducks and Commercial Meat-type Ducks Raised under Same Feeding and Rearing Conditions.

This study was conducted to compare carcass characteristics and physico-chemical meat quality in two different genotype ducks raised under identical feeding and rearing conditions. A total of ninety 1-d-old Korean native ducks (KND, n = 45) and commercial meat-type ducks (Grimaud, n = 45) were fed same experimental diets during 56 d and 42 d, respectively to obtain similar slaughter weights. The experimental diet for starter period contained 20% crude protein (CP) and 2,900 kcal nitrogen corrected true metabolizable energy (TMEn)/kg of diet and that for grower period contained 17% CP and 3,050 TMEn/kg of diet. Average daily gain and feed efficiency of KND were inferior to those of commercial meat-type ducks (p<0.05). Carcass weight was not different between two genetically different ducks, but carcass yield of KND was significantly higher (p<0.05) than that of commercial meat-type ducks. There were no significant differences in cooking loss and pH of breast meat between two genetically different ducks, but water holding capacity of KND was significantly higher than that of commercial meat-type ducks. The linoleic acid and total polyunsaturated fatty acid of breast meat from KND were significantly higher (p<0.05) than the corresponding part from commercial meat-type ducks. Significant differences were detected in water holding capacity and the content of linoleic acid and polyunsaturated fatty acid, which were significantly higher in KND, whereas growth performance tended to be superior in commercial ducks. At the market weight, the meat from KND was judged to have better qualities with regard to higher water holding capacity and greater content of polyunsaturated fatty acid compare with meat from commercial meat-type duck.

Comparison of growth performance, carcass characteristics and meat quality of korean local chickens and silky fowl.

This study was conducted to compare growth performance, carcass characteristics and meat quality of 4 breeds of local chicken. A total of 480 1-d-old chicks were distributed to 16 pens, with 4 treatments of breed, 4 replicates and 30 chicks per pen. Three Korean local breeds of white-mini broiler, Hanhyup-3-ho, and Woorimatdag, and a breed of silky fowl were raised under identical rearing and feeding conditions for 31-d, 37-d, 36-d, and 59-d, respectively. The BW and feed consumption on a pen basis were weekly measured for all pens, and ADFI, ADG and gain:feed were calculated for each pen. The ADFI and ADG of 3 breeds of Korean local chicken were greater than those of silky fowl (p<0.05). Within the Korean local breeds, ADFI of white-mini broiler was the highest (p<0.05), and ADG of Hanhyup-3-ho and white-mini broiler was the highest (p<0.05). Gain:feed of silky fowl was less than that of the 3 breeds of Korean local chicken. The carcass and breast yield of white-mini broiler were the greater than those of other breeds (p<0.05). The breast meat color (CIE L*, a*, and b*) of 3 breeds of Korean local chicken were higher than that of silky fowl (p<0.05). The breast meat of Hanhyup-3-ho had greater cooking loss (p<0.05), whereas water holding capacity and pH were less than those of other breeds (p<0.05). The color score of 3 breeds of Korean local chicken was higher than that of silky fowl (p<0.05). Woorimatdag had a higher score on tenderness (p<0.05), whereas flavor score was less than that of other breeds (p<0.05). In conclusion, 4 local breeds of chicken have some unique features and seem to have more advantages, and this information can help consumers who prefer healthy and premium chicken meat.

Growth performance and carcass characteristics of korean native ducks fed diets with varying levels of limiting amino acids.

There are multiple experiments conducted with male Korean native ducks (KND) to evaluate the optimal levels of limiting amino acids (AA). In Exp. 1, a total of 450 one-d-old male KNDs were divided into five groups with six replicates and fed experimental diets with varying levels of lysine, total sulfur amino acids (TSAA) and threonine (T1, 0.90/0.74/0.70%; T2, 1.00/0.82/0.77%; T3, 1.10/0.90/0.85%; T4, 1.20/0.98/0.93%; T5, 1.30/1.07/1.01%) to 21 d of age. In Exp. 2, one-d-old male KND were received and fed commercial starter diet from hatching to 21 d of age, and then divided into five groups with six replicates and fed one of five diets varying levels of lysine, TSAA, and threonine (T1, 0.73/0.62/0.54%; T2, 0.80/0.68/0.60%; T3, 0.87/0.74/0.65%; T4, 0.94/0.80/0.70%; T5, 1.01/0.86/0.75%) during 22 to 56 d of age, respectively. The BW gain was linearly increased as dietary limiting AA levels increased to 1.20% lysine, 0.98% TSAA and 0.93% threonine. There were no significant differences in feed intake, gain:feed and uniformity among groups. In Exp. 2, the BW gain and gain:feed were not affected by dietary limiting AA levels. There were no significant differences in carcass characteristics and meat quality among groups. The growth performance and carcass characteristics did not show the significant response to increasing dietary limiting AA levels in KND during 22 to 56 d of age. In conclusion, the levels of lysine, TSAA and threonine necessary to maximize growth for starter phase were at least 1.20%, 0.98%, and 0.93%, respectively. On the other hands, KND require relatively low levels of limiting AA for late growth and carcass yield. The dietary levels of 0.73% lysine, 0.62% TSAA and 0.54% threonine appear to be adequate during growing phase.

Identification of biomarkers that distinguish chemical contaminants based on gene expression profiles.

BACKGROUND: High throughput transcriptomics profiles such as those generated using microarrays have been useful in identifying biomarkers for different classification and toxicity prediction purposes. Here, we investigated the use of microarrays to predict chemical toxicants and their possible mechanisms of action. RESULTS: In this study, in vitro cultures of primary rat hepatocytes were exposed to 105 chemicals and vehicle controls, representing 14 compound classes. We comprehensively compared various normalization of gene expression profiles, feature selection and classification algorithms for the classification of these 105 chemicals into14 compound classes. We found that normalization had little effect on the averaged classification accuracy. Two support vector machine (SVM) methods, LibSVM and sequential minimal optimization, had better classification performance than other methods. SVM recursive feature selection (SVM-RFE) had the highest overfitting rate when an independent dataset was used for a prediction. Therefore, we developed a new feature selection algorithm called gradient method that had a relatively high training classification as well as prediction accuracy with the lowest overfitting rate of the methods tested. Analysis of biomarkers that distinguished the 14 classes of compounds identified a group of genes principally involved in cell cycle function that were significantly downregulated by metal and inflammatory compounds, but were induced by anti-microbial, cancer related drugs, pesticides, and PXR mediators. CONCLUSIONS: Our results indicate that using microarrays and a supervised machine learning approach to predict chemical toxicants, their potential toxicity and mechanisms of action is practical and efficient. Choosing the right feature and classification algorithms for this multiple category classification and prediction is critical.

Environmental assessment of depleted uranium used in military armor-piercing rounds in terrestrial systems.

Depleted uranium (DU) from the military testing and use of armor-piercing kinetic energy penetrators has been shown to accumulate in soils; however, little is known about the toxicity of DU geochemical species created through corrosion or weathering. The purpose of the present study was to assess the toxic effects and bioaccumulation potential of field-collected DU oxides to the model terrestrial invertebrates Eisenia fetida (earthworm) and Porcellio scaber (isopod). Earthworm studies were acute (72 h) dermal exposures or 28-d spiked soil exposures that used noncontaminated field-collected soils from the US Army's Yuma and Aberdeen Proving Grounds. Endpoints assessed in earthworm testing included bioaccumulation, growth, reproduction, behavior (soil avoidance), and cellular stress (neutral red uptake in coelomocytes). Isopod testing used spiked food, and endpoints assessed included bioaccumulation, survival, and feeding behavior. Concentration-dependent bioaccumulation of DU in earthworms was observed with a maximum bioaccumulation factor of 0.35; however, no significant reductions in survival or impacts to cellular stress were observed. Reproduction lowest-observed-effect concentrations (LOEC) of 158 mg/kg and 96 mg/kg were observed in Yuma Proving Ground and a Mississippi reference soil (Karnac Ferry), respectively. Earthworm avoidance of contaminated soils was not observed in 48-h soil avoidance studies; however, isopods were shown to avoid food spiked with 12.7% by weight DU oxides through digital tracking studies.

Conversion of residual organics in corn stover-derived biorefinery stream to bioenergy via a microbial fuel cell.

A biorefinery process typically uses about 4-10 times more water than the amount of biofuel generated. The wastewater produced in a biorefinery process contains residual sugars, 5-furfural, phenolics, and other pretreatment and fermentation byproducts. Treatment of the wastewater can reduce the need for fresh water and potentially add to the environmental benefits of the process. Use of microbial fuel cells (MFCs) for conversion of the complete range of phenolic compounds and furan aldehyde derivatives present in a postfermentation biorefinery stream is reported here. The consortium was capable of removing the molecules simultaneously with sugars, which were present at 2 orders of magnitude higher concentrations. Organic loading in a fed-batch MFC affected Coulombic efficiency, which decreased from 40% at 0.66 g/L loading to 1.8% at 66.4 g/L loading. Power density increased with loading reaching 1180 mW/m(2) at 5.3 g/L (8% dilution), but decreased thereafter. Excessive loading leads to poor electrogenic performance; therefore, operation of an MFC at an intermediate loading using dilution and recirculation of the process stream can enable effective treatment with bioenergy recovery.

Down-regulation of the caffeic acid O-methyltransferase gene in switchgrass reveals a novel monolignol analog.

BACKGROUND: Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography-mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors. RESULTS: GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples. CONCLUSIONS: Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations.

BACKGROUND: The model bacterium Clostridium cellulolyticum efficiently degrades crystalline cellulose and hemicellulose, using cellulosomes to degrade lignocellulosic biomass. Although it imports and ferments both pentose and hexose sugars to produce a mixture of ethanol, acetate, lactate, H2 and CO2, the proportion of ethanol is low, which impedes its use in consolidated bioprocessing for biofuels production. Therefore genetic engineering will likely be required to improve the ethanol yield. Plasmid transformation, random mutagenesis and heterologous expression systems have previously been developed for C. cellulolyticum, but targeted mutagenesis has not been reported for this organism, hindering genetic engineering. RESULTS: The first targeted gene inactivation system was developed for C. cellulolyticum, based on a mobile group II intron originating from the Lactococcus lactis L1.LtrB intron. This markerless mutagenesis system was used to disrupt both the paralogous L-lactate dehydrogenase (Ccel_2485; ldh) and L-malate dehydrogenase (Ccel_0137; mdh) genes, distinguishing the overlapping substrate specificities of these enzymes. Both mutations were then combined in a single strain, resulting in a substantial shift in fermentation toward ethanol production. This double mutant produced 8.5-times more ethanol than wild-type cells growing on crystalline cellulose. Ethanol constituted 93% of the major fermentation products, corresponding to a molar ratio of ethanol to organic acids of 15, versus 0.18 in wild-type cells. During growth on acid-pretreated switchgrass, the double mutant also produced four times as much ethanol as wild-type cells. Detailed metabolomic analyses identified increased flux through the oxidative branch of the mutant's tricarboxylic acid pathway. CONCLUSIONS: The efficient intron-based gene inactivation system produced the first non-random, targeted mutations in C. cellulolyticum. As a key component of the genetic toolbox for this bacterium, markerless targeted mutagenesis enables functional genomic research in C. cellulolyticum and rapid genetic engineering to significantly alter the mixture of fermentation products. The initial application of this system successfully engineered a strain with high ethanol productivity from cellobiose, cellulose and switchgrass.

Tauroursodeoxycholic acid enhances the pre-implantation embryo development by reducing apoptosis in pigs.

Apoptosis is an important determinant of the normal development of pre-implantation embryos in vitro. Recently, endoplasmic reticulum (ER) stress-mediated apoptosis has been extensively investigated in a wide variety of diseases. Efficient functioning of the ER is essential for most cellular activities and survival. Tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, has been reported to attenuate ER stress-mediated cell death by interrupting the classic pathways of apoptosis. Therefore, in this study, the anti-apoptotic effect of TUDCA on ER stress-induced apoptosis was examined in pre-implantation pig embryos. Also, tunicamycin was used to investigate the effects of ER stress on pig embryo development. After in vitro maturation and fertilization, presumptive pig embryos were cultured in NCSU-23 medium supplemented with TUDCA or TM for 6 days at 39 °C, 5% CO(2) in air. All data were analysed using one-way anova and Duncan's multiple range test in the statistical analysis system (SAS). In addition, we also determined the optimal TM and TUDCA concentrations. Samples were treated with TM at concentrations of 0, 1, 2 or 5 µm and with TUDCA at concentrations of 0, 100, 200 or 300 µm. When TM was used during in vitro culture, only 8.2% (8/97) of the embryos developed to the blastocyst stage when the treatment concentration was 1 µm compared with 27.4% (28/102) of the embryos in the control group (p < 0.05). In contrast, the frequency of blastocyst formation and the number of cells were higher when treated with 200 µm TUDCA compared with the control group (32.8% and 39.5 vs 22.2% and 35.6, p < 0.05). Moreover, the developmental rate to the blastocyst stage embryo in the group treated with TM and TUDCA was not significantly different from that of the control group (17.8%, 26/142 vs 24.9%, 36/145). Furthermore, the blastocyst cell number was enhanced (31.9 vs 36.9) and apoptosis reduced (TUNEL-positive nuclei number, 6.0 vs 3.2) by TUDCA treatment in pig embryos. In the real-time quantitative RT-PCR analysis, the expression of anti-apoptotic Bcl-XL gene was shown to be increased in the blastocyst stage because of TUDCA treatment, whereas expression of pro-apoptotic Bax was decreased. In addition, we also found that TUDCA decreased the rate of TM-induced apoptosis in the pre-implantation stage. Taken together, our results indicate that TUDCA improves the developmental competence of pig embryos by modulating ER stress-induced apoptosis during the pre-implantation stage.