Biology subjects

Kruger, A.

Publications and source records attributed to Kruger, A..

10 recordsLinked to original sources

bioRxiv2024

Multi-layered dosage compensation of the avian Z chromosome

Sex-chromosome dosage represents a challenge for heterogametic species to maintain correct proportion of gene products across chromosomes in each sex. While therian mammals (XX/XY system) achieve near-perfect balance of X-chromosome mRNAs through X-upregulation and X-inactivation, birds (ZW/ZZ system) have been found to lack efficient compensation at RNA level, challenging the necessity of resolving major gene-dosage discrepancies in avian cells. Through allele-resolved multiome analyses, we comprehensively examined dosage compensation in female (ZW), male (ZZ), and rare intersex (ZZW) chicken. Remarkably, this revealed that females exhibit upregulation of their single Z through increased transcriptional burst frequency similar to mammalian X-upregulation, and that Z-protein levels are further balanced via enhanced translation efficiency in females. Global analyses of transcriptional kinetics elements in birds demonstrate remarkable conservation of the genomic encoding of burst kinetics between mammals and birds. Our study uncovers new mechanisms for achieving sex-chromosome dosage compensation and highlights the importance of gene-dosage balance across diverse species.

molecular biology↗

bioRxiv2024

Molecular Cloning and Biochemical Characterisation of a Novel Acidic Laminarinase Derived from Jermuk Hot Spring Metagenome

Laminarinase, an enzyme with a specific affinity for laminarin--a complex polysaccharide found in the cell walls of brown algae and select marine organisms--was investigated in this study. We cloned and characterised a gene encoding a putative glycoside hydrolase family 16 (GH16) laminarinase from the Jermuk hot spring metagenome by heterologous expression in Escherichia coli. The resulting product, named Jermuk-LamM, represents a novel endo-1,3-{beta}-D-glucanase (EC 3.2.1.39) with only 48.1 % amino acid sequence similarity to previously characterised GH16 family members catalogued in the NCBI database. To date, this stands as the sole described endo-1,3-{beta}-D-glucanase within the Marinimicrobia phylum. Jermuk-LamM, identified as an acidic laminarinase, exhibits robust enzymatic activity at pH 5.0 and a temperature of 55 {degrees}C, maintaining its function for a duration of at least 7 hours. Notably, this enzyme effectively catalyses the hydrolysis of both soluble and insoluble (1,3)-{beta}-D-glucans, as well as (1,3;1,4)-{beta}-D-glucans, displaying a pronounced preference for laminarin. The specificity of Jermuk-LamM lies in its cleavage of 1,3-{beta}-D-glucosidic linkages, yielding monosaccharides, disaccharides, and oligosaccharides. These breakdown products hold the potential for conversion into energy carriers, including alcohols, methane, and hydrogen. The enzymes exceptional specific activities, coupled with its resistance to various additives, render Jermuk-LamM a promising candidate for various industrial applications, encompassing the realms of biofuel and pharmaceutical production.

biochemistry↗

bioRxiv2023

Evidence for deliberate burial of the dead by Homo naledi

In this study we describe new results of excavations in the Dinaledi Subsystem of the Rising Star cave system, South Africa. In two areas within the Hill Antechamber and the Dinaledi Chamber this work uncovered concentrations of abundant Homo naledi fossils including articulated, matrix-supported skeletal regions consistent with rapid covering by sediment prior to the decomposition of soft tissue. We additionally re-examine the spatial positioning of skeletal material and associated sediments within the Puzzle Box area, from which abundant H. naledi remains representing a minimum of six individuals were recovered in 2013 and 2014. Multiple lines of evidence exclude the hypothesis that skeletal remains from these three areas come from bodies that decomposed on the floor of the chamber or within a shallow depression prior to burial by sediments. The spatial positioning of skeletal material, the topography of the subsystem, and observations on sediments within and surrounding features exclude the hypothesis that rapid burial by sediment was a result of gravity-driven slumping or spontaneous movement of sediments. We present a minimal hypothesis of hominin cultural burial and test the evidence from all three areas, finding that this hypothesis is most compatible with the pattern of evidence. These results suggest that mortuary behavior including cultural burial was part of the repertoire of Homo naledi.

paleontology↗

bioRxiv2021

A high-throughput, automated, cell-free expression and screening platform for antibody discovery

Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this gap by developing an automated workflow combining cell-free DNA template generation, protein synthesis, and high-throughput binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to 119 published SARS-CoV-2 neutralizing antibodies and demonstrate rapid identification of the most potent antibody candidates.

synthetic biology↗

bioRxiv2021

Community science designed ribosomes with beneficial phenotypes

Functional design of ribosomes with mutant ribosomal RNA (rRNA) could expand opportunities for understanding molecular translation, building cells from the bottom-up, and engineering ribosomes with altered capabilities. However, such efforts have been hampered by cell viability constraints, an enormous combinatorial sequence space, and limitations on large-scale, 3D design of RNA structures and functions. To address these challenges, we developed an integrated community science and experimental screening approach for rational design of ribosomes. This approach couples Eterna, an online video game that crowdsources RNA sequence design to community scientists in the form of puzzles, with in vitro ribosome synthesis, assembly, and translation in multiple design-build-test-learn cycles. We applied our framework to discover mutant rRNA sequences that improve protein synthesis in vitro and cell growth in vivo, relative to wild type ribosomes, under diverse environmental conditions. This work provides new insights into ribosome rRNA sequence-function relationships, with implications for synthetic biology.

synthetic biology↗

bioRxiv2020

Magnetic Tweezers with Magnetic Flux Density Feedback Control

In this work, we present a single-pole magnetic tweezers (MT) device designed for integration with substrate deformation tracking microscopy (DTM) and/or traction force microscopy (TFM) experiments intended to explore extracellular matrix rheology and human epidermal keratinocyte mechanobiology. Assembled from commercially available off-the-shelf electronics hardware and software, the MT device is amenable to replication in the basic biology laboratory. In contrast to conventional solenoid current-controlled MT devices, operation of this instrument is based on real-time feedback control of the magnetic flux density emanating from the blunt end of the needle core using a cascade control scheme and a digital proportional-integral-derivative (PID) controller. Algorithms that compensate for an apparent spatially non-uniform remnant magnetization of the needle core that develops during actuation are implemented into the feedback control scheme. Through optimization of PID gain scheduling, the MT device exhibits magnetization and demagnetization response times of less than 100 ms without overshoot over a wide range of magnetic flux density setpoints. Compared to current-based control, magnetic flux density-based control allows for more accurate and precise magnetic actuation forces by compensating for temperature increases within the needle core due to heat generated by the applied solenoid currents. Near field calibrations validate the ability of the MT device to actuate 4.5 m-diameter superparamagnetic beads with forces up to 25 nN with maximum relative uncertainties of {+/-}30% for beads positioned between 2.5 and 40 m from the needle tip.

biophysics↗

bioRxiv2020

Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways

Gas fermentation by autotrophic bacteria, such as clostridia, offers a sustainable path to numerous bioproducts from a range of local, highly abundant, waste and low-cost feedstocks, such as industrial flue gases or syngas generated from biomass or municipal waste. Unfortunately, designing and engineering clostridia remains laborious and slow. The ability to prototype individual genetic parts, gene expression, and biosynthetic pathway performance in vitro before implementing them in cells could help address these bottlenecks by speeding up design. Unfortunately, a high-yielding cell-free gene expression (CFE) system from clostridia has yet to be developed. Here, we report the development and optimization of a high-yielding (236 {+/-} 24 {micro}g/mL) batch CFE platform from the industrially relevant anaerobe, Clostridium autoethanogenum. A key feature of the platform is that both circular and linear DNA templates can be applied directly to the CFE reaction to program protein synthesis. We demonstrate the ability to prototype gene expression, and quantitatively map cell-free metabolism in lysates from this system. We anticipate that the C. autoethanogenum CFE platform will not only expand the protein synthesis toolkit for synthetic biology, but also serve as a platform in expediting the screening and prototyping of gene regulatory elements in non-model, industrially relevant microbes.

synthetic biology↗

bioRxiv2020

Cell-free styrene biosynthesis at high titers

Styrene is an important petroleum-derived molecule that is polymerized to make versatile plastics, including disposable silverware and foamed packaging materials. Finding more sustainable methods, such as biosynthesis, for producing styrene is essential due to the increasing severity of climate change as well as the limited supply of fossil fuels. Recent metabolic engineering efforts have enabled the biological production of styrene in Escherichia coli, but styrene toxicity and volatility limit biosynthesis in cells. To address these limitations, we have developed a cell-free styrene biosynthesis platform. The cell-free system provides an open reaction environment without cell viability constraints, which allows exquisite control over reaction conditions and greater carbon flux toward product formation rather than cell growth. The two biosynthetic enzymes required for styrene production were generated via cell-free protein synthesis and mixed in defined ratios with supplemented L-phenylalanine and buffer. By altering the time, temperature, pH, and enzyme concentrations in the reaction, this approach increased the cell-free titer of styrene from 5.36 {+/-} 0.63 mM to 40.33 {+/-} 1.03 mM, an order of magnitude greater than cellular synthesis methods. Cell-free systems offer a complimentary approach to cellular synthesis of small molecules, which can provide particular benefits for producing toxic molecules. HighlightsA cell-free system for styrene biosynthesis was established. This in vitro system achieved styrene titers an order of magnitude greater than the highest reported concentration in vivo.

synthetic biology↗

bioRxiv2019

Muscle overexpression of Klf15 via an AAV8-Spc5-12 construct does not provide benefits in spinal muscular atrophy mice

Spinal muscular atrophy (SMA) is a neuromuscular disease caused by loss of the survival motor neuron (SMN) gene. While there are currently two approved gene-based therapies for SMA, availability, high cost, and differences in patient response indicate that alternative treatment options are needed. Optimal therapeutic strategies will likely be a combination of SMN-dependent and -independent treatments aimed at alleviating symptoms in the central nervous system and peripheral muscles. Kruppel-like factor 15 (KLF15) is a transcription factor that regulates key metabolic and ergogenic pathways in muscle. We have recently reported significant downregulation of Klf15 in muscle of pre-symptomatic SMA mice. Importantly, perinatal upregulation of Klf15 via transgenic and pharmacological methods resulted in improved disease phenotypes in SMA mice, including weight and survival. In the current study, we designed an adeno-associated virus serotype 8 (AAV8) vector to overexpress a codon-optimised Klf15 cDNA under the muscle-specific Spc5-12 promoter (AAV8-Klf15). Administration of AAV8-Klf15 to severe Taiwanese Smn-/-;SMN2 or intermediate Smn2B/- SMA mice significantly increased Klf15 expression in muscle. We also observed significant activity of the AAV8-Klf15 vector in liver and heart. AAV8-mediated Klf15 overexpression moderately improved survival in the Smn2B/- model but not in the Taiwanese mice. An inability to specifically induce Klf15 expression at physiological levels in a time- and tissue-dependent manner may have contributed to this limited efficacy. Thus, our work demonstrates that an AAV8-Spc5-12 vector induces high gene expression as early as P2 in several tissues including muscle, heart and liver, but highlights the challenges of achieving meaningful vector-mediated transgene expression of Klf15.

pathology↗

bioRxiv2019

Integrating past, present and future wheat research with Pretzel

MotivationMajor advances have been made in the assembly of complex genomes such as wheat. Remaining challenges include linking these new resources to legacy research, as well as lowering the bar to entry for members of the research community who may lack specific bioinformatics skills.\n\nResultsPretzel aims to solve these problems by providing an interactive, online environment for data visualisation and analysis which, when loaded with appropriately curated data, can enable researchers with no bioinformatics training to exploit the latest genomic resources. We demonstrate that Pretzel can be used to answer common questions asked by researchers as well as for advanced curation of pseudomolecule structure.\n\nAvailabilityPretzel is implemented in JavaScript and is freely available under a GPLv3 license online at: https://github.com/plantinformatics/pretzel.\n\nContactgabriel.keeble-gagnere@ecodev.vic.gov.au

bioinformatics↗