Synthetic biology approach to cellulose degradation

Cellulose, the most abundant biopolymer on earth, is composed of β – 1,4 – linked glucose units, which in turn form a highly ordered crystalline structure that is insoluble and recalcitrant to degradation. It is the world’s most attractive, abundant and renewable energy resource, representing the bioconversion of carbon dioxide into green plants. Cellulosic biomass, such as agricultural and forestry residues, waste paper and industrial waste can therefore be used as an inexpensive and abundantly available source of sugar for fermentation into fuel ethanol. The combustion of biofuels releases carbon dioxide which is thus recycled and hence the use of these fuels in transportation provides an alternative to fossil fuels, solving many environmental problems. The ability to degrade crystalline cellulose seems to be restricted to a specialized group of microorganisms which includes for example Clostridium, Cellulomonas, Cytophaga, Trichoderma etc. Hence the aim of this project was to create BioBricks using different cellulases from cellulose degraders and express them in different expression hosts like Escherichia coli, Bacillus subtilis, Citrobacter freundii etc., using two different promoters, spac and lac. It was observed that the expression of Cytophaga hutchinsonii cellulases (CHU_2103 and CHU_2802) and dehydrogenases (CHU_1944 and CHU_2315) was toxic to the E. coli host for some unknown reason. Therefore it was decided to use cellulases from Cellulomonas fimi, which are well characterized. BioBricks of cellulases (cenA and cex) from C. fimi were introduced into different expression hosts. It was observed that under our experimental conditions Citrobacter freundii SBS197 gave the best results. Both Pspac and Plac were functional in this organism with expression being higher when Pspac was used. When E. coli JM109 was used as an expression host, activity was only detected when the lac promoter was used to control the expression. Although the expression was higher when E. coli JM109 (containing Plac) was used as an expression host, almost all of this activity was residing within the cells, whereas when C. freundii SBS197 was used as an expression host, considerable activity was detected in the surrounding medium, which is essential for cellulose degradation. Growth curve studies were done to see if heterologous cellulases enable the host to use cellulosic substrates as a source of carbon. It was observed that C. freundii SBS197 expressing cenA and cex was able to use filter paper and Avicel as a source of carbon with maximum growth of up to 8.8×108 cfu/ml and 1.2×109 cfu/ml respectively. This was about 2 – 5 fold higher when compared to the control (vector and/or negative) strains. Filter paper completely disappeared within 3 – 4 days when C. freundii SBS197 was used. Slight degradation was observed when E. coli JM109 was used but there was no physical degradation seen when B. subtilis 168 was used as an expression host. Hence it was concluded that heterologous cellulases impart to C. freundii SBS197 with the ability to use cellulosic substrates as a source of carbon. The maximum growth obtained using these cultures is to our knowledge higher than what has been reported so far for recombinant organisms expressing heterologous cellulases using cellulosic substrates as a source of carbon

Turvatuotteiden käyttö veritapaturmien ennaltaehkäisyssä : Osastotunti akuuttiosaston henkilökunnalle

Turvatuotteiden käyttö vähentää merkittävästi veritapaturmia ja lisää työ- ja potilasturvallisuutta. Turvatuotteiden käyttöön ollaankin siirtymässä lisääntyvissä määrin terveydenhuollossa. Opinnäytetyön tarkoituksena oli perehdyttää akuuttiosaston henkilökunta turvatuotteiden käyttöön osastotunnilla. Opinnäytetyön tavoitteena oli kehittää henkilökunnan turvatuotteiden käytön ja veritapaturmien ennaltaehkäisyn osaamista. Lisäksi tavoitteena oli, että henkilökunta ymmärtää, miksi turvatuotteiden käyttöön oltiin siirtymässä akuuttiosastolla. Opinnäytetyön tilaajana toimi Valkeakosken terveyskeskussairaalan akuuttiosasto. Opinnäytetyö toteutettiin toiminnallisena opinnäytetyönä, jonka tuotoksena oli osastotunti. Lisäksi osastotunnilla käsitellyistä asioista tehtiin opinnäytetyön tilaajalle kirjallinen yhteenveto, joka laitettiin osaston perehdytyskansioon. Opinnäytetyön tietoperustassa käsiteltiin turvatuotteiden käyttöönottoa, veritapaturmien ennaltaehkäisyä työ- ja potilasturvallisuuden näkökulmasta sekä henkilökunnan perehdyttämistä turvatuotteiden käyttöön. Lähteinä käytettiin Suomen lainsäädäntöä, terveydenhuoltoalan kirjallisuutta ja kansainvälisiä tutkimuksia. Osastotunti koostui esittelyosuudesta, teoriaosuudesta, toiminnallisesta osuudesta, jossa harjoiteltiin turvatuotteiden käyttöä, ja palauteosuudesta. Opinnäytetyön toteutusta arvioitiin palautekyselyn vastausten perusteella. Johtopäätöksenä voitiin todeta, että osastotunti oli mielenkiintoinen ja henkilökunnan osaamista kehittävä. Lisäksi käytännön harjoittelu oli tärkeää turvatuotteiden käyttöön perehdyttämisessä. Henkilökunnan perehtyminen turvatuotteiden käyttöön jatkui vielä käytännön hoitotyössä osastotunnin jälkeen.Safety devices decrease sharp and needlestick injuries and increase work and patient safety. The use of safety devices is increasing in health care. The purpose of the Bachelor's thesis was to familiarize the staff on the ward with the use of safety devices during a ward meeting. The aim was to improve the staff's knowledge of safety devices and the prevention of sharp and needlestick injuries. The aim was that the staff understands why the ward was switching to using safety devices. The thesis was commissioned by Valkeakoski health center's acute ward. The product of this practice based thesis was the ward meeting. A summary was also written about the content of the ward meeting and given to the commissioner. The summary was put into the ward's education file. The theoretical basis of the thesis consisted of safety devices and familiarization with the safety devices. Sharp and needlestick prevention was discussed part of patient and work safety. The main sources of information were the Finnish legislation, health care literature and international researches. The ward meeting was divided into four parts including an introduction, a theoretical part, a practical part and feedback. In the practical part the staff got to practice the use of the safety devices. The ward meeting was evaluated based on the feedback. The conclusion was that the ward meeting was interesting, and it improved the staff's knowledge and skills. The hands-on practice was an important part of the familiarization with the safety devices. The staff continued to familiarize with the safety devices in nursing on the ward after the ward meeting

Status of the effectiveness of contact lens solutions against keratitis-causing pathogens

PurposeThe aim of this study was to assess the antimicrobial effects of marketed contact lens disinfecting solutions.MethodsUsing ISO 14729 Stand-Alone Test for disinfecting solutions, bactericidal, fungicidal and amoebicidal assays of eight different contact lens solutions including: ReNu MultiPlus, DuraPlus, Ultimate Plus, OptiFree Express, Kontex Clean, Kontex Normal, Kontex Multisol extra+, Kontex Soak were performed. The efficacy of contact lens solutions was determined against keratitis-causing microbes, namely: Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, Methicillin-resistant Staphylococcus aureus, Fusarium solani and Acanthamoeba castellanii.ResultsThe results revealed that ReNu MultiPlus, DuraPlus and OptiFree Express were effective in killing bacterial and fungal pathogens as per manufacturer's minimum recommended disinfection time. Ultimate Plus was effective against F. solani and MRSA but ineffective against P. aeruginosa, S. marcescens and S. aureus. Of concern however, is that none of the locally formulated contact lens disinfecting solutions from Pakistan, i.e., Kontex Clean, Kontex Normal, Kontex Multisol extra+ and Kontex Soak were effective against any of the keratitis-causing organisms tested. All eight contact lens disinfecting solutions were unable to destroy Acanthamoeba cysts.ConclusionsBecause such ineffective contact lens disinfection solutions present a major risk to public health, these findings are of great concern to the health officials and to the manufacturers of the contact lens disinfection solutions and effective solutions are needed, along with emphasis on proper hygiene for contact lens care and special guidelines for developing countries regarding the manufacture and storage of contact lens disinfecting solutions

Interactions of Pseudomonas aeruginosa and Corynebacterium spp. with non-phagocytic brain microvascular endothelial cells and phagocytic Acanthamoeba castellanii

Several lines of evidence suggest that Acanthamoeba interact with bacteria, which may aid in pathogenic bacterial transmission to susceptible hosts, and these interactions may have influenced evolution of bacterial pathogenicity. In this study, we tested if Gram-negative Pseudomonas aeruginosa and Gram-positive Corynebacterium spp. can associate/invade and survive inside Acanthamoeba castellanii trophozoites and cysts, as well as non-phagocytic human brain microvascular endothelial cells. The results revealed that both Corynebacterium spp. and P. aeruginosa were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. In contrast, P. aeruginosa exhibited higher association as well as invasion of non-phagocytic HBMEC compared with Corynebacterium spp. Notably, P. aeruginosa remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (74.54 bacteria per amoebae) compared with Corynebacterium spp. (2.69 bacteria per amoeba) (P < 0.05). As Acanthamoeba cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of P. aeruginosa to susceptible hosts. When bacterial-ridden amoebae were exposed to favourable (nutrient-rich) conditions, A. castellanii emerged as vegetative trophozoites and remained viable, and likewise viable P. aeruginosa were also observed but rarely any Corynebacterium spp. were observed. Correspondingly, P. aeruginosa but not Corynebacterium spp. exhibited higher cytotoxicity to non-phagocytic HBMEC, producing more than 75 % cell death in 24 h, compared to 20 % cell death observed with Corynebacterium spp. Additionally, it was observed that the bacterial conditioned medium had no negative effect on A. castellanii growth. Further characterization of amoebal and bacterial interactions will assist in identifying the role of Acanthamoeba in the transmission and evolution of pathogenic bacteria

The effect of environmental and physiological conditions on excystation of Acanthamoeba castellanii belonging to the T4 genotype

Excystation in Acanthamoeba is an important property for the onset of infection as well as infection recurrence, post-treatment. The overall aim of this study was to determine the effects of several environmental and physiological parameters on excystation in Acanthamoeba castellanii belonging to the T4 genotype. Cysts were prepared by inoculating A. castellanii trophozoites on non-nutrient agar plates for up to 2 weeks. To determine the effects of various conditions on excystation, A. castellanii cysts were inoculated in growth medium i.e. PYG and incubated at varying temperatures (4–40 °C), various pHs (4–9), artificial light/dark cycles and 5 % of CO2. Optimum excystation was observed when cysts were incubated at 30 °C in growth medium at neutral pH. Extremes of temperature and pH reduced excystation, while light/dark cycles had no effect on excystation of A. castellanii. On the other hand, 5 % of CO2 enhanced excystation and growth of excysting amoebae. To determine the effect of serum on A. castellanii excystation, assays were performed in the presence of varying concentrations of heat-inactivated foetal bovine serum (FBS) (5–100 %). The results revealed that FBS promoted excystation. The involvement of G proteins in excystation was also determined. Using propranolol hydrochloride, a G protein inhibitor, the results revealed that G proteins play a role in A. castellanii differentiation. Furthermore, organic solvents (methanol/ethanol) completely blocked excystation. None of the aforementioned conditions had any effect on the viability of A. castellanii. A complete understanding of excystation in A. castellanii will be of value to counter infection recurrence

The effect of environmental and physiological conditions on excystation of Acanthamoeba castellanii belonging to the T4 genotype

Excystation in Acanthamoeba is an important property for the onset of infection as well as infection recurrence, post-treatment. The overall aim of this study was to determine the effects of several environmental and physiological parameters on excystation in Acanthamoeba castellanii belonging to the T4 genotype. Cysts were prepared by inoculating A. castellanii trophozoites on non-nutrient agar plates for up to 2 weeks. To determine the effects of various conditions on excystation, A. castellanii cysts were inoculated in growth medium i.e. PYG and incubated at varying temperatures (4–40 °C), various pHs (4–9), artificial light/dark cycles and 5 % of CO2. Optimum excystation was observed when cysts were incubated at 30 °C in growth medium at neutral pH. Extremes of temperature and pH reduced excystation, while light/dark cycles had no effect on excystation of A. castellanii. On the other hand, 5 % of CO2 enhanced excystation and growth of excysting amoebae. To determine the effect of serum on A. castellanii excystation, assays were performed in the presence of varying concentrations of heat-inactivated foetal bovine serum (FBS) (5–100 %). The results revealed that FBS promoted excystation. The involvement of G proteins in excystation was also determined. Using propranolol hydrochloride, a G protein inhibitor, the results revealed that G proteins play a role in A. castellanii differentiation. Furthermore, organic solvents (methanol/ethanol) completely blocked excystation. None of the aforementioned conditions had any effect on the viability of A. castellanii. A complete understanding of excystation in A. castellanii will be of value to counter infection recurrence

Interactions of Pseudomonas aeruginosa and Corynebacterium spp with non-phagocytic brain microvascular endothelial cells and phagocytic Acanthamoeba castellanii

Several lines of evidence suggest that Acanthamoeba interact with bacteria, which may aid in pathogenic bacterial transmission to susceptible hosts, and these interactions may have influenced evolution of bacterial pathogenicity. In this study, we tested if Gram-negative Pseudomonas aeruginosa and Gram-positive Corynebacterium spp. can associate/invade and survive inside Acanthamoeba castellanii trophozoites and cysts, as well as non-phagocytic human brain microvascular endothelial cells. The results revealed that both Corynebacterium spp. and P. aeruginosa were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. In contrast, P. aeruginosa exhibited higher association as well as invasion of non-phagocytic HBMEC compared with Corynebacterium spp. Notably, P. aeruginosa remained viable during the encystment process and exhibited higher levels of recovery from mature cysts (74.54 bacteria per amoebae) compared with Corynebacterium spp. (2.69 bacteria per amoeba) (P\u3c0.05). As Acanthamoeba cysts can be airborne, these findings suggest that Acanthamoeba is a potential vector in the transmission of P. aeruginosa to susceptible hosts. When bacterial-ridden amoebae were exposed to favourable (nutrient-rich) conditions, A. castellanii emerged as vegetative trophozoites and remained viable, and likewise viable P. aeruginosa were also observed but rarely any Corynebacterium spp. were observed. Correspondingly, P. aeruginosa but not Corynebacterium spp. exhibited higher cytotoxicity to non-phagocytic HBMEC, producing more than 75% cell death in 24 h, compared to 20% cell death observed with Corynebacterium spp. Additionally, it was observed that the bacterial conditioned medium had no negative effect on A. castellanii growth. Further characterization of amoebal and bacterial interactions will assist in identifying the role of Acanthamoeba in the transmission and evolution of pathogenic bacteria

Cellulose degradation: a therapeutic strategy in the improved treatment of Acanthamoeba infections

Acanthamoeba is an opportunistic free-living amoeba that can cause blinding keratitis and fatal brain infection. Early diagnosis, followed by aggressive treatment is a pre-requisite in the successful treatment but even then the prognosis remains poor. A major drawback during the course of treatment is the ability of the amoeba to enclose itself within a shell (a process known as encystment), making it resistant to chemotherapeutic agents. As the cyst wall is partly made of cellulose, thus cellulose degradation offers a potential therapeutic strategy in the effective targeting of trophozoite encased within the cyst walls. Here, we present a comprehensive report on the structure of cellulose and cellulases, as well as known cellulose degradation mechanisms with an eye to target the Acanthamoeba cyst wall. The disruption of the cyst wall will make amoeba (concealed within) susceptible to chemotherapeutic agents, and at the very least inhibition of the excystment process will impede infection recurrence, as we bring these promising drug targets into focus so that they can be explored to their fullest