Understanding and Control of Calcium Carbonate Crystal Growth on Model Substrates in Dishwasher

This thesis presents the understanding and control of calcium carbonate crystal growth on model substrates under dishwasher conditions. The study was conducted to understand why calcium carbonate crystals grow on the surface of kitchenware under dishwasher conditions, especially the severe deposition observed on plastic samples. Chapter 1 gives reviews on the current crystallization theories, with a particular focus on the current understanding of surface crystallization and mineral crystal growth control on the surface. Chapter 2 and Chapter 3 present the underlying reasons that promote the mineral growth under dishwasher conditions. Chapter 2 focuses on assessing how the different ingredients in the prototype dishwasher formula affect the ‘shine’ on the surface. Chapter 3 further explores the reason of shine loss by looking into the system parameters (pH, temperature and phosphate ions). Poly (methyl methacrylate) slide and soda-lime glass slides are used as two main model substrates. It is observed that the dishwasher conditions (high pH, high temperature, high Ca concentration), in any way, promotes the formation of calcium carbonate growth. The polymer, one of the ingredients in the prototype dishwasher formulation, encourages this process, especially for plastic samples. Chemical inhibition methods are still the most efficient way of limiting mineral formation on the surface. Chapter 4 examines the X-ray crystal structures of a range of newly synthesized calcium compounds with oligocarboxylate candidate inhibitors so as to understand the exact structure and calcium binding mode of each potential inhibitor. Chapter 5 presents the screening results of inhibition performance for thirty-nine different phosphate-free inhibitors, in order to find an alternative phosphorus-free inhibitor to the currently used phosphate-containing inhibitor 1-hydroxyethane 1,1-diphosphoric acid (HEDP). Two phosphate-free, cyclic poly(carboxylic acids) inhibitors were found to be effective replacements. Chapter 6 summarizes the main findings and conclusions towards the CaCO3 crystallization under dishwasher conditions and the structural characteristics of an efficient CaCO3 inhibitor. It was concluded that an effective inhibitor should achieve a critical charge density. While the exact structure requirements of an efficient inhibitor for CaCO3 is still unknown, factors such as balanced flexibility and rigidity in the backbone are desirable

Gated Multi-head Attention Pooling for Weakly Labelled Audio Tagging

Multiple instance learning (MIL) has recently been used for weakly labelled audio tagging, where the spectrogram of an audio signal is divided into segments to form instances in a bag, and then the low-dimensional features of these segments are pooled for tagging. The choice of a pooling scheme is the key to exploiting the weakly labelled data. However, the traditional pooling schemes are usually fixed and unable to distinguish the contributions, making it difficult to adapt to the characteristics of the sound events. In this paper, a novel pooling algorithm is proposed for MIL, named gated multi-head attention pooling (GMAP), which is able to attend to the information of events from different heads at different positions. Each head allows the model to learn information from different representation subspaces. Furthermore, in order to avoid the redundancy of multi-head information, a gating mechanism is used to fuse individual head features. The proposed GMAP increases the modeling power of the single-head attention with no computational overhead. Experiments are carried out on Audioset, which is a large-scale weakly labelled dataset, and show superior results to the non-adaptive pooling and the vanilla attention pooling schemes

Weakly labelled audio tagging via convolutional networks with spatial and channel-wise attention

Multiple instance learning (MIL) with convolutional neural networks (CNNs) has been proposed recently for weakly labelled audio tagging. However, features from the various CNN filtering channels and spatial regions are often treated equally, which may limit its performance in event prediction. In this paper, we propose a novel attention mechanism, namely, spatial and channel-wise attention (SCA). For spatial attention, we divide it into global and local submodules with the former to capture the event-related spatial regions and the latter to estimate the onset and offset of the events. Considering the variations in CNN channels, channel-wise attention is also exploited to recognize different sound scenes. The proposed SCA can be employed into any CNNs seamlessly with affordable overheads and is end-to-end trainable fashion. Extensive experiments on weakly labelled dataset Audioset show that the proposed SCA with CNNs achieves a state-of-the-art mean average precision (mAP) of 0.390

Calcium cyclic carboxylates as structural models for calcium carbonate scale inhibitors

Cyclic oligocarboxylic acids are the most commonly explored phosphate-free inhibitors for calcium carbonate scale deposition. The structural chemistry of calcium complexes of candidate inhibitors has the potential to give insight into inhibitor mode of action and design. We report a series of calcium compounds of cyclic oligocarboxylic acids of (1α,3α,5α)-1,3,5,-cyclohexanetricarboxylic acid (CHTCA); cyclohexane-1,2,4,5-tetracarboxylic acid (CHTTCA); 1,2,3,4,5,6-cyclohexanehexacarboxylic acid (CHHCA); 1,1-cyclohexandiacetic acid (CHDAA) and cis,cis,cis,cis-1,2,3,4-cyclopentanetetracarboxylic acid (CPTCA) to understand the relationship between ligand stereochemistry and calcium ion coordination mode

Phosphate-Free Inhibition of Calcium Carbonate Dishwasher Deposits

This paper reports the characterization of the composition and morphology of mineral formation on glass and plastic (polymethylmethacrylate) substrates in a dishwasher environment and the identification of suitable phosphate-free mineral crystallization inhibitors as environmentally benign candidates to replace the currently used phosphate-containing inhibitor 1-hydroxyethane 1,1-diphosphoric acid (HEDP). Screening of the calcium carbonate crystallization inhibition performance of twenty-eight different compounds resulted in the identification of two phosphate-free, cyclic polycarboxylic acid inhibitors, which were found in combination to be effective replacements. Each inhibitor proved to be highly substrate specific with all-cis-cyclohexane-1,2,3,4,5,6-hexacarboxylic acid (CHHCA) preventing deposition on glass (where calcite is the dominant polymorph) and cis,cis,cis,cis-cyclopentane-1,2,3,4-tetracarboxylic acid (CPTCA) inhibiting aragonite deposition on polymethylmethacrylate (PMMA). When used in combination, these two species prevented all forms of calcium carbonate deposition on both substrate types. The underlying inhibition mechanism and structural requirements of an efficient calcium carbonate inhibitor are also discussed

Enhancement of recombinant human IL-24 (rhIL-24) protein production from site-specific integrated engineered CHO cells by sodium butyrate treatment

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 +/- 1.5% (**p < 0.01), 57.49 +/- 2.4% (**p < 0.01), and 20.17 +/- 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 +/- 0.5% (**p < 0.01) and 15.0 +/- 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis

Adaptation process of engineered cell line FCHO/IL-24 stably secreted rhIL-24 in serum-free suspension culture

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 & PLUSMN; 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 x 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium EdenTM-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes

Adaptation process of engineered cell line FCHO/IL-24 stably secreted rhIL-24 in serum-free suspension culture

Interleukin-24 (IL-24) displays tumor cell-specific proliferation inhibition in vitro and in vivo. Recombinant human IL-24 (rhIL-24) has significantly higher activity, yet significantly lower expression level in mammalian cells than in bacteria. To further realize therapeutic potential of IL-24, we enhanced rhIL-24 expression in mammalian cell systems by adapting engineered Flp-InTMCHO/IL-24 (FCHO/IL-24) cells (adherent cultured in Ham's F12 medium with 10% serum) to serum-free suspension culture. First, MTT assay showed that among four different media (F12, DMEM/F12, 1640 and DMEM), DMEM/F12 medium was the most suitable media for lower-serum adherent culture. Then, cells were adherently cultured in DMEM/F12 with serum concentration reduced from 10% to 0.5% in a gradient manner. Compared to cells in 10% serum, cells in 0.5% serum displayed significantly lower relative cell viability by 40%, increased G0/G1 phase arrest (8.5 & PLUSMN; 2.4%, p < 0.05), decreased supernatant rhIL-24 concentration by 73%, and altered metabolite profiles, such as glucose, lactate and ammonia concentration. Next, the cells were directly adapted to 0.5% serum suspension culture in 125 mL shake flask at 119 rpm with the optimal cell seeding density of 5 x 105 cells/mL (3.3 times higher than that of adherent culture), under which the concentration of rhIL-24 in culture medium was stable at 3.5 ng/mL. Finally, cells adapted to 0.5% serum proliferated better in serum-free medium EdenTM-B300S with higher rhIL-24 expression level compared to CDM4CHO. The successful adaptation of engineered cells FCHO/IL-24 laid foundation for adapting cells from adherent culture to suspension serum-free culture to mass produce rhIL-24 protein for therapeutic purposes

Enhancement of recombinant human IL-24 (rhIL-24) protein production from site-specific integrated engineered CHO cells by sodium butyrate treatment

Interleukin-24 (IL-24) has specific inhibitory effects on the proliferation of various tumor cells with almost no toxicity to normal cells. The antitumor activity of recombinant human IL-24 protein produced in mammalian cells is much higher than that of bacteria, but its expression level is extremely low. Sodium butyrate (NaBu) was utilized as a media additive to increase protein expression in Chinese hamster ovary cells. The site-specific integrated engineered cells FCHO/IL-24 were treated with NaBu under different culture conditions (10% and 0.5% serum adherent culture, 0.5% serum suspension culture). First, 3 days of 1 mmol/L NaBu treatment significantly increased rhIL-24 expression level in FCHO/IL-24 cells by 119.94 +/- 1.5% (**p < 0.01), 57.49 +/- 2.4% (**p < 0.01), and 20.17 +/- 3.03% (*p < 0.05) under the above culture conditions. Second, NaBu has a time- and dose-dependent inhibitory effect on FCHO/IL-24 proliferation and induces G0/G1 phase arrest. Under 10% and 0.5% serum adherent culture, G0/G1 phase cells were increased by 11.3 +/- 0.5% (**p < 0.01) and 15.0 +/- 2.6% (**p < 0.01), respectively. No induction of apoptosis was observed under a high dosage of NaBu treatment. These results suggest that NaBu increases rhIL-24 secretion via inhibiting cell cycle progression, thereby trapping cells in the highly productive G0/G1 phase. Finally, with increasing NaBu dose, glucose concentration increased (**p < 0.01) while lactic acid and ammonia concentrations reduced significantly (**p < 0.01) in 10% and 0.5% serum adherent culture supernatant. RNA-seq showed that NaBu treatment affected multiple tumor and immune-related pathways. In conclusion, NaBu treatment dramatically promoted rhIL-24 production in engineered FCHO/IL-24 cells by altering downstream pathways and inducing G0/G1 cell arrest with little effect on apoptosis