HiCRes: a computational method to estimate and predict the genomic resolution of Hi-C libraries

Three-dimensional (3D) conformation of the chromatin is crucial to stringently regulate gene expression patterns and DNA replication in a cell-type specific manner. Hi-C is a key technique for measuring 3D chromatin interactions genome wide. Estimating and predicting the resolution of a library is an essential step in any Hi-C experimental design. Here, we present the mathematical concepts to estimate the resolution of a dataset and predict whether deeper sequencing would enhance the resolution. We have developed HiCRes, a docker pipeline, by applying these concepts to several Hi-C libraries.     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Aulonemia bogotensis (Poaceae: Bambusoideae), a new species from the Cordillera Oriental of Colombia

A new species of Aulonemia from the páramos of the Cordillera Oriental, Cundinamarca, Colombia, is described and illustrated. Aulonemia bogotensis is distinguished by its diminutive size, abaxially tomentose foliage leaf blades, and spikelets with muticous lemmas. Two similar Colombian endemics, A. pumila and A. trianae, are compared and contrasted with the new species.     

Sexual Differences in the Behavioral Response of Túngara Frogs, Physalaemus pustulosus, to Cues Associated with Increased Predation Risk

Engaging in mating behaviors usually increases exposure to predators for both males and females. Anti‐predator strategies during reproduction may have important fitness consequences for prey. Previous studies have shown that individuals of several species adjust their reproductive behavior according to their assessment of predation risk, but few studies have explored potential sexual differences in these strategies. In this study, we investigate whether the acoustic cues associated with predatory attacks or those associated with predators themselves affect the mating behavior of female and male túngara frogs, Physalaemus pustulosus. We compared the responses of females approaching a mate and those of calling males when exposed to mating calls associated with sounds representing increased hazard. When presented with mating calls that differed only in whether or not they were followed by a predation‐related sound, females preferentially approached the call without predation‐related sounds. In contrast to females, calling males showed greater vocal response to calls associated with increased risk than to a call by itself. We found significant differences in the responses of females and males to several sounds associated with increased hazard. Females behaved more cautiously than males, suggesting that the sexes balance the risk of predation and the cost of cautious mating strategies differently.     

Characterization of a Novel Alkalophilic Lipase From <Emphasis Type="Italic">Aneurinibacillus thermoaerophilus</Emphasis>: Lid Heterogeneity and Assignment to Family I.5

Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase α/β hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 °C under neutral pH, but its maximal activity was detected at pH 10 and 45 °C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 °C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure–function relationships in lipases.     

The development of Alternaria alternata is prevented by chitinases and beta-1,3-glucanases from Citrus limon seedlings

In addition to phytoalexin synthesis, the defense response of intact Citrus limon seedlings against Alternaria alternata involves both constitutive and induced enzyme activities such as chitinases (Ch) and beta-1,3-glucanases (Glu). A alternata conidial germination was prevented by protein extracts from inoculated lemon seedlings, but also by extracts from mock-inoculated specimens. On the other hand, degradation of mycelia was accomplished only by protein extracts from inoculated seedlings. The presence of six Ch isoenzymes and of four Glu isoenzymes was detected in protein extracts from mock-inoculated seedlings. As a result of fungal inoculation, the isoenzyme pattern of Ch and Glu changed, making possible the detection of a new Ch isoenzyme and of three new Glu. Also, some constitutive Ch and Glu increased their enzyme activity, and those Ch that increased their activity also showed a broadening of their substrate specificity. These changes were prevented by alpha-amanitin and cycloheximide, suggesting that the presence of new Ch and Glu isoenzymes was due to de novo synthesis of proteins. Results suggest that constitutive Ch and Glu could act as pre-formed defense molecules in Citrus limon preventing A. alternata germination, while those induced after fungal inoculation of lemon seedlings could act along with the former, to produce lysis of fungal mycelia, resulting in a more efficient control of A. alternata development.     

Protection against experimental malaria associated with AMA-1 peptide analogue structures

We have identified, using site-directed mutagenesis, a proline located at position 13 of Baxalpha (Bax) as crucial for the maintenance of its cytosolic conformation. The substitution of this proline by a valine results in a strong binding of Bax to mitochondria and to conformational changes monitored by a decreased sensitivity of Bax to mild proteolysis and the enhancement of its oligomerization state. Deletion of the C-terminus of Bax does not modify its intracellular localization. On the other hand, the pro-apoptotic activity of Bax is enhanced by a deletion of the C-terminus in the absence of the N-terminus but is decreased in its presence. These results suggest that both extremities functionally interact to control the activity but not the subcellular localization of Bax.     

Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus

Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.     

Next-Generation Sequencing of Human Mitochondrial Reference Genomes Uncovers High Heteroplasmy Frequency

We describe methods for rapid sequencing of the entire human mitochondrial genome (mtgenome), which involve long-range PCR for specific amplification of the mtgenome, pyrosequencing, quantitative mapping of sequence reads to identify sequence variants and heteroplasmy, as well as de novo sequence assembly. These methods have been used to study 40 publicly available HapMap samples of European (CEU) and African (YRI) ancestry to demonstrate a sequencing error rate <5.63×10⁻⁴, nucleotide diversity of 1.6×10⁻³ for CEU and 3.7×10⁻³ for YRI, patterns of sequence variation consistent with earlier studies, but a higher rate of heteroplasmy varying between 10% and 50%. These results demonstrate that next-generation sequencing technologies allow interrogation of the mitochondrial genome in greater depth than previously possible which may be of value in biology and medicine.