Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase

The CRISPR-Cas9 system is a powerful and revolutionary genome-editing tool for eukaryotic genomes, but its use in bacterial genomes is very limited. Here, we investigated the use of the Streptococcus pyogenes CRISPR-Cas9 system in editing the genome of Clostridium cellulolyticum, a model microorganism for bioenergy research. Wild-type Cas9-induced double-strand breaks were lethal to C. cellulolyticum due to the minimal expression of nonhomologous end joining (NHEJ) components in this strain. To circumvent this lethality, Cas9 nickase was applied to develop a single-nick-triggered homologous recombination strategy, which allows precise one-step editing at intended genomic loci by transforming a single vector. This strategy has a high editing efficiency (>95%) even using short homologous arms (0.2 kb), is able to deliver foreign genes into the genome in a single step without a marker, enables precise editing even at two very similar target sites differing by two bases preceding the seed region, and has a very high target site density (median interval distance of 9 bp and 95.7% gene coverage in C. cellulolyticum). Together, these results establish a simple and robust methodology for genome editing in NHEJ-ineffective prokaryotes.     

The dichotomy in the DNA-binding behaviour of ruthenium(II) complexes bearing benzoxazole and benzothiazole groups

The substituted tris(bipyridine)ruthenium(II) complexes {[Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) [where bpy=2,2'-bipyridine and bbob=bis(benzoxazol-2-yl)-2,2'-bipyridine] have been prepared and compared to the previously studied complex [Ru(bpy)(2)(4,4'-bbtb)](2+) [where bbtb=bis(benzothiazol-2-yl)-2,2'-bipyridine]. From the UV/VIS titration studies, Delta-[Ru(bpy)(2)(4,4'-bbob)](2+) displays a stronger association than the Lambda-isomer with calf-thymus DNA (ct-DNA). For [Ru(bpy)(2)(5,5'-bbob)](2+), there appears to be minimal interaction with ct-DNA. The results of fluorescence titration studies suggest that [Ru(bpy)(2)(4,4'-bbob)](2+) gives an increase in emission intensity with increasing ct-DNA concentrations, with an enantiopreference for the Delta isomer, confirmed by membrane dialysis studies. The fluorescent intercalation displacement studies revealed that [Ru(bpy)(2)(4,4'-bbob)](2+) and [Ru(bpy)(2)(5,5'-bbob)](2+) display a preference for more open DNA structures such as bulge and hairpin sequences. While Lambda-[Ru(bpy)(2)(4,4'-bbtb)](2+) has shown the most significant affinity for all the oligonucleotides sequences screened in previous studies, it is the Delta isomer of the comparable benzoxazole ruthenium(II) complex (Delta-[Ru(bpy)(2)(4,4'-bbob)](2+)) that preferentially binds to DNA.     

Grazing lucerne as fattening management for young bulls: technical and economic performance and diet authentication

Three fattening systems were evaluated from weaning to slaughter in order to find alternatives to grain feeding in young bulls, and to test the reliability of carcass subcutaneous fat colour to discriminate among them. After weaning (224 kg), one group of animals was fed concentrates and straw until they reached the target slaughter weight (450 kg; Feedlot), another group grazed rotationally on lucerne supplemented with 1.8 kg DM/day barley until slaughter (LUC), and the third group had the same management as LUC animals for 3 months (period 1) and thereafter was finished on concentrates and straw until slaughter (period 2; LUC + Feedlot). Animals were weighed weekly and sampled monthly for serum IGF-I and leptin, and plasma non-esterified fatty acids and carotenoid pigment concentration analyses. Carcass characteristics and subcutaneous fat colour were recorded after slaughter. In period 1, Feedlot animals had slightly greater weight gains than their grazing counterparts (P < 0.10), and at the end of period 1 they had 66% greater IGF-I and 35% greater leptin concentration (P < 0.01). Plasma carotenoid pigments were undetectable in Feedlot animals, but increased during grazing in LUC and LUC + Feedlot treatments. In period 2, weight gains were lowest for LUC, intermediate for Feedlot and greatest for LUC + Feedlot animals (P < 0.001), conditioning the time taken to reach slaughter weight (73, 58 and 47 days, respectively; P < 0.05). Leptin and IGF-I concentrations increased in all management systems during period 2. Plasma carotenoid pigment concentration reached its maximum at the end of period 2 in LUC animals, but it decreased sharply in LUC + Feedlot animals in this period. Management did not affect carcass traits except for subcutaneous fat colour. Yellowness, Chroma (C*) and the value of the integral of the translated reflectance spectrum (SUM), estimator of carotenoid pigment content in fat, were higher in LUC than in LUC + Feedlot and Feedlot animals (P < 0.001). Two logistic regressions were obtained to discriminate carcasses from LUC treatment: P (LUC) = (1 + e(18.8-5.6 × lightness-36.9 × redness + 0.3 × SUM + 29.8 × C*))(-1) and LUC + Feedlot treatment: P (LUC + Feedlot)=(1 + e(833.7-11.8 × lightness + 4.7 × redness + 0.2 × SUM-2.5 × C*))(-1). The economic margin, calculated as income achieved minus costs, was greatest for LUC, intermediate for LUC + Feedlot and lowest for Feedlot treatment. Therefore, grazing lucerne supplemented with barley was an interesting alternative for fattening young bulls in these conditions, producing carcasses of similar quality, which could be accurately traced by measuring subcutaneous fat colour.     

Metabolism of some amino acids in relation to the photorespiratory nitrogen cycle of pea leaves

¹⁵N-labelled (amino group) asparagine (Asn), glutamate (Glu), alanine (Ala), aspartate (Asp) and serine (Ser) were used to study the metabolic role and the participation of each compound in the photorespiratory N cycle ofPisum sativum L. leaves. Asparagine was utilised as a nitrogen source by either deamidation or transamination, Glu was converted to Gln through NH₃ assimilation and was a major amino donor for transamination, and Ala was utilised by transamination to a range of amino acids. Transamination also provided a pathway for Asp utilisation, although Asp was also used as a substrate for Asn synthesis. In the photorespiratory synthesis of glycine (Gly), Ser, Ala, Glu and Asn acted as sources of amino-N, contributing, in the order given, 38, 28, 23, and 7% of the N for glycine synthesis; Asp provided less than 4% of the amino-N in glycine. Calculations based on the incorporation of¹⁵N into Gly indicated that about 60% (Ser), 20% (Ala), 12% (Glu) and 11% (Asn) of the N metabolised from each amino acid was utilised in the photorespiratory nitrogen cycle.Abbreviations Ala alamine - Asn asparagine - Asp aspartate - Glu glutamate - MOA methoxylamine - Ser serine     

Performances of improved cassava (Manihot esculenta Crantz) cultivars against root rot disease and yield in cassava-maize intercropping systems under natural infection

This study examined the performances of 21 cassava cultivars in two cropping seasons on the field against root rot disease and the yield in cassava-maize intercrop. Data were collected on number of root/plant, weight of root (t/ha) and disease severity (DS) on rotted roots at 12 and 16 months after planting (MAP), respectively. There were significant (P ≤ 0.05) differences for DS at 12 and 16 MAP in both seasons with cultivar TMS 97/JW2 having the least DS score. TMS 97/JW2 was resistant to the root rot pathogen, while eleven other cultivars were moderately resistant to the disease at 16 MAP. There was no consistency in the roots weight for the cultivars over the two cropping seasons but higher roots weight was recorded at 16 MAP than 12 MAP with different cultivars having highest roots weight at these periods. Intercropping maize with cassava does not have any management potential on root rot development.     

Targeted discovery of quantitative trait loci for resistance to northern leaf blight and other diseases of maize

To capture diverse alleles at a set of loci associated with disease resistance in maize, heterogeneous inbred family (HIF) analysis was applied for targeted QTL mapping and near-isogenic line (NIL) development. Tropical maize lines CML52 and DK888 were chosen as donors of alleles based on their known resistance to multiple diseases. Chromosomal regions (“bins”; n = 39) associated with multiple disease resistance (MDR) were targeted based on a consensus map of disease QTLs in maize. We generated HIFs segregating for the targeted loci but isogenic at ~97% of the genome. To test the hypothesis that CML52 and DK888 alleles at MDR hotspots condition broad-spectrum resistance, HIFs and derived NILs were tested for resistance to northern leaf blight (NLB), southern leaf blight (SLB), gray leaf spot (GLS), anthracnose leaf blight (ALB), anthracnose stalk rot (ASR), common rust, common smut, and Stewart’s wilt. Four NLB QTLs, two ASR QTLs, and one Stewart’s wilt QTL were identified. In parallel, a population of 196 recombinant inbred lines (RILs) derived from B73 × CML52 was evaluated for resistance to NLB, GLS, SLB, and ASR. The QTLs mapped (four for NLB, five for SLB, two for GLS, and two for ASR) mostly corresponded to those found using the NILs. Combining HIF- and RIL-based analyses, we discovered two disease QTLs at which CML52 alleles were favorable for more than one disease. A QTL in bin 1.06–1.07 conferred resistance to NLB and Stewart’s wilt, and a QTL in 6.05 conferred resistance to NLB and ASR.     

Characterizing the glycome of the mammalian immune system

The outermost layer of all immune cells, the glycocalyx, is composed of a complex mixture of glycoproteins, glycolipids and lectins, which specifically recognize particular glycan epitopes. As the glycocalyx is the cell's primary interface with the external environment many biologically significant events can be attributed to glycan recognition. For this reason the rapidly expanding glycomics field is being increasingly recognized as an important component in our quest to better understand the functioning of the immune system. In this review, we highlight the current status of immune cell glycomics, with particular attention being paid to T- and B-lymphocytes and dendritic cells. We also describe the strategies and methodologies used to define immune cell glycomes.     

Kinetic characterization of yeast pyruvate carboxylase isozyme pyc1

Yeast (Saccharomyces cerevisiae) is unusual in being the only organism thus far identified as having two genes for pyruvate carboxylase. The expression of the two isozymes Pyc1 and Pyc2 appears to be differentially regulated, and since both are expressed cytoplasmically, this suggests that they have different properties. To the present, little has been done to characterize these isozymes, and almost all of the published kinetic information on yeast pyruvate carboxylase comes from measurements of enzyme prepared from bakers' yeast which is likely to be a mixture of both isozymes. Here we have measured basic kinetic parameters for Pyc1 and found that the K(a) of this isozyme for acetyl CoA is in the order of 8-10-fold higher than previously recorded, suggesting that Pyc1 and Pyc2 may be differentially regulated by this effector. Pyc1 is highly dependent on the presence of acetyl CoA for activity and in this respect is similar to chicken liver pyruvate carboxylase. However, unlike the chicken liver enzyme, the quaternary structure of the enzyme is quite stable in the absence of acetyl CoA, and the major locus of action of this effector appears to lie outside of the stimulation of the biotin carboxylation reaction.     

A synthetic peptide analog of in silico-predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits

Development of a serotyping-capable dengue detection test is hampered by the absence of an identified unique marker that can detect specific dengue virus (DENV) serotype. In the current commercially available antibody-capture diagnostic methods, immobilized nonstructural 1 (NS1) antigen indiscriminately binds and detects immunoglobulin M or immunoglobulin G against any serotype, thus limiting its capability to distinguish existing serotypes of dengue. Identification of dengue serotype is important because certain serotypes are associated with severe forms of dengue as well as dengue hemorrhagic fever. In this study, we aimed to identify an immunogenic epitope unique to DENV2 NS1 antigen and determine the binding specificity of its synthetic peptide mimotope to antibodies raised in animal models. Selection of a putative B-cell epitope from the reported DENV2 NS1 antigen was done using Kolaskar and Tongaonkar Antigenicity prediction, Emini surface accessibility prediction, and Parker hydrophilicity prediction available at the immune epitope database and analysis resource. Uniqueness of the B-cell epitope to DENV2 was analyzed by BLASTp. Immunogenicity of the synthetic peptide analog of the predicted immunogenic epitope was tested in rabbits. The binding specificity of the antibodies raised in animals and the synthetic peptide mimotope was tested by indirect ELISA. A synthetic peptide analog comprising the unique epitope of DENV2 located at the 170th–183rd position of DENV2 NS1 was found to be immunogenic in animal models. The antipeptide antibody produced in rabbits showed specific binding to the synthetic peptide mimotope of the predicted unique DENV2 NS1 immunogenic epitope.     

Integrating spatial and temporal variability into the analysis of fish food web linkages in Tijuana Estuary

Our understanding of fish feeding interactions at Tijuana Estuary was improved by incorporating estimates of spatial and temporal variability into diet analyses. We examined the stomach contents of 7 dominant species (n=579 total fish) collected between 1994 and 1999. General feeding patterns pooled over time produced a basic food web consisting of 3 major trophic levels: (1) primary consumers (Atherinops affinis, Mugil cephalus) that ingested substantial amounts of plant material and detritus; (2) benthic carnivores (Clevelandia ios, Hypsopsetta guttulata, Gillichthys mirabilis, and Fundulus parvipinnis) that ingested high numbers of calanoid copepods and exotic amphipods (Grandidierella japonica); and (3) piscivores (Paralichthys californicus and Leptocottus armatus) that often preyed on smaller gobiids. Similarity-based groupings of individual species' diets were identified using nonmetric multidimensional scaling to characterize their variability within and between species, and in space and time. This allowed us identify major dietary shifts and recognize events (i.e., modified prey abundance during 1997–98 El Ni no floods) that likely caused these shifts.