Identification of a mutation that causes exon skipping during collagen pre-mRNA splicing in an Ehlers-Danlos syndrome variant

Recent biochemical studies have shown that the fibroblasts from a patient with Ehlers-Danlos Syndrome Type VIIB produce nearly equal amounts of normal and shortened pro-alpha 2(I) collagen chains (Wirtz, M.K., Glanville, R. W., Steinmann, B., Rao, V. H., and Hollister, D. (1987) J. Biol. Chem. 262, 16376-16385). Compositional and sequencing studies of the abnormal pro-alpha 2(I) chain identified an interstitial deletion of 18 residues corresponding to the N-telopeptide of the collagen molecule. Since this region is encoded by a 54-base pair exon, number 6, the protein defect could have been caused by gene deletion, abnormal pre-mRNA splicing, or both. Here, in order to elucidate the molecular nature of this mutation we have analyzed the sequences of pro-alpha 2(I) collagen cDNA and genomic clones obtained from RNA and DNA of the patient's fibroblasts. Using oligomer-specific cloning we identified a cDNA that contains a 54-base pair deletion corresponding precisely to the sequence of exon 6. Identification of the normal gene was based on the finding of an identical sequence polymorphism in a normal cDNA and in the genomic clone derived from one of the two collagen alleles. The other gene, instead, displayed a base substitution (T to C) in the obligatory GT dinucleotide of the 5' splice-site sequence of intron 6. Analysis of nearly 100 base pairs immediately 5' to exons 5, 6, and 7, and 3' to exons 5 and 7 did not reveal any additional change. Therefore, the data strongly suggest that the observed GT-to-GC transition at the splice donor site of intron 6 generates an abnormally spliced mRNA in which the sequence of exon 5 is joined to the sequence of exon 7. Since skipping of exon 6 does not interfere with the coding frame of the mRNA, the resulting shortened polypeptide, albeit utilized in the assembly of a procollagen trimer, ultimately causes the Ehlers-Danlos Syndrome Type VII phenotype.     

Effects of Number of Animals Monitored on Representations of Cattle Group Movement Characteristics and Spatial Occupancy

The number of animals required to represent the collective characteristics of a group remains a concern in animal movement monitoring with GPS. Monitoring a subset of animals from a group instead of all animals can reduce costs and labor; however, incomplete data may cause information losses and inaccuracy in subsequent data analyses. In cattle studies, little work has been conducted to determine the number of cattle within a group needed to be instrumented considering subsequent analyses. Two different groups of cattle (a mixed group of 24 beef cows and heifers, and another group of 8 beef cows) were monitored with GPS collars at 4 min intervals on intensively managed pastures and corn residue fields in 2011. The effects of subset group size on cattle movement characterization and spatial occupancy analysis were evaluated by comparing the results between subset groups and the entire group for a variety of summarization parameters. As expected, more animals yield better results for all parameters. Results show the average group travel speed and daily travel distances are overestimated as subset group size decreases, while the average group radius is underestimated. Accuracy of group centroid locations and group radii are improved linearly as subset group size increases. A kernel density estimation was performed to quantify the spatial occupancy by cattle via GPS location data. Results show animals among the group had high similarity of spatial occupancy. Decisions regarding choosing an appropriate subset group size for monitoring depend on the specific use of data for subsequent analysis: a small subset group may be adequate for identifying areas visited by cattle; larger subset group size (e.g. subset group containing more than 75% of animals) is recommended to achieve better accuracy of group movement characteristics and spatial occupancy for the use of correlating cattle locations with other environmental factors.     

Allosteric Inhibition of Human Porphobilinogen Synthase

Porphobilinogen synthase (PBGS) catalyzes the first common step in tetrapyrrole (e.g. heme, chlorophyll) biosynthesis. Human PBGS exists as an equilibrium of high activity octamers, low activity hexamers, and alternate dimer configurations that dictate the stoichiometry and architecture of further assembly. It is posited that small molecules can be found that inhibit human PBGS activity by stabilizing the hexamer. Such molecules, if present in the environment, could potentiate disease states associated with reduced PBGS activity, such as lead poisoning and ALAD porphyria, the latter of which is associated with human PBGS variants whose quaternary structure equilibrium is shifted toward the hexamer (Jaffe, E. K., and Stith, L. (2007) Am. J. Hum. Genet. 80, 329–337). Hexamer-stabilizing inhibitors of human PBGS were identified using in silico prescreening (docking) of ∼111,000 structures to a hexamer-specific surface cavity of a human PBGS crystal structure. Seventy-seven compounds were evaluated in vitro; three provided 90–100% conversion of octamer to hexamer in a native PAGE mobility shift assay. Based on chemical purity, two (ML-3A9 and ML-3H2) were subjected to further evaluation of their effect on the quaternary structure equilibrium and enzymatic activity. Naturally occurring ALAD porphyria-associated human PBGS variants are shown to have an increased susceptibility to inhibition by both ML-3A9 and ML-3H2. ML-3H2 is a structural analog of amebicidal drugs, which have porphyria-like side effects. Data support the hypothesis that human PBGS hexamer stabilization may explain these side effects. The current work identifies allosteric ligands of human PBGS and, thus, identifies human PBGS as a medically relevant allosteric enzyme.     

Post-Hypoxic Recovery of Respiratory Rhythm Generation Is Gender Dependent

The preBötzinger complex (preBötC) is a critical neuronal network for the generation of breathing. Lesioning the preBötC abolishes respiration, while when isolated in vitro, the preBötC continues to generate respiratory rhythmic activity. Although several factors influence rhythmogenesis from this network, little is known about how gender may affect preBötC function. This study examines the influence of gender on respiratory activity and in vitro rhythmogenesis from the preBötC. Recordings of respiratory activity from neonatal mice (P10–13) show that sustained post-hypoxic depression occurs with greater frequency in males compared to females. Moreover, extracellular population recordings from the preBötC in neonatal brainstem slices (P10–13) reveal that the time to the first inspiratory burst following reoxygenation (TTFB) is significantly delayed in male rhythmogenesis when compared to the female rhythms. Altering activity of ATP sensitive potassium channels (K_ATP) with either the agonist, diazoxide, or the antagonist, tolbutamide, eliminates differences in TTFB. By contrast, glucose supplementation improves post-hypoxic recovery of female but not male rhythmogenesis. We conclude that post-hypoxic recovery of respiration is gender dependent, which is, in part, centrally manifested at the level of the preBötC. Moreover, these findings provide potential insight into the basis of increased male vulnerability in a variety of conditions such as Sudden Infant Death Syndrome (SIDS).     

A high incidence of prophage carriage among natural isolates of Streptococcus pneumoniae.

The majority (591 of 791, or 76%) of Streptococcus pneumoniae clinical isolates examined showed the presence of two or more chromosomal SmaI fragments that hybridized with the lytA-specific DNA probe. Only one of these fragments, frequently having an approximate molecular size of 90 kb, was shown to carry the genetic determinant of the pneumococcal autolysin (N-acetylmuramic acid-L-alanine amidase). Strains carrying multiple copies of lytA homologues included both antibiotic-susceptible and -resistant isolates as well as a number of different serotypes and strains recovered from geographic sites on three continents. Mitomycin C treatment of strains carrying several lytA-hybridizing fragments caused the appearance of extrachromosomal DNA hybridizing to the lytA gene, followed by lysis of the bacteria. Such lysates contained phage particles detectable by electron microscopy. The findings suggest that the lytA-hybridizing fragments in excess of the host lytA represent components of pneumococcal bacteriophages. The high proportion of clinical isolates carrying multiple copies of lytA indicates the widespread occurrence of lysogeny, which may contribute to genetic variation in natural populations of pneumococci.     

Diversity and distribution of the tick-borne relapsing fever spirochete Borrelia turicatae

Borrelia turicatae is a causative agent of tick-borne relapsing fever (TBRF) in the subtropics and tropics of the United States and Latin America. Historically, B. turicatae was thought to be maintained in enzootic cycles in rural areas. However, there is growing evidence that suggests the pathogen has established endemic foci in densely populated regions of Texas. With the growth of homelessness in the state and human activity in city parks, it was important to implement field collection efforts to identify areas where B. turicatae and its vector circulate. Between 2017 and 2020 we collected Ornithodoros turicata ticks in suburban and urban areas including public and private parks and recreational spaces. Ticks were fed on naïve mice and spirochetes were isolated from the blood. Multilocus sequence typing (MLST) was performed on eight newly obtained isolates and included previously reported sequences. The four chromosomal loci targeted for MLST were 16S ribosomal RNA (rrs), flagellin B (flaB), DNA gyrase B (gyrB), and the intergenic spacer (IGS). Given the complexity of Borrelia genomes, plasmid diversity was also evaluated. These studies indicate that the IGS locus segregates B. turicatae into four genomic types and plasmid diversity is extensive between isolates. Furthermore, B. turicatae and its vector have established endemic foci in parks and recreational areas in densely populated settings of Texas.     

Entomological outcomes of cluster-randomised,community-driven dengue vector-suppression interventions in Kampong Cham province,Cambodia

Cambodia has one of the highest dengue infection rates in Southeast Asia. Here we report quantitative entomological results of a large-scale cluster-randomised trial assessing the impact on vector populations of a package of vector control interventions including larvivorous guppy fish in household water containers, mosquito trapping with gravid-ovitraps, solid waste management, breeding-container coverage through community education and engagement for behavioural change, particularly through the participation of school children. These activities resulted in major reductions in Container Index, House Index, Breteau Index, Pupal Index and Adult Index (all p-values 0.002 or lower) in the Intervention Arm compared with the Control Arm in a series of household surveys conducted over a follow-up period of more than one year, although the project was not able to measure the longer-term sustainability of the interventions. Despite comparative reductions in Adult Index between the study arms, the Adult Index was higher in the Intervention Arm in the final household survey than in the first household survey. This package of biophysical and community engagement interventions was highly effective in reducing entomological indices for dengue compared with the control group, but caution is required in extrapolating the reduction in household Adult Index to a reduction in the overall population of adult Aedes mosquitoes, and in interpreting the relationship between a reduction in entomological indices and a reduction in the number of dengue cases. The package of interventions should be trialled in other locations.     

A Comparative Study between Spanish and British SARS-CoV-2 Variants

The study of the interaction between the SARS-CoV-2 spike protein and the angiotensin-converting enzyme 2 (ACE2) receptor is key to understanding binding affinity and stability. In the present report, we sought to investigate the differences between two already sequenced genome variants (Spanish and British) of SARS-CoV-2. Methods: In silico model evaluating the homology, identity and similarity in the genome sequence and the structure and alignment of the predictive spike by computational docking methods. Results: The identity results between the Spanish and British variants of the Spike protein were 28.67%. This close correspondence in the results between the Spanish and British SARS-CoV-2 variants shows that they are very similar (99.99%). The alignment obtained results in four deletions. There were 23 nucleotide substitutions also predicted which could affect the functionality of the proteins produced from this sequence. The interaction between the binding receptor domain from the spike protein and the ACE2 receptor produces some of the mutations found and, therefore, the energy of this ligand varies. However, the estimated antigenicity of the British variant is higher than its Spanish counterpart. Conclusions: Our results indicate that minimal mutations could interfere in the infectivity of the virus due to changes in the fitness between host cell recognition and interaction proteins. In particular, the N501Y substitution, situated in the RBD of the spike of the British variant, might be the reason for its extraordinary infective potential.     

Phase transition characteristics of diphosphatidyl-glycerol (cardiolipin) and stereoisomeric phosphatidyldiacylglycerol bilayers. Mono- and divalent metal ion effects

Synthesis and phase transition chaaracteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature (Tm) and the enthalpy of transition (delta H) increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; Tm for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric sn-3,3- and sn-1,1-phosphatidyldiacylglycerol and for the diastereomeric, meso-sn-1,3-phosphatidyldiacylglycerol are approximately the same. The molar enthalpy for the transition of cardiolipin-NH+4 bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C16-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e., the highest Tm is observed for Ca2+-cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest Tm is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding.