Phylodynamic Inference of Bacterial Outbreak Parameters Using Nanopore Sequencing

Nanopore sequencing and phylodynamic modeling have been used to reconstruct the transmission dynamics of viral epidemics, but their application to bacterial pathogens has remained challenging. Cost-effective bacterial genome sequencing and variant calling on nanopore platforms would greatly enhance surveillance and outbreak response in communities without access to sequencing infrastructure. Here, we adapt random forest models for single nucleotide polymorphism (SNP) polishing developed by Sanderson and colleagues (2020. High precision Neisseria gonorrhoeae variant and antimicrobial resistance calling from metagenomic nanopore sequencing. Genome Res. 30(9):1354–1363) to estimate divergence and effective reproduction numbers (R_e) of two methicillin-resistant Staphylococcus aureus (MRSA) outbreaks from remote communities in Far North Queensland and Papua New Guinea (PNG; n = 159). Successive barcoded panels of S. aureus isolates (2 × 12 per MinION) sequenced at low coverage (>5× to 10×) provided sufficient data to accurately infer genotypes with high recall when compared with Illumina references. Random forest models achieved high resolution on ST93 outbreak sequence types (>90% accuracy and precision) and enabled phylodynamic inference of epidemiological parameters using birth–death skyline models. Our method reproduced phylogenetic topology, origin of the outbreaks, and indications of epidemic growth (R_e > 1). Nextflow pipelines implement SNP polisher training, evaluation, and outbreak alignments, enabling reconstruction of within-lineage transmission dynamics for infection control of bacterial disease outbreaks on portable nanopore platforms. Our study shows that nanopore technology can be used for bacterial outbreak reconstruction at competitive costs, providing opportunities for infection control in hospitals and communities without access to sequencing infrastructure, such as in remote northern Australia and PNG.     

Relationships of morphological and phototextural attributes of presumptive ovine zygotes and early embryos to their developmental competence in vitro: a preliminary assessment using time-lapse imaging

The assessment of morphology and digital image opacity may provide valuable information on the present embryo quality. Time-lapse imaging has been employed in research to establish a means of monitoring the dynamic nature of preimplantation embryo development. The aim of present study was to use time-lapse imaging for assessing various prospective morphometric and phototextural markers of the developmental potential of in vitro-derived ovine embryos. Oocytes were obtained by scarification of ovaries from nine Polish Longwool ewes. After in vitro maturation (IVM) and fertilization (IVF) of oocytes with fresh ram semen, the development of embryos to the blastocyst stage was monitored and evaluated using Primo Vision time-lapse imaging technology. Commercially available Image-Pro^® Plus software was used to measure zona pellucida thickness, embryo diameter, total area of the perivitelline space, cellular grey-scale pixel intensity and cellular pixel heterogeneity. Statistical assessment of all attributes was done at various time points during embryo development (i.e., presumptive zygote stage: t(0); first cleavage detected at t(2) or t(3); and second cleavage detected at t(4) or t(6)). Out of thirty-seven zygotes analyzed in this study, five did not divide, 26 arrested before and six developed to the blastocyst stage. Our present results indicate that most parameters analyzed did not differ among embryos varying in their developmental fate except for the perivitelline space area that was greater (P<0.05) for non-dividing zygotes than future blastocysts at the presumptive zygote stage (4040±1850 vs. 857±262 µm², respectively; means±SEM). Consequently, the measurement of perivitelline space at t(0) can potentially be used to prognosticate developmental potential of in vitro-produced ovine embryos albeit further confirmational studies are needed.     

Limnological control of brine shrimp population dynamics and cyst production in the Great Salt Lake, Utah

In the Great Salt Lake of Utah, the brine shrimp Artemia franciscanaKellogg is an important food resource for birds and they produce dormant cysts that are harvested and used extensively in the aquaculture industry. We analyzed the limnological factors controlling Artemia growth and cyst production over 12 months in 1994 and 1995. Laboratory experiments showed that inter-brood intervals were highly dependent on temperature and slightly on food level. At optimal temperatures and nutritious food, juveniles reached reproductive size within 7 d in the laboratory. In winter when temperatures were less than 3 °C, Artemia were absent from the lake, phytoplankton abundance was high (13 Chl a g l^–1), and the dominant grazers were ciliated protozoans. In the spring, cysts hatched when phytoplankton was abundant (15–30 g Chl a l^–1), and the Artemia grew and produced large clutches of ovoviviparous eggs. Estimated naupliar production from these eggs was 80 l^–1 from April to May. Despite the high production of nauplii, Artemia densities declined to 8 l^–1by June and the growing shrimp population grazed down the phytoplankton resource to <1 g Chl a l^–1. With the depleted phytoplankton food resource during the summer, Artemia growth slowed, lipid indices decreased, clutch sizes declined, and females switched primarily to oviparous cyst production. During the summer, there was limited production of ovoviviparous eggs, and limited recruitment of juveniles, probably due to low food. Although oviparous reproduction began in June, more than 90% of the cysts were produced after July when female densities had declined to 1.5 l^–1, but nearly all of them were producing cysts. Estimated cyst production was 650000 m^–2, or 4.54 × 10⁶ kg dry weight for the entire lake. The reported commercial harvest took 21% of the 1994 cyst production. That harvest had little impact on the subsequent year's population, as Artemia densities were ultimately controlled by algal production in the lake.     

Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D

We have investigated the mechanism by which the complement protease, Factor D, achieves its high specificity for the cleavage of Factor B in complex with C3(H2O). Kinetic experiments showed that Factor B and C3(H2O) associate with a KD of >/=2.5 microM and that Factor D acts on this complex with a second-order rate constant of kcat/KM >/= 2 x 10(6) M-1 s-1, close to the rate of a diffusion-controlled reaction for proteins of this size. In contrast, Factor D, which is a member of the trypsin family of serine proteases, was 10(3)-10(4)-fold less active than trypsin toward both thioester and p-nitroanilide substrates containing an arginine at P1. Furthermore, peptides spanning the Factor B cleavage site were not detectably cleaved by Factor D (kcat/KM /=9 kcal/mol of binding energy to stabilize the transition state for reaction. In support of this, we demonstrate that chemical modification of Factor D at a single lysine residue that is distant from the active site abolishes the activity of the enzyme toward Factor B while not affecting activity toward small synthetic substrates. We propose that Factor D may exemplify a special case of the induced fit mechanism in which the requirement for conformational activation of the enzyme results in a substantial increase in substrate specificity.     

Functional design space of single-veined leaves: role of tissue hydraulic properties in constraining leaf size and shape

BACKGROUND AND AIMS: Morphological diversity of leaves is usually quantified with geometrical characters, while in many cases a simple set of biophysical parameters are involved in constraining size and shape. One of the main physiological functions of the leaf is transpiration and thus one can expect that leaf hydraulic parameters can be used to predict potential morphologies, although with the caveat that morphology in turn influences physiological parameters including light interception and boundary layer thickness and thereby heat transfer and net photosynthesis. METHODS: An iterative model was used to determine the relative sizes and shapes that are functionally possible for single-veined leaves as defined by their ability to supply the entire leaf lamina with sufficient water to prevent stomatal closure. The model variables include the hydraulic resistances associated with vein axial and radial transport, as well as with water movement through the mesophyll and the leaf surface. KEY RESULTS: The four parameters included in the model are sufficient to define a hydraulic functional design space that includes all single-veined leaf shapes found in nature, including scale-, awl- and needle-like morphologies. This exercise demonstrates that hydraulic parameters have dissimilar effects: surface resistance primarily affects leaf size, while radial and mesophyll resistances primarily affect leaf shape. CONCLUSIONS: These distinctions between hydraulic parameters, as well as the differential accessibility of different morphologies, might relate to the convergent evolutionary patterns seen in a variety of fossil lineages concerning overall morphology and anatomical detail that frequently have evolved in linear and simple multi-veined leaves.     

Intrinsic factor-mediated modulation of cyanocobalamin-N-sulfonyl-acridinium-9-carboxamide chemiluminescence

Two cyanocobalamin-N-sulfonyl-acridinium-9-carboxamides with linkage through the N(10) or 9-position were prepared from B(12)-e-carboxylic acid. The noncovalent association of intrinsic factor with these ligands resulted in specific modulation of the associated chemiluminescence signal either by quenching or changing the emission profile. Either effect was sufficient to formulate a homogeneous assay to detect vitamin B(12) in buffer.     

Only neutral polymorphisms found in the TIGR/myocilin gene of 45 Polish patients with primary open-angle glaucoma

The aim of the study was to identify mutations of the TIGR gene in Polish patients with primary open-angle glaucoma (POAG) and to define possible genotype-phenotype correlations. The study included 45 patients with a verified diagnosis of POAG. The PCR amplification of all three exons of the TIGR gene and screening for the sequence changes by CSGE analysis was done for every patient. The probes with identified heteroduplexes were sequenced. Altogether 315 PCR products were obtained. The CSGE analysis detected 60 possible changes of the sequence in 28 patients. 34 heteroduplexes were chosen for sequencing, including 29 unique changes and 5 changes representative of identical heteroduplexes. Direct sequencing enabled detection of only four different changes in the TIGR gene sequence. Three of them: 5'UTR -83G-->A (in 14 patients), +227 exon 1 G-->A, Arg76Lys (in 14 patients) and +311 exon 3 T-->C, Tyr347Tyr (in 4 patients) have already been described in the literature as neutral polymorphisms of the gene. Only one change in the promoter, 5'UTR -126T-->C (in 2 patients), has not been described in the literature to date. However, this change does not alter directly the sequence of amino acids in myocilin, so it is difficult to conclude on its pathogenetic role. Thus our study showed only neutral polymorphisms of the TIGR gene. This suggests that the patients probably have mutations in other genes, so other loci that predispose to POAG must be analyzed.     

Primary ciliary dyskinesia: genes, candidate genes and chromosomal regions

Primary ciliary dyskinesia (PCD) is a multisystem disease characterized by recurrent respiratory tract infections, sinusitis, bronchiectasis and male subfertility, associated in about 50% patients with situs inversus totalis (the Kartagener syndrome). The disease phenotype is caused by ultrastructural defects of respiratory cilia and sperm tails. PCD is a heterogenetic disorder, usually inherited as an autosomal recessive trait. So far, mutations in two human genes have been proved to cause the disease. However, the pathogenetics of most PCD cases remains unsolved. In this review, the disease pathomechanism is discussed along with the genes that are or may be involved in the pathogenesis of primary ciliary dyskinesia and the Kartagener syndrome.     

Apparent X-linked primary ciliary dyskinesia associated with retinitis pigmentosa and a hearing loss

Three brothers, one 10-year-old and a pair of 14-year-old dizygotic twins--expressed the classical, early-onset retinitis pigmentosa (RP) with typical ophthalmoscopic findings, night blindness, visual field constricted to 10 degrees and flat ERG response. All three brothers were also diagnosed with primary ciliary dyskinesia (PCD) and had recurrent respiratory infections, chronic sinusitis and bronchiectasis. In all of them, resection of the middle lobe of the right lung was performed. A similar clinical picture of coexisting RP and PCD was noted in the brother of the probands' mother. All probands displayed situs solitus. Consistent with the X-linked mode of RP inheritance, there were also three obligatory female carriers of the disorder in this family: the mother of the affected boys, her mother and a daughter of her brother. In all of them, retinitis pigmentosa "sine pigmento" was found with milder but clinically significant symptoms (mild night blindness, visual field constricted to 30 degrees, and scotopic and photopic ERG responses reduced to 30-60%). No extraocular symptoms were detected in any of the heterozygous female carriers. This family presents an example of two rare phenomena: X-linked dominant retinitis pigmentosa (with milder expression in females) and a rare combination of RP with recurrent respiratory infections due to PCD.