Browsing by Subject "stability"

In recent years, exceptionally large bacteriophages with genome sizes over 500 kilobase pairs (kbp), called megaphages, have been discovered from sequence data, but no previous publications discussing megaphage isolates have been published. In 2011, a phage infecting a Flavobacterium strain was isolated from the Kymijoki river. The phage, named FKy-1, was determined to have a genome size of 643 kbp, based on yet unpublished results, making it the first described megaphage isolate. In this study, we focused on characterizing megaphage FKy-1, by observing the virus morphology, determining the type and length of its life cycle, and measuring its stability in different temperatures and conditions. Purification of the phage by precipitation and ultracentrifugation in a sucrose density gradient resulted in separation of both virion and phage subcomplexes. Based on transmission electron microscopy and cryogenic electron microscopy, FKy-1 was observed to have typical myovirus morphology, with a large icosahedral head of around 160 nm in diameter, and a tail of around 180 nm in length. Molecular masses of the major proteins present in the virion and phage subcomplexes were estimated using sodium dodecyl sulfate polyacrylamide gel electrophoresis to be 50-70 kDa for the major capsid protein, 60-70 kDa and 150-200 kDa for the major tail proteins. Digestion attempts with restriction endonucleases proved unsuccessful, indicating possible phage genome modifications or other defensive mechanisms. The phage was determined to have a lytic life cycle which takes over 3 h to cause cell lysis, resulting in the release of around 10 progenies per infected host cell. The phage proved to be quite stable, with minimal impact on infectivity measured at a temperature range of -20 °C to +40 °C, and in minimal buffer conditions. In summary, we proved that the purification method used here is well suited for megaphages, and that FKy-1 is of myovirus morphology, produces a low number of progenies per host, and is relatively stable. As no other publications regarding megaphages exist, this study acts as a good basis for future research regarding megaphage morphology, infection cycle and stability.

Hexanal is a highly volatile liquid that can prolong the shelf life of fruits and vegetables (Paliyath and Subramanian 2008). Previously, Lehtonen et al. (2020) introduced a freeze-dried active packaging material which was continuously releasing hexanal through the naturally occurring lipid oxidation reactions of sunflower oil. In this study, nine different sunflower oil-based matrices were characterized at different stages of their preparation process. They contained crystallization modifiers (acylglycerols, phytosterols, or their blend), coating materials (gum Arabic and maltodextrin), and were produced by spray-drying of an emulsion. The crystallization modifiers in the matrix may enable the control over the start of the reaction for hexanal release. The emulsions with crystallization modifiers were significantly unstable, their viscosity increased with an increasing proportion of gum Arabic, and they contained crystalline regions from the first day of preparation. All tested emulsions formed a white powder after spray drying and the powder particle sizes were characterized. The differential scanning calorimetry (DSC) did not reveal any melting point of the lipid phase in majority of the powder samples. However, the DSC results showed that the melting temperatures of corresponding oleogels with acylglycerols and acylglycerol/phytosterol blend were 64 and 56.6 °C, respectively. In this thesis, emulsions containing acylglycerols and/or phytosterols were characterized and shown to be suitable for the encapsulation by the spray drying technique. Further studies focusing on the hexanal release from the matrix need to be performed since no hexanal release was detected from the powder samples.

The aim of the study. Working memory (WM) is a cognitive function that relies on the neurotransmitter dopamine. WM has multiple subfunctions: maintaining relevant information, ignoring distractors, and updating the information when needed. Information maintenance and ignoring irrelevant stimuli are associated with brain activity in the prefrontal cortex and updating information with the striatum. A connection between polymorphisms within the dopaminergic genes COMT (COMT Val158Met polymorphism) and DRD2/ANKK1 (Taq1A polymorphism), and WM performance has been established in previous studies, and these genes seem to impact dopamine signaling in the prefrontal cortex and striatum, respectively. The present study investigates associations between dopaminergic gene polymorphisms and WM performance. The main research questions include the main effects and interactions that the genes have to WM on performance overall and WM stability and flexibility in particular. The study investigates also whether obesity, diet, and age affect WM performance, as there are indications for these from previous studies. Methods. Data from three separate cross-sectional studies with a total of 244 participants was used. A computer-based visual working memory task was used to assess the WM performance measures accuracy and reaction time. Other measurements included blood measures, Body Mass Index (BMI), and the Dietary Fat and free Sugar Questionnaire (DFS). Results and conclusion. In WM accuracy analyses, there was a significant main effect for the task condition as well as interactions between the COMT*task condition and COMT*Taq1A*task condition. In the three-way interaction analysis for WM accuracy, the most beneficial combination was Met/Met & A1- in the task condition measuring the stability, and Val/Met & A1+ in the task condition measuring the flexibility. This result gives support for established knowledge about how Met/Met combined with A1- is beneficial especially for WM stability and maintenance. Also, the inverted-U shape theory and previous studies' results of A1+ being beneficial in executive functions updating tasks, give support to the result regarding WM flexibility. In the three-way interaction reaction time analysis, the combination of Val/Met & A1+ was the fastest in every task condition. However, the significant differences were mainly between A1- and A1+ genotypes when combined with Val/Met and between Met/Met and Val/Met genotypes when combined with A1-. The two-way interaction between COMT Val158Met and task condition did not remain significant in pairwise comparisons. In reaction time analyses, COMT Val158Met and Taq1A had a significant main effect where reaction times followed the order: Met/Met < Val/Val < Val/Met, with Val/Met being significantly slower than the others and in Taq1A A1+ < A1-, with a significant difference. Adding age, BMI, and DFS to the analyses did not affect the significant/non- significant main effects or interactions in the analyses. The results of this thesis converge with the previous knowledge about these genotypes having an interacting effect on working memory stability and flexibility. However, these effects are complex to interpret as the results and their directions differed between the task conditions and outcome measurements. In the main outcome variable (accuracy), the result regarding the WM stability is well in line with previous literature about the Met/Met & A1- combination being beneficial to WM performance, and the result about Val/Met & A1+ being beneficial for WM flexibility gets preliminary support from executive function studies, but also opens new research avenues regarding the WM flexibility subfunction.

Infant formulas are breast milk substitutes for 0 to 12-month-old infants. Addition of milk fat to infant formulas leads to an increase in solid fat content. Infant formulas are oil-in- water emulsions in which oil is dispersed into a continuous aqueous phase. Milk fat crystallization leads to partial coalescence and creaming which are types of instability in emulsions. Partial coalescence occurs when two droplets containing crystals collide and they partially coalesce by making contact between their oil phases. The aim of this thesis was to study if milk fat crystallization leads to partial coalescence which leads to a higher rate of creaming or the increasing density of crystallizing droplets leads to a lower rate of creaming. Also, the aim was to study the effect of interfacial layer on partial coalescence. Milk fat crystallization and emulsion stability were investigated from four series of oil-in- water emulsions containing different oils in different concentrations and either whey protein or lecithin as stabilizer. The samples were stored at 5, 20 and 40 °C and measured after 0, 7 and 28 days of storage. The crystallization of bulk fat and oil in emulsions were studied with melting enthalpies measured with differential scanning calorimetry. Emulsion stability was examined with particle size distribution, instability index and creaming velocity measurements. According to the results combining vegetable oil and butter oil, and emulsifying the fat decreases the total enthalpy change and crystallization onset temperature of oils. Mixing vegetable oil and butter oil before homogenization decreased crystallization onset temperature and total enthalpy change because the oils were in same droplet, and made the emulsions behave more like vegetable oil emulsions. Mixing the oils after homogenization made the emulsions behave similarly to butter oil emulsions. Whey protein was found to be a better stabilizer of emulsions than lecithin at 5 and 20 °C, but at 40 °C some Maillard reaction was noticed. It was suspected that the concentration of lecithin in emulsions was insufficient. Based on the results it could be stated that the increasing density due to crystallizing droplets made the emulsions more stable against creaming. Best stability of emulsions containing oil mixtures would be achieved if butter oil and vegetable oil were mixed after homogenization and whey protein used as emulsifier.

Avopurennassa vastakkaisten hammaskaarten hampaat eivät ole kontaktissa toisiinsa yhteen purtaessa vaan niiden väliin jää vertikaalinen aukko. Etualueen avopurennan esiintyvyys suomalaisessa aikuisväestössä on 1-2%, maitohampaistossa kuitenkin jopa 6-7%. Tämän kirjallisuuskatsauksen tavoitteena on esittää olemassa olevan tiedon ja tutkimusnäytön perusteella, mitkä ovat etualueen avopurennan hoitomuodot sen etiologia huomioiden, sekä arvioida hoidon pysyvyyttä. Tutkimusaineistona käytettiin tutkielman aiheesta laadittuja tieteellisiä julkaisuja. Julkaisujen hakemiseen käytettiin Medline-tietokannan Pubmed-käyttöliittymää. Etualueen avopurenta voidaan luokitella hampaistolliseksi ja luustolliseksi purentavirheeksi. Hampaistollinen avopurenta kehittyy varhaislapsuudessa aiheutuen tyypillisesti pitkittyneestä tutin tai peukalon imemisestä, suuhengityksestä tai kielen ja huulten virheellisistä funktioista. Tutista tulisi luopua 2-3 vuoden iässä. Avopurennan varhaishoidossa käytettäviä oikomiskojeita ovat mm. quad-helix, kiinteä kielieste, sekä myofunktionaaliset kojeet. Myös suurten risakudosten poisto on indikoitu. Haitalliset tavat yhdistettynä perinnöllisiin tekijöihin aiheuttavat usein luustollista etualueen avopurentaa. Myös leukanivelen kasvuhäiriöt ja sairaudet, kuten reuma, voivat olla avopurennan taustalla. Luustollisen avopurennan erityispiirteet ovat suuri goniaalikulma ja etukasvokorkeus, pieni takakasvokorkeus ja taaksepäin kallistunut kondyyli. Luustollisen avopurennan hoitoperiaatteita ovat molaarien puhkeamisen esto ekstraoraalivedolla tai luustoankkuroidulla palatinaalikaarella. Molaareita voidaan myös aktiivisesti intrudoida luustoankkureiden avulla tai posteriorisilla purentaesteillä. Alaleuan kasvusuuntaan pyritään vaikuttamaan vertikaalisella leukakapalla. Avopurennan palautumisluvut ovat suuria. Useiden tutkimusten mukaan hoitotuloksen pysyvyys on parhaimmillaan silloin, kun hoito on aloitettu varhain, ennen avopurennan kehittymistä luustolliseksi. Lukuisilla eri hoitomenetelmillä on saatu hyviäkin tuloksia avopurennan hoidossa, mutta pitkäaikaistutkimuksia on vähän. Tarvitaan pidempiä seurantajaksoja, jotta voidaan arvioida hoitotuloksien palautuvuutta tarkemmin. Jokaiselle potilaalle tulee tehdä yksilöllinen hoitosuunnitelma, jossa huomioidaan avopurennan etiologiset syyt. (228 sanaa)

Plant-based milks can be unstable during storage due to the colloidal structure of these drinks. In general, e-coded additives are used to increase the viscosity of the drinks and hence, improve stability. Oat beta-glucan, microbially or enzymatically produced exopolysaccharides, such as dextran, and flaxseed mucilage are natural hydrocolloids that are known to have the ability to increase viscosity. These hydrocolloids could potentially be used to replace e-coded additives. The aim of this thesis was to study the stabilizing potential of oat beta-glucan, exopolysaccharides, and flaxseed mucilage in oat drinks. Oat drinks with added hydrocolloids were compared to control samples that were oat drink without added hydrocolloids and oat drinks with pectin at concentrations of 0.2% and 0.4%. Oat beta-glucan, exopolysaccharides, and flaxseed mucilage were added to the prepared drinks by replacing 10% of water with the extracts. Additionally, stabilizing properties of isolated and freeze-dried microbially produced dextran was studied by adding it to the drinks at concentrations of 1.5%, 1.0% and 0.5%. Stability of the oat drinks was assessed during a 14−day storage period by measuring the viscosity, pH, and phase separation. Results of this study demonstrated the stabilizing potential of the natural hydrocolloids. The addition of hydrocolloids increased the viscosity in all samples. The inclusion of natural hydrocolloids led to a similar stability, and in some cases also significantly lower viscosity, compared to pectin added drinks. Oat beta-glucan had a remarkable positive impact on the stability of oat drinks. This was most likely due to the network formation of oat beta-glucan molecules, which decreased the sedimentation of colloidal particles. Enzymatically produced dextran also improved the stability of oat drinks, which might be attributed to the viscosity-increasing -capacity of dextran. However, the stabilizing role of microbially produced exopolysaccharides was not shown. For future study, the molecular interactions of natural hydrocolloids in the colloidal solution should be investigated.

Some problems in dry powder inhaler formulation include low dose efficiency and changes in dispersibility during storage. For lung deposition particles should have aerodynamic size of 1 - 5µm. Poor dispersion of drug particles from carriers' surface is thought to be the main reason for low dose efficacy. A tertiary component of small particles has been generally added to formulation to improve fine particle dose. Small particles are usually manufactured by micronization. This may induce crystal defects and amorphous sites on the surface of crystals. Amorphous sites are metastable and they may crystallize during storing. Changes in particles crystallinity may have an action on efficiency and stability of dry powder inhalers. Conditioning is designated as stabilisation of particles surface by mixture of solvent vapour and inert gas. Vapour may also dissolve surface roughness. This is called deliquescence. Ostwald ripening is phenomenon whereby small particles dissolves and recrystallizes onto larger crystals. This can be extended for surface asperities. Amorphous materials have also better solubility than crystalline materials so amorphous sites may also dissolve and recrystallize onto crystalline surface. Amorphous sites may crystallize spontaneously by absorbing plasticizing agents from vapour phase or by influence of temperature. The purpose of this work was to study process variables in conditioning and their effect on modification of surface roughness and stabilization of micronized α-lactose monohydrate and test drug substance. The purpose was also to study how surface modification and stabilization effects on powders flowability and stability of dry powder inhaler. The dry powder inhaler contained two different vicinity of lactose and two different drug substances. Conditioning was based on evaporation of liquid from open surface. Studied process variables were temperature of powder, temperature of bath of liquid phase and flow rate of nitrogen gas. The aim of this study was to form a process design for conditioning of new substances, to improve powders flowability and to remove changes in fine particle dose during storage. Surface roughness was studied by laser diffraction analysis and specific surface area measurements and also by electron microscopy. Specific surface area was measured by nitrogen adsorption method. Stabilization of amorphous sites ware studied by dynamic vapour sorption. Flowability was measured by angle of repose and with FlowPro device. Fine particle dose was measured with next generator impactor device. The study showed that increasing the amount of solvent in vapour increases surface smoothness and stabilization. Also increase of temperature of sample increased stabilization. Influence of temperature on surface smoothness was not as clear. Changes in temperature may have altered adsorption and kinetic of crystallization of dissolved molecules. Flowability of lactose was significantly improved. Condition did not improve dry powder inhalers fine particle dose, but there was significant difference between different process conditions. This was concluded to be caused of surface modification. It was also shown that different process conditions affected on formulations stability.

Nowadays growing number of new active pharmaceutical ingredients (API) have large molecular weight and are hydrophobic. The energy of their crystal lattice is bigger and polarity has decreased. This leads to weakened solubility and dissolution rate of the drug. These properties can be enhanced for example by amorphization. Amorphous form has the best dissolution rate in the solid state. In the amorphous form drug molecules are randomly arranged, so the energy required to dissolve molecules is lower compared to the crystalline counterpart. The disadvantage of amorphous form is that it is unstable. Amorphous form tends to crystallize. Stability of amorphous form can be enhanced by adding an adjuvant to drug product. Adjuvant is usually a polymer. Polymers prevent crystallization both by forming bonds with API molecules and by steric hindrance. The key thing in stabilizing amorphous form is good miscibility between API and polymer. They have to be mixed in a molecular level so that the polymer is able to prevent crystallization. The aim of this work was to study miscibility of drug and polymer and stability of their dispersion with different analytical methods. Amorphous dispersions were made by rotary evaporator and freeze dryer. Amorphicity was confirmed with X-ray powder diffraction (XRPD) right after preparation. Itraconazole and theophylline were the chosen molecules to be stabilized. Itraconazole was expected to be easier and theophylline more difficult to stabilize. Itraconazole was stabilized with HPMC and theophylline was stabilized with PVP. Miscibility was studied with XRPD and differential scanning calorimetry (DSC). In addition it was studied with polarized light microscope if miscibility was possible to see visually. Dispersions were kept in stressed conditions and the crystallization was analyzed with XRPD. Stability was also examined with isothermal microcalorimetry (IMC). The dispersion of itraconazole and theophylline 40/60 (w/w) was completely miscible. It was proved by linear combination of XRPD results and single glass transition temperature in DSC. Homogenic well mixed film was observed with light microscope. Phase separation was observed with other compositions. Dispersions of theophylline and PVP mixed only partly. Stability of itraconazole dispersions were better than theophylline dispersions which were mixed poorer. So miscibility was important thing considering stability. The results from isothermal microcalorimetry were similar to results from conventional stability studies. Complementary analytical methods should be used when studying miscibility so that the results are more reliable. Light microscope is one method in addition to mostly used XRPD and DSC. Analyzing light microscope photos is quite subjective but it gives an idea of miscibility. Isothermal microcalorimetry can be one option for conventional stability studies. If right conditions can be made where the crystallization is not too fast, it may be possible to predict stability with isothermal microcalorimetry.

Proteins are endogenous molecules that carry out most biological functions in vivo. They are called as the biological workhorses. Proteins are made up of polypeptide chains that usually fold in the three dimensional space to adopt a native stable conformation. Stability of proteins is dependent on the interplay of environmental factors (pH, temperature, ionic strength). For most proteins, the biological function closely relates to the structural attributes of the protein. Misfolding or unfolding of proteins often result in aggregation. Protein aggregation in vivo is known to cause debilitating and fatal diseases such as Alzheimer's, Huntington's, Parkinson's and age related macular degeneration (AMD). Instability (physical and chemical) of proteins in vitro is believed to result in aggregation. This is a huge concern for the biopharmaceutical industry as it not only limits the effectiveness of the manufacturing process but also poses a great risk of fatality in vivo due to the immunogenic nature of the aggregates. Mechanisms of protein aggregation are complex and not well understood. Regulatory requirements for patient safety in biopharmaceutical products require characterization and analysis of aggregates in protein drug formulations. This review provides an overview of protein aggregation in general and highlights the different analytical methods used to characterize protein aggregates in biopharmaceuticals. Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of CNTF necessitate a recombinant expression system to produce large amounts of functional protein. Previous studies have reported that recombinant expression of CNTF in Escherichia coli (E. coli) was limited by low yields and the need to refold the protein from inclusion bodies. In this report, we describe a strategy to effectively screen fusion constructs and expression conditions for soluble hCNTF production in E. coli. Most conditions tested with the codon optimized hCNTF sequence in fusion with soluble tags resulted in soluble expression of the protein. The construct 6-His-CNTF showed soluble expression in all the conditions tested. Our results suggest that codon optimization of the hCNTF sequence is sufficient for soluble expression in E. coli. The recombinant hCNTF was found to bind to CNTFRα with an EC50 = 36 nM.

Formulation development for protein drugs should base on the knowledge of the mechanism of protein degradation. Excipients and formulation can be chosen to stabilize the protein and prevent decomposition. Stability testing is important to identify the likely degradation routes and provide information for formulation development and stability-indicating analytical method development. Gonadotropin-releasing hormone (GnRH) is a neuropeptide hormone that regulates the synthesis and release of gonadotropins: luteinizing hormone (LH) and follicle-stimulating hormone (FHS). Analogs of the endogenous GnRH have been developed to achieve more potent and longer-acting agonists or antagonists. GnRH agonists degrade in several pathways. The primary degradation routes are hydrolysis/backbone cleavage, oxidation, isomerization and aggregation. The stability of GnRH agonists in solid dosage forms has not been studied as excessively as in solutions. The objective of this study was to evaluate the stability of a GnRH agonist (API) at different storage conditions in powder form and in tablet formulations with maize starch or hydroxypropyl methylcellulose (HPMC). The samples were stored for three months at 5 °C (common refrigerator conditions) 25 °C/58 %RH (long-term conditions), and 40 °C/75 %RH (accelerated storage conditions). The samples were analyzed using high performance liquid chromatography. Additionally, the mechanical properties of the formulations and tablets were studied. The stability of API was confirmed in tablet dosage form, when maize starch or HPMC were used as excipients. No degradation products of API were found. As a pure powder API did not degrade either, but it did not stay physically stable at 40 °C/75 %RH. Stressed conditions could be used to find out degradation products in solid state that were not found in this study. Further, the formulations were not ideal, because neither of the studied excipient produced tablets with desirable properties.