Late Quaternary palaeolimnology and environmental change in the South Wollo Highlands, Ethiopia

Lake Hayq is a closed, freshwater basin on the eastern margin of the north-central highlands, Ethiopia. Using a sediment core extracted from the northern basin, this thesis aims to provide a high-resolution, detailed palaeolimnological reconstruction of changes to the environment and climate in the region since the late Pleistocene. A multi-proxy approach was applied, utilising diatoms, photosynthetic pigments and X-ray fluorescence (XRF) spectrometry. Lithological and chronological analyses were also performed, as well as the development of a transfer function to model diatom-inferred conductivity, and other quantitative analyses. Between ~ 15.6 15.1 cal kyr BP, Lake Hayq experienced a lowstand, synchronous with the timing of Heinrich Event 1 and an intense drought across East Africa. At ~ 15.1 cal kyr BP a lake began to develop at the core site in response to wetter, more humid conditions, most likely caused by the reactivation of the African-Indian monsoonal circulation. This was abruptly ended however at ~ 14.7 cal kyr BP, as the climate shifted back towards aridity and Lake Hayq shallowed, in contrast to the majority of other East African lakes, which continued to refill. This most likely reflects changes to the Indian Ocean monsoon system caused by variability in the Atlantic Meridional Overturning Circulation at this time, in conjunction with site-specific mechanisms affecting the delivery of precipitation to Lake Hayq. At ~ 12.3 cal kyr BP the African Humid Period resumed over Lake Hayq and the lake refilled, reaching maximum water depth between ~ 12.0 10.0 cal kyr BP. The lake was dominated by planktonic diatom taxa and photosynthetic pigments indicate it was meromictic. Lake level gradually declined throughout the Holocene, culminating in the termination of the African Humid Period. A high-resolution study of the period tentatively suggests that climate flickering , in the form of oscillations between dominant diatom taxa, occurred in the build up to the major climatic shift. The termination spanned ~ 600 cal years between ~ 5.2 4.6 cal kyr BP. A lowstand occurred between ~ 3.9 2.2 cal kyr BP, during which the lake became occasionally subsaline. In the late Holocene, ~ 2.2 1.3 cal kyr BP, Lake Hayq became deep and fresh again, although there is evidence of lake level variability. The palaeo-record from Lake Hayq indicates that it broadly experienced the same high-latitude, glacial-interglacial dynamics and sub-millennial shifts in climate found in other palaeolimnological records from across East Africa. The precise timing and expression of these climatic events is not always synchronous between Lake Hayq and other East African waterbodies however, most likely caused by local, site-specific positive feedback mechanisms and variability in lake morphometry. This highlights the heterogeneous pattern of climate across the region and the significance of regional drivers. This palaeo-record, spanning the late Quaternary, will help bridge gaps in current knowledge and understanding of the underrepresented, climatically sensitive and vulnerable north Ethiopian highlands. This is vital for future climate change modelling and regional downscaling, and may also inform ethnographic-archaeological research in a region considered to be the cradle of humankind

Solution structure and dynamics of DNA duplexes containing the universal base analogues 5-nitroindole and 5-nitroindole 3-carboxamide

Universal bases hybridize with all other natural DNA or RNA bases, and have applications in PCR and sequencing. We have analysed by nuclear magnetic resonance spectroscopy the structure and dynamics of three DNA oligonucleotides containing the universal base analogues 5-nitroindole and 5-nitroindole-3-carboxamide. In all systems studied, both the 5-nitroindole nucleotide and the opposing nucleotide adopt a standard anti conformation and are fully stacked within the DNA duplex. The 5-nitroindole bases do not base pair with the nucleotide opposite them, but intercalate between this base and an adjacent Watson–Crick pair. In spite of their smooth accommodation within the DNA double-helix, the 5-nitroindole-containing duplexes exist as a dynamic mixture of two different stacking configurations exchanging fast on the chemical shift timescale. These configurations depend on the relative intercalating positions of the universal base and the opposing base, and their exchange implies nucleotide opening motions on the millisecond time range. The structure of these nitroindole-containing duplexes explains the mechanism by which these artificial moieties behave as universal bases

Automatic speech recognition and the transcription of indistinct forensic audio: how do the new generation of systems fare?

This study provides an update on an earlier study in the “Capturing Talk” research topic, which aimed to demonstrate how automatic speech recognition (ASR) systems work with indistinct forensic-like audio, in comparison with good-quality audio. Since that time, there has been rapid technological advancement, with newer systems having access to extremely large language models and having their performance proclaimed as being human-like in accuracy. This study compares various ASR systems, including OpenAI’s Whisper, to continue to test how well automatic speaker recognition works with forensic-like audio. The results show that the transcription of a good-quality audio file is at ceiling for some systems, with no errors. For the poor-quality (forensic-like) audio, Whisper was the best performing system but had only 50% of the entire speech material correct. The results for the poor-quality audio were also generally variable across the systems, with differences depending on whether a .wav or .mp3 file was used and differences between earlier and later versions of the same system. Additionally, and against expectations, Whisper showed a drop in performance over a 2-month period. While more material was transcribed in the later attempt, more was also incorrect. This study concludes that forensic-like audio is not suitable for automatic analysis

Lethal Mutagenesis of Poliovirus Mediated by a Mutagenic Pyrimidine Analogue

Lethal mutagenesis is the mechanism of action of ribavirin against poliovirus (PV) and numerous other RNA viruses. However, there is still considerable debate regarding the mechanism of action of ribavirin against a variety of RNA viruses. Here we show by using T7 RNA polymerase mediated production of PV genomic RNA, PV polymerase-catalyzed primer extension and cell-free PV synthesis that a pyrimidine ribonucleoside triphosphate analogue (rPTP) with ambiguous basepairing capacity is an efficient mutagen of the PV genome. The in vitro incorporation properties of rPTP are superior to ribavirin triphosphate. We observed a log-linear relationship between virus titer reduction and the number of rPMP molecules incorporated. A PV genome encoding a high-fidelity polymerase was more sensitive to rPMP incorporation, consistent with diminished mutational robustness of high-fidelity PV. The nucleoside (rP) did not exhibit antiviral activity in cell culture owing to the inability of rP to be converted to rPMP by cellular nucleotide kinases. rP was also a poor substrate for herpes simplex virus thymidine kinase. The block to nucleoside phosphorylation could be bypassed by treatment with the P nucleobase, which exhibited both antiviral activity and mutagenesis, presumably a reflection of rP nucleotide formation by a nucleotide salvage pathway. These studies provide additional support for lethal mutagenesis as an antiviral strategy, suggest that rPMP prodrugs may be highly efficacious antiviral agents, and provide a new tool to determine the sensitivity of RNA virus genomes to mutagenesis as well as interrogation of the impact of mutational load on the population dynamics of these viruses

Lethal Mutagenesis of Picornaviruses with N-6-Modified Purine Nucleoside Analogues

RNA viruses exhibit extraordinarily high mutation rates during genome replication. Nonnatural ribonucleosides that can increase the mutation rate of RNA viruses by acting as ambiguous substrates during replication have been explored as antiviral agents acting through lethal mutagenesis. We have synthesized novel N-6-substituted purine analogues with ambiguous incorporation characteristics due to tautomerization of the nucleobase. The most potent of these analogues reduced the titer of poliovirus (PV) and coxsackievirus (CVB3) over 1,000-fold during a single passage in HeLa cell culture, with an increase in transition mutation frequency up to 65-fold. Kinetic analysis of incorporation by the PV polymerase indicated that these analogues were templated ambiguously with increased efficiency compared to the known mutagenic nucleoside ribavirin. Notably, these nucleosides were not efficient substrates for cellular ribonucleotide reductase in vitro, suggesting that conversion to the deoxyriboucleoside may be hindered, potentially limiting genetic damage to the host cell. Furthermore, a high-fidelity PV variant (G64S) displayed resistance to the antiviral effect and mutagenic potential of these analogues. These purine nucleoside analogues represent promising lead compounds in the development of clinically useful antiviral therapies based on the strategy of lethal mutagenesis

Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm

One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes

Chemical synthesis, DNA incorporation and biological study of a new photocleavable 2′-deoxyadenosine mimic

The phototriggered cleavage of chemical bonds has found numerous applications in biology, particularly in the field of gene sequencing through photoinduced DNA strand scission. However, only a small number of modified nucleosides that are able to cleave DNA at selected positions have been reported in the literature. Herein, we show that a new photoactivable deoxyadenosine analogue, 3-nitro-3-deaza-2′-deoxyadenosine (d(3-NiA)), was able to induce DNA backbone breakage upon irradiation (λ > 320 nm). The d(3-NiA) nucleoside was chemically incorporated at desired positions into 40-mer oligonucleotides as a phosphoramidite monomer and subsequent hybridization studies confirmed that the resulting modified duplexes display a behaviour that is close to that of the related natural sequence. Enzymatic action of the Klenow fragment exonuclease free revealed the preferential incorporation of dAMP opposite the 3-NiA base. On the other hand, incorporation of the analogous 3-NiA triphosphate to a primer revealed high enzyme efficiency and selectivity for insertion opposite thymine. Furthermore, only the enzymatically synthesized base pair 3-NiA:T was a substrate for further extension by the enzyme. All the hybridization and enzymatic data indicate that this new photoactivable 3-NiA triphosphate can be considered as a photochemically cleavable dATP analogue

The /el/-/æl/ merger in Australian English:Acoustic and articulatory insights

This paper investigates a merger-in-progress of /e/-/æ/ in prelateral contexts for speakers of Australian English in Victoria. Twelve participants (7F, 5M) were recorded producing a wordlist resulting in acoustic and concurrent articulatory data via stabilised mid-sagittal ultrasound tongue imaging. Focusing on a subset of the data comprising short front vowels /ɪ, e, æ/ in /hVt/ and /hVl/ contexts, findings show that there are robust acoustic differences between /e/ and /æ/ preceding /t/, as anticipated. However, individual differences emerge for /e/ and /æ/ preceding /l/, with highly gradient production patterns across the speakers, ranging from speakers who exhibit merger behaviour to those who maintain categorical distinctions. The evidence for merging behaviour across speakers is similar, but does not map directly, across both the acoustic and articulatory data, and illustrates the value of incorporating a range of data types in investigating a merger-in-progress

Insights into substrate stabilization from snapshots of the peptidyl transferase center of the intact 70S ribosome

Protein synthesis is catalyzed in the peptidyl transferase center (PTC), located in the large (50S) subunit of the ribosome. No high-resolution structure of the intact ribosome has contained a complete active site including both A- and P-site tRNAs. In addition, although past structures of the 50S subunit have found no ordered proteins at the PTC, biochemical evidence suggests that specific proteins are capable of interacting with the 3′ ends of tRNA ligands. Here we present structures, at 3.6-Å and 3.5-Å resolution respectively, of the 70S ribosome in complex with A- and P-site tRNAs that mimic pre- and post-peptidyl-transfer states. These structures demonstrate that the PTC is very similar between the 50S subunit and the intact ribosome. They also reveal interactions between the ribosomal proteins L16 and L27 and the tRNA substrates, helping to elucidate the role of these proteins in peptidyl transfer

Template-Directed Ligation of Tethered Mononucleotides by T4 DNA Ligase for Kinase Ribozyme Selection

Background: In vitro selection of kinase ribozymes for small molecule metabolites, such as free nucleosides, will require partition systems that discriminate active from inactive RNA species. While nucleic acid catalysis of phosphoryl transfer is well established for phosphorylation of 59 or 29 OH of oligonucleotide substrates, phosphorylation of diffusible small molecules has not been demonstrated. Methodology/Principal Findings: This study demonstrates the ability of T4 DNA ligase to capture RNA strands in which a tethered monodeoxynucleoside has acquired a 59 phosphate. The ligation reaction therefore mimics the partition step of a selection for nucleoside kinase (deoxy)ribozymes. Ligation with tethered substrates was considerably slower than with nicked, fully duplex DNA, even though the deoxynucleotides at the ligation junction were Watson-Crick base paired in the tethered substrate. Ligation increased markedly when the bridging template strand contained unpaired spacer nucleotides across from the flexible tether, according to the trends: A2.A1.A3.A4.A0.A6.A8.A10 and T2.T3.T4.T6<T1.T8.T10. Bridging T’s generally gave higher yield of ligated product than bridging A’s. ATP concentrations above 33 mM accumulated adenylated intermediate and decreased yields of the gap-sealed product, likely due to re-adenylation of dissociated enzyme. Under optimized conditions, T4 DNA ligase efficiently (.90%) joined a correctly paired, or T:G wobble-paired, substrate on the 39 side of the ligation junction while discriminating approximately 100-fold against most mispaire