Biology subjects

Wallace, J.

Publications and source records attributed to Wallace, J..

26 records · Page 2Linked to original sources

bioRxiv2020

Interplay between intraocular and intracranial pressure effects on the optic nerve head in vivo

Intracranial pressure (ICP) has been proposed to play an important role in the sensitivity to intraocular pressure (IOP) and susceptibility to glaucoma. However, the in vivo effects of simultaneous, controlled, acute variations in ICP and IOP have not been directly measured. We quantified the deformations of the anterior lamina cribrosa (ALC) and scleral canal at Bruchs membrane opening (BMO) under acute elevation of IOP and/or ICP. Four eyes of three monkeys were imaged in vivo with OCT under four pressure conditions: IOP and ICP either at baseline or elevated. The BMO and ALC were reconstructed from manual delineations. From these, we determined canal area at the BMO (BMO area), BMO aspect ratio and planarity, and ALC median depth relative to the BMO plane. To better account for the pressure effects on the imaging, we also measured ALC visibility as a percent of the BMO area. Further, ALC depths were analyzed only in regions where the ALC was visible in all pressure conditions. Bootstrap sampling was used to obtain mean estimates and confidence intervals, which were then used to test for significant effects of IOP and ICP, independently and in interaction. Response to pressure manipulation was highly individualized between eyes, with significant changes detected in a majority of the parameters. Significant interactions between ICP and IOP occurred in all measures, except ALC visibility. On average, ICP elevation expanded BMO area by 0.17mm2 at baseline IOP, and contracted BMO area by 0.02 mm2 at high IOP. ICP elevation decreased ALC depth by 10m at baseline IOP, but increased depth by 7 m at high IOP. ALC visibility decreased as ICP increased, both at baseline (-10%) and high IOP (-17%). IOP elevation expanded BMO area by 0.04 mm2 at baseline ICP, and contracted BMO area by 0.09 mm2 at high ICP. On average, IOP elevation caused the ALC to displace 3.3 m anteriorly at baseline ICP, and 22 m posteriorly at high ICP. ALC visibility improved as IOP increased, both at baseline (5%) and high ICP (8%). In summary, changing IOP or ICP significantly deformed both the scleral canal and the lamina of the monkey ONH, regardless of the other pressure level. There were significant interactions between the effects of IOP and those of ICP on LC depth, BMO area, aspect ratio and planarity. On most eyes, elevating both pressures by the same amount did not cancel out the effects. Altogether our results show that ICP affects sensitivity to IOP, and thus that it can potentially also affect susceptibility to glaucoma. Research Highlights- In vivo ONH deformations caused by acute, controlled, simultaneous changes in IOP and/or ICP can be directly visualized and measured in the monkey eye using OCT. - Acute changes of either IOP or ICP significantly deformed both the scleral canal and the lamina cribrosa, regardless of the other pressure level. - Pressures interacted, meaning that the effects of one pressure depended significantly on the level of the other pressure. - Elevating both pressures did not cancel out the effects of one of them being elevated. - Our results show that ICP affects sensitivity to IOP, and thus that it can potentially also affect susceptibility to glaucoma.

bioengineering↗

bioRxiv2020

Comparing DNA Extraction and 16s Amplification Methods for Plant-Associated Bacterial Communities

Plant-associated microbes play important roles in global ecology and agriculture. The most common method to profile these microbial communities is amplicon sequencing of the bacterial 16s rRNA gene. Both the DNA extraction and PCR amplification steps of this process are subject to bias, especially since the latter requires some way to exclude DNA from plant organelles, which would otherwise dominate the sample. We compared several common DNA extraction kits and 16s rRNA amplification protocols to determine the relative biases of each and to make recommendations for plant microbial researchers. For DNA extraction, we found that, as expected, kits optimized for soil were the best for soil, though each still included a distinct "fingerprint" of its own biases. Plant samples were less clear, with different species having different "best" options. For 16s amplification, we find that using peptide nucleic acid (PNA) clamps provides the least taxonomic distortion, while chloroplast-discriminating primers are easy and inexpensive but present significant bias in the results. We do not recommend blocking oligos, as they involved a more complex protocol and showed significant taxonomic bias in the results. Further methods development will hopefully result in protocols that are even more reliable and less biased.

microbiology↗

bioRxiv2020

Nef enhances HIV-1 replication and infectivity independently of SERINC3 and SERINC5 in CEM T cells

The lentiviral nef gene encodes several discrete activities aimed at co-opting or antagonizing cellular proteins and pathways to defeat host defenses and maintain persistent infection. Primary functions of Nef include downregulation of CD4 and MHC class-I from the cell surface, disruption or mimicry of T-cell receptor signaling, and enhancement of viral infectivity by counteraction of the host antiretroviral proteins SERINC3 and SERINC5. In the absence of Nef, SERINC5 incorporates into virions and inhibits viral fusion with target cells, decreasing infectivity. However, whether Nefs counteraction of SERINC5 is the cause of its positive influence on viral growth-rate in CD4-positive T cells is unclear. Here, we utilized CRISPR/Cas9 to knockout SERINC3 and SERINC5 in a leukemic CD4-positive T cell line (CEM) that displays relatively robust nef-related infectivity and growth-rate phenotypes. As previously reported, viral replication was attenuated in CEM cells infected with HIV-1 lacking Nef (HIV-1{Delta}Nef). This attenuated growth-rate phenotype was observed regardless of whether the coding regions of the serinc3 or serinc5 genes were intact. Moreover, knockout of serinc3 or serinc5 failed to restore the infectivity of HIV1{Delta}Nef virions produced from infected CEM cells. Taken together, our results corroborate a similar study using another T-lymphoid cell line (MOLT-3) and indicate that the antagonism of SERINC3 and SERINC5 cannot fully explain the virology of HIV-1 lacking Nef.

microbiology↗

bioRxiv2020

Characterization of gene repression by designed transcription activator-like effector dimer proteins

Gene regulation by control of transcription initiation is a fundamental property of living cells. Much of our understanding of gene repression originated from studies of the E. coli lac operon switch, where DNA looping plays an essential role. To validate and generalize principles from lac for practical applications, we previously described artificial DNA looping driven by designed Transcription Activator-Like Effector Dimer (TALED) proteins. Because TALE monomers bind the idealized symmetrical lac operator sequence in two orientations, our prior studies detected repression due to multiple DNA loops. We now quantitatively characterize gene repression in living E. coli by a collection of individual TALED loops with systematic loop length variation. Fitting of a thermodynamic model allows unequivocal demonstration of looping and comparison of the engineered TALED repression system with the natural lac repressor system. Statement of SignificanceWe are designing and testing in living bacteria artificial DNA looping proteins engineered based on principles learned from studies of the E. coli lac repressor. The engineered proteins are based on artificial dimers of Transcription Activator-Like Effector (TALE) proteins that have programmable DNA binding specificities. The current work is the first to create unique DNA repression loops using this approach. Systematic study of repression as a function of loop size, with data fitting to a thermodynamic model, now allows this system to be compared in detail with lac repressor loops, and relevant biophysical parameters to be estimated. This approach has implications for the artificial regulation of gene expression.

biophysics↗

bioRxiv2020

Translocation of flexible and tensioned ssDNA through in silico de-signed hydrophobic nanopores with two constrictions.

Protein-inspired nanopores with hydrophobic constriction regions have previously been shown to offer some promise for DNA sequencing. Here we explore a series of pores with two hydrophobic constrictions. The impact of nanopore radius, the nature of residues that define the constriction region and the flexibility of the ssDNA is explored. Our results show that aromatic residues slow down DNA translocation, and in the case of short DNA strands, they cause deviations from a linear DNA conformation. When DNA is under tension, translocation is once again slower when aromatic residues are present in the constriction. However, the lack of flexibility in the DNA backbone provides a narrower window of opportunity for the DNA bases to be retained inside the pore via interaction with the aromatic residues, compared to more flexible strands. Consequently, there is more variability in translocation rates for strands under tension. DNA entry into the pores is correlated to pore width, but no such correlation between width and translocation rate is observed.

biophysics↗

bioRxiv2019

Biofilm Associated Genotypes of Multidrug-Resistant Pseudomonas aeruginosa

Pseudomonas aeruginosa is a ubiquitous environmental microorganism that is also a common cause of nosocomial infections that vary in severity from chronic wound infections to pneumonia, bloodstream infections and sepsis. Its ability to survive in many different environments and persistently colonize human tissue is linked to its presence within biofilms that form on indwelling device surfaces such as plastics and stainless steel. Biofilm promotes bacterial adhesion and survival on surfaces, reduces susceptibility to desiccation, and the actions of antibiotics and disinfectants. Recent genome sequencing studies demonstrate that P. aeruginosa is a highly diverse species with a very large pan-genome consistent with its adaptability to differing environments. However, most MDR infections are caused by a small number of \"high-risk\" clones or lineages that recently emerged and spread globally.\n\nIn our 2017 study of the resistome of P. aeruginosa we confirmed the power of genome-wide association (GWAS) techniques to explore the genetic basis of several antibiotic resistance phenotypes and discovered 46 novel putative resistance polymorphisms. In this study we sought to examine genetic associations within a subset of these isolates with simple biofilm phenotypes. We examined the genetic basis for biofilm production on polystyrene at room temperature (22{degrees}C) and body temperature (37{degrees}C) within a total of 280 isolates. 69% of isolates (n=193) produced more biofilm mass at 22{degrees}C, whilst those producing more biofilm at 37{degrees}C had reduced optical density 540 variation. We found statistically significant associations with IpxO and other genes associated with arsenic resistance to be significantly associated with this trait. IpxO which encodes a lipid A hydroxylase and arsenic reduction genes have previously been found to be associated with biofilm production in this species. We analyzed 260 ST111 and ST235 genomes and found considerable genetic variation between isolates in their content of genes previously found associated with biofilm production. This is indicative of a highly variable and flexible population within these clades with frequent emergence of successful sub-lineages. Analysis of 48 of these isolates ability to form biofilm on stainless steel surfaces showed that a good biofilm-forming phenotype had significant intra-clone variation, independent of core genome phylogeny with pan-genome analysis, suggesting a possible association and involvement of components of the type IV secretion system. However, GWAS and pan-GWAS analyses yielded weaker statistical significance. This study confirms GWAS and pan-GWAS trait associations can be performed for biofilm phenotype and produce data in agreement with each other. This panel of 280 study isolates, matched to genomic data has potential for the investigation of other phenotypes in P. aeruginosa perhaps as part of a growing database / collection. A representative, curated, genome sequenced collection should increase in usefulness as it grows offering increasing statistical power.\n\nImportanceP. aeruginosa is a major cause of multiply antibiotic infections worldwide but it is also found in many hospital and natural environments, especially aquatic ones. In this study we examined genetic polymorphism associated with biofilm production at room temperature and at body temperature, the biofilm associated gene repertoire of two major MDR clones and also genetic polymorphisms associated with biofilm production on stainless steel. Using these genome-wide and pan-genome wide association methods we identified / confirmed potential key genes involved in biofilm production and survival of P. aeruginosa. The study demonstrates the potential usefulness of large, genome sequenced isolate collections such as ours, to better understand the genetics underlying phenotypic diversity in this species.

genomics↗

bioRxiv2019

Bund removal to re-establish tidal flow, remove aquatic weeds and restore coastal wetland services - North Queensland, Australia

The shallow tidal and freshwater coastal wetlands adjacent to the Great Barrier Reef lagoon provide a vital nursery and feeding complex that supports the life cycles of marine and freshwater fish, important native vegetation and vital bird habitat. Urban and agricultural development threaten these wetlands, with many of the coastal wetlands becoming lost or changed due to the construction of artificial barriers (e.g. bunds, roads, culverts and floodgates). Infestation by weeds has become a major issue within many of the wetlands that were modified (bunded) for ponded pasture growth last century. A range of expensive chemical and mechanical control methods has been used to try and restore some of these coastal wetlands, with limited success. This study describes an alternative approach to those methods, investigating the impact of tidal reinstatement after bund removal on weed infestation, associated changes in water quality, and fish biodiversity, in the Boolgooroo lagoon region of the Mungalla wetlands, East of Ingham in North Queensland. High resolution remote sensing, electrofishing and in-water logging was used to track changes over time - 1 year before and 4 years after removal of an earth bund. With tides only penetrating the wetland a few times yearly, gross changes towards a more natural system occurred within a relatively short timeframe, leading to a reduction in weed infestation, reappearance of native vegetation, improvements in water quality, and a tripling of fish diversity. Weed abundance and water quality does appear to oscillate however, dependent on summer rainfall, as changes in hydraulic pressure stops or allows tidal ingress (fresh/saline cycling). With an estimated 30% of coastal wetlands bunded in the Great Barrier Reef region, a passive remediation method such as reintroduction of tidal flow by removal of an earth bund or levee could provide a more cost effective and sustainable means of controlling freshwater weeds and improving coastal water quality into the future.

ecology↗

bioRxiv2018

Genome-wide Population Structure Analyses of Three Minor Millets: Kodo Millet, Little Millet, and Proso Millet

Millets are a diverse group of small-seeded grains that are rich in nutrients but have received relatively little advanced plant breeding research. Millets are important to smallholder farmers in Africa and Asia because of their short growing season, good stress tolerance, and high nutritional content. To advance the study and use of these species, we present a genome-wide marker datasets and population structure analyses for three minor millets: kodo millet (Paspalum scrobiculatum), little millet (Panicum sumatrense), and proso millet (Panicum miliaceum). We generated genome-wide marker data sets for 190 accessions of each species with genotyping-by-sequencing (GBS). After filtering, we retained between 161 and 165 accessions of each species, with 3461, 2245, and 1882 single-nucleotide polymorphisms (SNPs) for kodo, proso, and little millet, respectively. Population genetic analysis revealed 7 putative subpopulations of kodo millet and 8 each of proso millet and little millet. To confirm the accuracy of this genetic data, we used public phenotype data on a subset of these accessions to estimate the heritability of various agronomically relevant phenotypes. Heritability values largely agree with the prior expectation for each phenotype, indicating that these SNPs provide an accurate genome-wide sample of genetic variation. These data represent one of first genome-wide population genetics analyses, and the most extensive, in these species and the first genomic analyses of any sort for little millet and kodo millet. These data will be a valuable resource for researchers and breeders trying to improve these crops for smallholder farmers.

genetics↗