title: Application-Level QoS: Improving video conferencing quality through sending the best packet next

authors: McDonald, Ian

subjects: computer science

abstract: In a traditional network stack, data from an application is transmitted in the order that it is received. An algorithm is proposed where information about the priority of packets and expiry times is used by the transport layer to reorder or discard packets at the time of transmission to optimise the use of available bandwidth. This can be used for video conferencing to prioritise important data. This scheme is implemented and compared to unmodified datagram congestion control protocol (DCCP). This algorithm is implemented as an interface to DCCP and tested using traffic modelled on video conferencing software. The results show improvement can be made to video conferencing during periods of congestion - substantially more audio packets arrive on time with the algorithm, which leads to higher quality video conferencing. In many cases video packet arrival rate also increases and adopting the algorithm gives improvements to video conferencing that are better than using unmodified queuing for DCCP. The algorithm proposed is implemented on the server only, so benefits can be obtained on the client without changes being required to the client.

text: Application-Level QoS: Improving video conferencing quality through sending the best packet next In a traditional network stack, data from an application is transmitted in the order that it is received. An algorithm is proposed where information about the priority of packets and expiry times is used by the transport layer to reorder or discard packets at the time of transmission to optimise the use of available bandwidth. This can be used for video conferencing to prioritise important data. This scheme is implemented and compared to unmodified datagram congestion control protocol (DCCP). This algorithm is implemented as an interface to DCCP and tested using traffic modelled on video conferencing software. The results show improvement can be made to video conferencing during periods of congestion - substantially more audio packets arrive on time with the algorithm, which leads to higher quality video conferencing. In many cases video packet arrival rate also increases and adopting the algorithm gives improvements to video conferencing that are better than using unmodified queuing for DCCP. The algorithm proposed is implemented on the server only, so benefits can be obtained on the client without changes being required to the client.

year: 2008

title: The structural and mechanistic characterisation of Thermophilic RNA Ligases

authors: Kamal, Aliyah

subjects: RNA Ligase

abstract: MicroRNAs (miRNAs) are short regulatory RNAs that play critical roles in post-transcriptional gene regulation. Dysregulation of miRNA expression can be implicated in numerous diseases, including cancer, making miRNAs valuable diagnostic and prognostic biomarkers. Next-generation sequencing (NGS) enables quantitative profiling of miRNA populations, but a key limitation lies in the adapter ligation step required for library preparation. Current commercial RNA ligases, typically active between 14-37 ℃, display strong biases based on RNA sequence and secondary structure resulting in a skewed representation of miRNA populations. At low temperatures, miRNA secondary structures can obscure 5’-phosphate, and 3’-hydroxyl ends, preventing efficient ligation. Thermophilic RNA ligases with high thermostability and distinct substrate preferences may therefore reduce ligation bias and improve NGS accuracy. The archaeal Paleococcus pacificus RNA ligase (PpaRnl) represents a valuable model for exploring the structural and mechanistic features of thermophilic RNA ligases. 
This study aimed to further investigate the substrate preference of a PpaRnl mutant; K238G, a point mutation of lysine at position 238, a key residue in the coordination of the AMP and in turn the binding of the Mg2+ ions in PpaRnl. Alongside the structural and mechanistic characterisation of several PpaRnl mutants to explore the role of a newly identified Mg2+ binding site (Mg2+ B) close to the active site. Key residues for coordinating the Mg2+B ion were mutated in the PpaRnl enzyme (D94A, D248A, E155A, and the double mutant D94A-D248A) as characterisation of these mutants would allow for a deeper understanding of this mechanism. 
Mutants were expressed in Escherichia coli and purified using a combination of immobilised metal affinity chromatography (IMAC) and gel-filtration chromatography. Expression and purification outcomes varied significantly between constructs, while some mutants were successfully obtained in sufficient yields, others demonstrated poor or inconsistent expression, often not even seeing improvement after optimisation of growth conditions and large-scale culture volumes. In particular, the E155A mutant displayed extremely slow growth, while the D94A-D248A double mutant produced negligible results across expression trials. Further characterisation of the mutants, K238G, D94A, and D248A, had shown that these enzymes retain robust activity between 60-65 ℃, with measurable activity extending up to 80 ℃.  Distinct sequence and cofactor preferences were observed. When combined with adapters, these mutants efficiently produced RNA ligation products with minimal side reactions and, in several cases, displayed improved activity compared to the wild-type enzyme. 
The structures of PpaRnl D94A and D248A were solved via X-ray crystallography and although there was similarity in the structures compared to that of the wild-type enzyme and K238G, it was found that a number of the residues coordinating the ligands present within the active-site had changed their orientation. This resulted in the Mg2+B ion not being present in D94A, and no AMP cofactor, Mg2+A or Mg2+B present within the D248A mutant.
Overall, this work highlighted the variability in expression and crystallisation outcomes among the second Mg2+ binding site mutants of PpaRnl with notable differences observed in expression, activity, and structure, highlighting that the Mg2+B ion and its coordination have an impact of the efficiency of the enzyme. This unique mechanism is still not fully understood but its importance and impact has been highlighted within this research, reiterating the importance of construct specific optimisation in structural studies and the potential utility of engineered thermophilic RNA ligases, as tools to reduce ligation bias in NGS based miRNA sequencing. Allowing for the possible discovery of biomarkers within viral diseases, and cancer.

text: The structural and mechanistic characterisation of Thermophilic RNA Ligases MicroRNAs (miRNAs) are short regulatory RNAs that play critical roles in post-transcriptional gene regulation. Dysregulation of miRNA expression can be implicated in numerous diseases, including cancer, making miRNAs valuable diagnostic and prognostic biomarkers. Next-generation sequencing (NGS) enables quantitative profiling of miRNA populations, but a key limitation lies in the adapter ligation step required for library preparation. Current commercial RNA ligases, typically active between 14-37 ℃, display strong biases based on RNA sequence and secondary structure resulting in a skewed representation of miRNA populations. At low temperatures, miRNA secondary structures can obscure 5’-phosphate, and 3’-hydroxyl ends, preventing efficient ligation. Thermophilic RNA ligases with high thermostability and distinct substrate preferences may therefore reduce ligation bias and improve NGS accuracy. The archaeal Paleococcus pacificus RNA ligase (PpaRnl) represents a valuable model for exploring the structural and mechanistic features of thermophilic RNA ligases. 
This study aimed to further investigate the substrate preference of a PpaRnl mutant; K238G, a point mutation of lysine at position 238, a key residue in the coordination of the AMP and in turn the binding of the Mg2+ ions in PpaRnl. Alongside the structural and mechanistic characterisation of several PpaRnl mutants to explore the role of a newly identified Mg2+ binding site (Mg2+ B) close to the active site. Key residues for coordinating the Mg2+B ion were mutated in the PpaRnl enzyme (D94A, D248A, E155A, and the double mutant D94A-D248A) as characterisation of these mutants would allow for a deeper understanding of this mechanism. 
Mutants were expressed in Escherichia coli and purified using a combination of immobilised metal affinity chromatography (IMAC) and gel-filtration chromatography. Expression and purification outcomes varied significantly between constructs, while some mutants were successfully obtained in sufficient yields, others demonstrated poor or inconsistent expression, often not even seeing improvement after optimisation of growth conditions and large-scale culture volumes. In particular, the E155A mutant displayed extremely slow growth, while the D94A-D248A double mutant produced negligible results across expression trials. Further characterisation of the mutants, K238G, D94A, and D248A, had shown that these enzymes retain robust activity between 60-65 ℃, with measurable activity extending up to 80 ℃.  Distinct sequence and cofactor preferences were observed. When combined with adapters, these mutants efficiently produced RNA ligation products with minimal side reactions and, in several cases, displayed improved activity compared to the wild-type enzyme. 
The structures of PpaRnl D94A and D248A were solved via X-ray crystallography and although there was similarity in the structures compared to that of the wild-type enzyme and K238G, it was found that a number of the residues coordinating the ligands present within the active-site had changed their orientation. This resulted in the Mg2+B ion not being present in D94A, and no AMP cofactor, Mg2+A or Mg2+B present within the D248A mutant.
Overall, this work highlighted the variability in expression and crystallisation outcomes among the second Mg2+ binding site mutants of PpaRnl with notable differences observed in expression, activity, and structure, highlighting that the Mg2+B ion and its coordination have an impact of the efficiency of the enzyme. This unique mechanism is still not fully understood but its importance and impact has been highlighted within this research, reiterating the importance of construct specific optimisation in structural studies and the potential utility of engineered thermophilic RNA ligases, as tools to reduce ligation bias in NGS based miRNA sequencing. Allowing for the possible discovery of biomarkers within viral diseases, and cancer.

year: 2025-09-14

title: Alum dosing of Waitangi Stream: Biomonitoring of aluminium 2021-2023

authors: Tempero, Grant Wayne

abstract: Continuous alum dosing of the Waitangi Stream at Lake Rotoehu commenced in 2011 to reduce concentrations of dissolved reactive phosphorus in this inflow to the lake and improve water quality. The Bay of Plenty Regional Council contracted the University of Waikato to undertake monitoring of biota in the Waitangi Stream for potential impacts due to alum dosing. Macroinvertebrate community analysis and tissue aluminium concentrations in kōura (Paranephrops planifrons) and goldfish (Carassius auratus) have been undertaken annually since 2013 to determine the impacts and bioaccumulation of aluminium.

text: Alum dosing of Waitangi Stream: Biomonitoring of aluminium 2021-2023 Continuous alum dosing of the Waitangi Stream at Lake Rotoehu commenced in 2011 to reduce concentrations of dissolved reactive phosphorus in this inflow to the lake and improve water quality. The Bay of Plenty Regional Council contracted the University of Waikato to undertake monitoring of biota in the Waitangi Stream for potential impacts due to alum dosing. Macroinvertebrate community analysis and tissue aluminium concentrations in kōura (Paranephrops planifrons) and goldfish (Carassius auratus) have been undertaken annually since 2013 to determine the impacts and bioaccumulation of aluminium.

year: 2024