title: Towards automated configuration of stream clustering algorithms

authors: Carnein, Matthias

subjects: computer science

abstract: Clustering is an important technique in data analysis which can reveal hidden patterns and unknown relationships in the data. A common problem in clustering is the proper choice of parameter settings. To tackle this, automated algorithm configuration is available which can automatically find the best parameter settings. In practice, however, many of our today’s data sources are data streams due to the widespread deployment of sensors, the internet-of-things or (social) media. Stream clustering aims to tackle this challenge by identifying, tracking and updating clusters over time. Unfortunately, none of the existing approaches for automated algorithm configuration are directly applicable to the streaming scenario. In this paper, we explore the possibility of automated algorithm configuration for stream clustering algorithms using an ensemble of different configurations. In first experiments, we demonstrate that our approach is able to automatically find superior configurations and refine them over time.

text: Towards automated configuration of stream clustering algorithms Clustering is an important technique in data analysis which can reveal hidden patterns and unknown relationships in the data. A common problem in clustering is the proper choice of parameter settings. To tackle this, automated algorithm configuration is available which can automatically find the best parameter settings. In practice, however, many of our today’s data sources are data streams due to the widespread deployment of sensors, the internet-of-things or (social) media. Stream clustering aims to tackle this challenge by identifying, tracking and updating clusters over time. Unfortunately, none of the existing approaches for automated algorithm configuration are directly applicable to the streaming scenario. In this paper, we explore the possibility of automated algorithm configuration for stream clustering algorithms using an ensemble of different configurations. In first experiments, we demonstrate that our approach is able to automatically find superior configurations and refine them over time.

year: 2019

title: Thermostable proteases from thermophilic microorganisms

authors: Toogood, Helen S.

abstract: Two metallo-proteases, EA1 and YP-T proteases from Bacillus st. EA1 and B. caldolyticus st. YP-T, respectively, differ in amino acid sequence by only one residue (Val61 = Gly61 in EA1 and YP-T respectively). Yet EA1 protease has a higher thermostability than YP-T protease. An analysis of the half lives of the two proteases at 85°C at different Ca²⁺ concentrations shows that at low Ca²⁺ levels, autolysis is the most significant cause of loss of activity, while at higher Ca²⁺ levels denaturation is most significant.

A comparison of the sequences of the two proteases and thermolysin shows that this amino acid difference is located within the third Ca²⁺ binding site (based on the known structure of thermolysin). The residues are not directly involved in binding to the Ca²⁺, but are located within this pocket. Molecular modelling studies of EA1 and YP-T proteases, based on the structure of thermolysin, propose that the stabilisation of EA1 over YP-T protease could be due to extra interaction/s of the side chain of Val 61 of EA1 protease with the benzene ring of Tyr 27. YP-T protease has Gly in this position. As the “side chain” of Gly is only a hydrogen, there is unlikely to be any such interaction with Tyr 27. This possible extra interaction/s could increase the bonding of the amino acids within the calcium binding pocket, which may stabilise the pocket.

Bacillus st. Ak.1 produces a thermostable serine protease (subtilisin). The 16S rRNA analysis closely groups Bacillus st. Ak.1 and B. thermoglucosidasius. However, the organisms have different growth requirements. Bacillus st. Ak.1 has a requirement for glutamate, while B. thermoglucosidasius has a requirement for a compound found in yeast extract and tryptone, but not glutamate (possibly maltose). RAPD-PCR analysis of twelve organisms show that the banding patterns of the organisms are all unique, though the closest two were between Bacillus st. EA1 and B. caldolyticus. Escherichia coli clone PB5517 produces Ak.1 protease constitutively at 35°C. a 10 1 fermentor run was conducted, and 51 mg of the protease was purified to homogeneity.

Ak.1 protease is dramatically stabilised by Ca²⁺ ions. The half life at 70°C increases by four orders of magnitude in the presence of 5 mM Ca²⁺, as compared to the  thermostability in the absence of Ca²⁺. As the concentration of Ca²⁺ ions increased, the degree of denaturation decreased. At high Ca²⁺ concentrations, the major cause of loss of activity was due to autolysis.

Based on the structural and enzymatic data, the extra degree of stabilisation of Ak.1 protease by Ca²⁺ above that of thermitase-Ca²⁺ could be due to the presence of an extra Ca²⁺-binding site in Ak.1 protease. This new site is located close to Ca(1), and could therefore change the binding properties of this site also. Another possibility is that the binding constants of one or more of the Ca²⁺-binding sites could be higher for Ak.1 protease, as compared to thermitase. 

Lanthanide ions stabilised the protease, though to a much smaller degree than Ca²⁺. Like Ca²⁺, they stabilised the protease by the prevention of denaturation. Other cations stabilised the protease to a small degree, especially Sr²⁺. Different cations had different effects on the stability of the enzyme. Other significant stabilisers were 90% solutions of sorbitol, trehalose and glycerol. At 105°C, 90% sorbitol increased the thermostability of the protease from <<1 minute to 104 minutes. It did so by the prevention of autolysis.

The protease has a limited substrate specificity, preferring to cleave substrates containing neutral or hydrophobic amino acids, such as valine, alanine or phenylalanine, at the P₁ site. It has a preference for proline at the P₂ site, and alanine at the P₁-P₄ sites. It also has esterase activity, being able to cleave methyl, ethyl and p-nitrophenyl esters. Studies with Suc-Alaₙ-pNA substrates (n=2-5) shows that the specific activity of the protease increases with increasing chain length, though such a substrate containing 5 alanine residues appears to be being cleaved significantly at more than one site. A comparison of the Kₘ and Vₘₐₓ of the protease to the substrates Suc-Ala-Ala-Pro-Xaa-pNA, where Xaa = Phe, Leu or Ala, shows that the larger and more hydrophobic the P₁ amino acid is, the higher the specificity of the protease for that substrate.

An analysis of the effect of temperature on the Kₘ and Vₘₐₓ of Ak.1 protease with several substrates revealed that the specificity of the protease (Vₘₐₓ/Kₘ) changes with temperature. The Kₘ and Vₘₐₓ decreased with decreasing temperature, but not to the same degree with all substrates. If the protease is assayed with substrates in the presence of 50% methanol, the Kₘ tends to increase dramatically. This is due to hydrophobic partitioning of the solvent. The Vₘₐₓ of the protease decreases under these conditions.

The active site is a cleft, composed of hydrophobic amino acids m the substrate-binding cleft. It is similar to other subtilisins, but differs in the presence of a disulphide bond. The space-filling model of the protease with the substrate SAAPFpNA in the active site shows the cleft ‘bends’ at the P₂ site. This is easily accomidated for by proline at this position, as proline causes a bend in the substrate. This can explain the preference for proline at the P₂ site.

The sequence of Ak.1 protease indicates the presence of two cysteine residues, separated by only one amino acid. The 3D structure showed that these cysteine residues exist in a disulphide bond. Tests (e.g. Ellman assay) confirmed the presence of not only two cysteine residues, but that in native conditions (i.e. in solution) these residues exist as a disulphide bond.

Reductants, such as dithiothreitol (DTI) typically reduce disulphide bonds into their constituent cysteine residues. The lower thermostability in the presence of DTT indicates that it appears to have opened a disulphide bond as disulphide bonds are known to increase the thermostability of proteins. It is proposed that the reduction of the disulphide bond causes a localised opening of the substrate binding cleft at the P₄ site, due to the location of the disulphide bond in this position. This was supported by the results that showed that the larger the substrate, the greater the effect of DTT on the Kₘ of the protease. With that substrate. For example, the Kₘ was unchanged with DTT with a substrate occupying only sites P₂-P₁', while a substrate which occupies sites P₅-P₁' showed a significant increase of the Kₘ, suggesting it binds weaker to the binding cleft.

Heavy metals such as Hg²⁺ and Pb²⁺ bind to Ak.1 protease, causing a decrease in the specific activity of the protease. The effects of the heavy metals on activity is much smaller than with DTT. In general, the Kₘ and Vₘₐₓ were only changed to a small degree. Fluorescein mercuric acetate (FMA) binds to Ak.1 protease, causing the inherent fluorescence of FMA to increase. The presence of DTT caused a decrease in the thermostability of Ak.1 protease of 9.3 fold at 85°C.

In conclusion, the disulphide bond has a dual role, that of maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease.

text: Thermostable proteases from thermophilic microorganisms Two metallo-proteases, EA1 and YP-T proteases from Bacillus st. EA1 and B. caldolyticus st. YP-T, respectively, differ in amino acid sequence by only one residue (Val61 = Gly61 in EA1 and YP-T respectively). Yet EA1 protease has a higher thermostability than YP-T protease. An analysis of the half lives of the two proteases at 85°C at different Ca²⁺ concentrations shows that at low Ca²⁺ levels, autolysis is the most significant cause of loss of activity, while at higher Ca²⁺ levels denaturation is most significant.

A comparison of the sequences of the two proteases and thermolysin shows that this amino acid difference is located within the third Ca²⁺ binding site (based on the known structure of thermolysin). The residues are not directly involved in binding to the Ca²⁺, but are located within this pocket. Molecular modelling studies of EA1 and YP-T proteases, based on the structure of thermolysin, propose that the stabilisation of EA1 over YP-T protease could be due to extra interaction/s of the side chain of Val 61 of EA1 protease with the benzene ring of Tyr 27. YP-T protease has Gly in this position. As the “side chain” of Gly is only a hydrogen, there is unlikely to be any such interaction with Tyr 27. This possible extra interaction/s could increase the bonding of the amino acids within the calcium binding pocket, which may stabilise the pocket.

Bacillus st. Ak.1 produces a thermostable serine protease (subtilisin). The 16S rRNA analysis closely groups Bacillus st. Ak.1 and B. thermoglucosidasius. However, the organisms have different growth requirements. Bacillus st. Ak.1 has a requirement for glutamate, while B. thermoglucosidasius has a requirement for a compound found in yeast extract and tryptone, but not glutamate (possibly maltose). RAPD-PCR analysis of twelve organisms show that the banding patterns of the organisms are all unique, though the closest two were between Bacillus st. EA1 and B. caldolyticus. Escherichia coli clone PB5517 produces Ak.1 protease constitutively at 35°C. a 10 1 fermentor run was conducted, and 51 mg of the protease was purified to homogeneity.

Ak.1 protease is dramatically stabilised by Ca²⁺ ions. The half life at 70°C increases by four orders of magnitude in the presence of 5 mM Ca²⁺, as compared to the  thermostability in the absence of Ca²⁺. As the concentration of Ca²⁺ ions increased, the degree of denaturation decreased. At high Ca²⁺ concentrations, the major cause of loss of activity was due to autolysis.

Based on the structural and enzymatic data, the extra degree of stabilisation of Ak.1 protease by Ca²⁺ above that of thermitase-Ca²⁺ could be due to the presence of an extra Ca²⁺-binding site in Ak.1 protease. This new site is located close to Ca(1), and could therefore change the binding properties of this site also. Another possibility is that the binding constants of one or more of the Ca²⁺-binding sites could be higher for Ak.1 protease, as compared to thermitase. 

Lanthanide ions stabilised the protease, though to a much smaller degree than Ca²⁺. Like Ca²⁺, they stabilised the protease by the prevention of denaturation. Other cations stabilised the protease to a small degree, especially Sr²⁺. Different cations had different effects on the stability of the enzyme. Other significant stabilisers were 90% solutions of sorbitol, trehalose and glycerol. At 105°C, 90% sorbitol increased the thermostability of the protease from <<1 minute to 104 minutes. It did so by the prevention of autolysis.

The protease has a limited substrate specificity, preferring to cleave substrates containing neutral or hydrophobic amino acids, such as valine, alanine or phenylalanine, at the P₁ site. It has a preference for proline at the P₂ site, and alanine at the P₁-P₄ sites. It also has esterase activity, being able to cleave methyl, ethyl and p-nitrophenyl esters. Studies with Suc-Alaₙ-pNA substrates (n=2-5) shows that the specific activity of the protease increases with increasing chain length, though such a substrate containing 5 alanine residues appears to be being cleaved significantly at more than one site. A comparison of the Kₘ and Vₘₐₓ of the protease to the substrates Suc-Ala-Ala-Pro-Xaa-pNA, where Xaa = Phe, Leu or Ala, shows that the larger and more hydrophobic the P₁ amino acid is, the higher the specificity of the protease for that substrate.

An analysis of the effect of temperature on the Kₘ and Vₘₐₓ of Ak.1 protease with several substrates revealed that the specificity of the protease (Vₘₐₓ/Kₘ) changes with temperature. The Kₘ and Vₘₐₓ decreased with decreasing temperature, but not to the same degree with all substrates. If the protease is assayed with substrates in the presence of 50% methanol, the Kₘ tends to increase dramatically. This is due to hydrophobic partitioning of the solvent. The Vₘₐₓ of the protease decreases under these conditions.

The active site is a cleft, composed of hydrophobic amino acids m the substrate-binding cleft. It is similar to other subtilisins, but differs in the presence of a disulphide bond. The space-filling model of the protease with the substrate SAAPFpNA in the active site shows the cleft ‘bends’ at the P₂ site. This is easily accomidated for by proline at this position, as proline causes a bend in the substrate. This can explain the preference for proline at the P₂ site.

The sequence of Ak.1 protease indicates the presence of two cysteine residues, separated by only one amino acid. The 3D structure showed that these cysteine residues exist in a disulphide bond. Tests (e.g. Ellman assay) confirmed the presence of not only two cysteine residues, but that in native conditions (i.e. in solution) these residues exist as a disulphide bond.

Reductants, such as dithiothreitol (DTI) typically reduce disulphide bonds into their constituent cysteine residues. The lower thermostability in the presence of DTT indicates that it appears to have opened a disulphide bond as disulphide bonds are known to increase the thermostability of proteins. It is proposed that the reduction of the disulphide bond causes a localised opening of the substrate binding cleft at the P₄ site, due to the location of the disulphide bond in this position. This was supported by the results that showed that the larger the substrate, the greater the effect of DTT on the Kₘ of the protease. With that substrate. For example, the Kₘ was unchanged with DTT with a substrate occupying only sites P₂-P₁', while a substrate which occupies sites P₅-P₁' showed a significant increase of the Kₘ, suggesting it binds weaker to the binding cleft.

Heavy metals such as Hg²⁺ and Pb²⁺ bind to Ak.1 protease, causing a decrease in the specific activity of the protease. The effects of the heavy metals on activity is much smaller than with DTT. In general, the Kₘ and Vₘₐₓ were only changed to a small degree. Fluorescein mercuric acetate (FMA) binds to Ak.1 protease, causing the inherent fluorescence of FMA to increase. The presence of DTT caused a decrease in the thermostability of Ak.1 protease of 9.3 fold at 85°C.

In conclusion, the disulphide bond has a dual role, that of maintaining the integrity of the substrate-binding cleft and increasing the thermostability of the protease.

year: 1998

title: Ngā Hua a Tāne Rore: The Benefits of Kapa Haka.

authors: Pihama, Leonie

abstract: This report presentsthe findings of scoping research that is focused on developing a research agenda that will enable a greater understanding of the value and contribution of kapa haka to Aotearoa New Zealand society, and which explores the multiple ways in which we may view the value and contribution of kapa haka within cultural, social and economic contexts.
• There was a unanimous and resounding view that kapa haka does indeed make a valuable contribution to Aotearoa New Zealand society, but that its value is not fully understood or acknowledged within Aotearoa New Zealand.
• The most important components of kapa haka are its intrinsic link to culture and Māori identity, and the essential element of whanaungatanga, the importance of people and connectedness.
• Kapa haka has a dynamic role as a vehicle for the revitalisation and retention of te reo, tikanga, ritual processes and histories.
• It is perceived as a gateway into the culture for Māori who are disengaged from their marae/hapū/iwi, as well as as a safe, inclusive activity through which all New Zealanders can engage with Māori culture.
• Kapa haka is seen as a medium for fostering a richer, more cohesive and inclusive society in Aotearoa New Zealand. As such, it makes a major contribution to building and strengthening New Zealand’s nationhood.
• Kapa haka makes a significant contribution to New Zealand’s national identity and how we are represented and viewed internationally. It provides an effective platform for creating meaningful connections with other nations and peoples.
• Māori culture/kapa haka is increasingly utilised to add value to many forums, both formal and informal, within the public and government sectors. This was seen by some to reflect a more embracing attitude toward Māori cultural protocols as a normal part of New Zealand’s social fabric.
• Kapa haka is not, however, being given the status or respect it deserves as a cultural icon. Because its significance is not well understood by non-Māori, particularly at the level of government, it is often treated in a tokenistic way.
• A major component of kapa haka is its power to effect wellbeing, and to positively transform the lives of individuals and communities. A strength of the movement towards increased health and fitness within kapa haka is that it is achieved collectively.
• An important social benefit of kapa haka is that it provides a positive, disciplined, strength-based environment for rangatahi.
• Kapa haka is having a powerful, transformative impact on social outcomes through the BMPA (Māori performing arts degree), which enables people involved in kapa haka who would otherwise never have considered tertiary education as an option, to embark on that journey and exceed their
own expectations.
• An overall feeling of optimism about the future of kapa haka was tempered by the conviction that there was much more potential to be explored within the realm of kapa haka, particularly in terms of its social benefits.
• The myriad benefits of kapa haka within school environments, including improved learning outcomes, were clearly attested to. However, some barriers to realising those improved outcomes were also identified.
• Some participants were very sure ofthe economic value of kapa haka to Aotearoa. Whilemost of them were less able to articulate specific economic outcomes, they nonetheless had a definite sense of the link between the cultural, social and economic, and that that was ofsignificant value.
• The immense value of ‘culture-to-culture’ relationships was identified, with Maōri culture, including kapa haka, playing a significant role in laying the foundations for lasting trade relationships with other nations, and other cultures.
• Two significant and largely unacknowledged areas of economic contribution were identified: the ‘silent’ economy generated by kapa haka activities; and the extensive ‘productivity’ activity around voluntary workers involved in kapa haka.
• There was an overall view that the economic potential of kapa haka is underestimated and needs to be explored more fully, including the potential for engagement with New Zealand’s private sector.
• A recurrent theme that emerged around the value of kapa haka was to do with how we should go about measuring the value of culture, knowledge and art, and how ‘value’ itselfshould be defined and measured.
• Economic challenges were identified, especially in terms of financial hardship for those involved in kapa haka at the competitive level. It was also noted, however, that kapa haka communities are naturally adept and endlessly resourceful at generating funds.
• The issue of sustainability was raised by some participants who were concerned that kapa haka should be supported at every level into the future, and not just at the high-end competitive level.
• Of fundamental importance was that the growth and development of kapa haka should take place within the context of the acceptance and embracing of Māori culture as an integral component of New Zealand identity and nationhood.

text: Ngā Hua a Tāne Rore: The Benefits of Kapa Haka. This report presentsthe findings of scoping research that is focused on developing a research agenda that will enable a greater understanding of the value and contribution of kapa haka to Aotearoa New Zealand society, and which explores the multiple ways in which we may view the value and contribution of kapa haka within cultural, social and economic contexts.
• There was a unanimous and resounding view that kapa haka does indeed make a valuable contribution to Aotearoa New Zealand society, but that its value is not fully understood or acknowledged within Aotearoa New Zealand.
• The most important components of kapa haka are its intrinsic link to culture and Māori identity, and the essential element of whanaungatanga, the importance of people and connectedness.
• Kapa haka has a dynamic role as a vehicle for the revitalisation and retention of te reo, tikanga, ritual processes and histories.
• It is perceived as a gateway into the culture for Māori who are disengaged from their marae/hapū/iwi, as well as as a safe, inclusive activity through which all New Zealanders can engage with Māori culture.
• Kapa haka is seen as a medium for fostering a richer, more cohesive and inclusive society in Aotearoa New Zealand. As such, it makes a major contribution to building and strengthening New Zealand’s nationhood.
• Kapa haka makes a significant contribution to New Zealand’s national identity and how we are represented and viewed internationally. It provides an effective platform for creating meaningful connections with other nations and peoples.
• Māori culture/kapa haka is increasingly utilised to add value to many forums, both formal and informal, within the public and government sectors. This was seen by some to reflect a more embracing attitude toward Māori cultural protocols as a normal part of New Zealand’s social fabric.
• Kapa haka is not, however, being given the status or respect it deserves as a cultural icon. Because its significance is not well understood by non-Māori, particularly at the level of government, it is often treated in a tokenistic way.
• A major component of kapa haka is its power to effect wellbeing, and to positively transform the lives of individuals and communities. A strength of the movement towards increased health and fitness within kapa haka is that it is achieved collectively.
• An important social benefit of kapa haka is that it provides a positive, disciplined, strength-based environment for rangatahi.
• Kapa haka is having a powerful, transformative impact on social outcomes through the BMPA (Māori performing arts degree), which enables people involved in kapa haka who would otherwise never have considered tertiary education as an option, to embark on that journey and exceed their
own expectations.
• An overall feeling of optimism about the future of kapa haka was tempered by the conviction that there was much more potential to be explored within the realm of kapa haka, particularly in terms of its social benefits.
• The myriad benefits of kapa haka within school environments, including improved learning outcomes, were clearly attested to. However, some barriers to realising those improved outcomes were also identified.
• Some participants were very sure ofthe economic value of kapa haka to Aotearoa. Whilemost of them were less able to articulate specific economic outcomes, they nonetheless had a definite sense of the link between the cultural, social and economic, and that that was ofsignificant value.
• The immense value of ‘culture-to-culture’ relationships was identified, with Maōri culture, including kapa haka, playing a significant role in laying the foundations for lasting trade relationships with other nations, and other cultures.
• Two significant and largely unacknowledged areas of economic contribution were identified: the ‘silent’ economy generated by kapa haka activities; and the extensive ‘productivity’ activity around voluntary workers involved in kapa haka.
• There was an overall view that the economic potential of kapa haka is underestimated and needs to be explored more fully, including the potential for engagement with New Zealand’s private sector.
• A recurrent theme that emerged around the value of kapa haka was to do with how we should go about measuring the value of culture, knowledge and art, and how ‘value’ itselfshould be defined and measured.
• Economic challenges were identified, especially in terms of financial hardship for those involved in kapa haka at the competitive level. It was also noted, however, that kapa haka communities are naturally adept and endlessly resourceful at generating funds.
• The issue of sustainability was raised by some participants who were concerned that kapa haka should be supported at every level into the future, and not just at the high-end competitive level.
• Of fundamental importance was that the growth and development of kapa haka should take place within the context of the acceptance and embracing of Māori culture as an integral component of New Zealand identity and nationhood.

year: 2014