Syanobakteerien antileukeemisten yhdisteiden tunnistus ja bioaktiivisuuksien seulonta

Syanobakteerit tuottavat lukuisia sekundäärimetaboliitteja, jotka voidaan jakaa rakenteensa ja synteesimekanisminsa mukaan laajempiin ryhmiin, kuten peptideihin polyketideihin ja alkaloideihin. Vaikka näiden yhdisteiden synteesien merkitys niitä muodostaville syanobakteereille on kiistanalaista, joillakin yhdisteillä on havaittu olevan bioaktiivisuutta eli ne vaikuttavat muiden eliöiden soluihin. Tunnetuimpia näistä yhdisteistä ovat ihmisille ja eläimille vaaralliset toksiinit, kuten mikrokystiinit ja saksitoksiinit, mutta osalla yhdisteistä voisi olla muokattuina myönteinen vaikutus ihmisten terveyteen. Tällaiset yhdisteet voivat mm. estää haitallisten patogeenien kasvua elimistössä tai vahingoittaa syöpäsoluja. Niinpä syanobakteerit ovat nousseet kiinnostavimpien eliöiden joukkoon uusia lääkkeeksi kelpaavia yhdisteitä etsittäessä. Kuitenkin vain harvasta syanobakteerikannasta on löydetty lääketieteelle potentiaalisia yhdisteitä ja niiden osuus on silloinkin vähäinen kyseisen kannan kaikista sadoista metabolian tuotteista. Näiden yhdisteiden löytäminen ja eristäminen edellyttävät tehokkaita seulonta- ja puhdistusmenetelmiä. Tutkimuksessa pyrittiin eristämään antileukeemisia fraktioita syanobakteerikannoista, joilla oli aiemmin todettu sytotoksisia ominaisuuksia solutesteissä. Fraktioista pyrittäisiin sitten tunnistamaan ja eristämään aktiivisuuden aiheuttama yhdiste tai yhdisteet. Fraktioinnissa käytettiin kiinteäfaasiuuttoa ja korkean erotuskyvyn nestekromatografiaa. Fraktioiden antileukeemisuus testattiin alun perin ihmispotilaasta eristetyillä leukemiasoluilla. Edellä mainittuja menetelmiä käyttäen löydettiin yhdestä syanobakteerikannasta sytotoksisilta vaikuttavia yhdisteitä, jotka muistuttivat ominaisuuksiltaan karotenoideja. Tutkimuskannoilla suoritettiin myös maljadiffuusioon perustuvia seulontatestejä, joilla pyrittiin selvittämään syanobakteerikantojen metaboliittien kykyä estää erilaisten mikrobien kasvua sekä aiheuttaa hemolyysiä. Yksi tutkimuksessa mukana olleista kannoista ilmensi α-hemolyyttistä aktiivisuutta.Cyanobacteria produce several secondary metabolites which can be assorted into larger groups like peptides, polyketides and alkaloids based on their structure and synthesis pathways. Though the importance of these compounds to the cyanobacteria is under discussion, some compounds are detected to express bioactivity towards the cells of other organisms. The most known of these compounds are probably toxins like microcystins and saxitoxins which pose a health risk to humans and animals. However, some compounds, when modified, might have a positive affect towards human health. These compounds could for example inhibit the growth of pathogens in human body or harm cancer cells. Therefore, cyanobacteria have risen among the most promising organisms when it comes to finding new drug leads. Unfortunately only a few cyanobacteria strains seem to produce compounds with high medical potential and these compounds of interest are among the hundreds of others metabolites produced by the strain. Therefore, efficient screening and purification methods are needed. This study aimed to isolate antileukemic fractions from cyanobacteria strains, which had expressed cytotoxic activity in previous studies. The compounds of interest would be identified and purified out of these fractions. Fractions were made using solid phase extraction and high performance liquid chromatography. The antileukemic activity of the fractions were tested using human leukemia patient-derived cell line. Using described methods we found apparently cytotoxic compounds resembling carotenoids from one of the strains. We also performed screening based on plate diffusion. The tests were done in order to determine whether the compounds of cyanobacteria strains would be able to cause hemolysis or prevent growth of different microbes. One of the strains tested seemed to express α-hemolytic activity

Understanding interparticle interactions in dry powder inhalation: glass beads as an innovative model carrier system

Delivery of drugs via the pulmonary route is the most common approach to treat diseases of the respiratory system, e.g. asthma bronchiale. Here, the active pharmaceutical ingredient is generally formulated in a so-called interactive mixture with a coarse and inert carrier. This enhances flowability and therefore dose metering and dispersibility. Interparticle interactions between carrier and drug govern aerosolisation behaviour of the blend and consequently the efficacy of the drug deposition in the lungs. These interactions, for their part, are influenced by a variety of factors including chemical and topographical properties of the carrier surface. To investigate their effect, glass beads were utilized as a model carrier system. Different modification techniques were applied for surface alteration including silanisation, treatment with hydrofluoric acid and ball milling with tungsten carbide. Chemical properties proved to have different effects for drugs with varying hydrophilicity on pulmonary deposition. For the surface topography, same trends could be observed for the aerodynamic performance of blends with budesonide or formoterol fumarate. Indentations on micro-scale significantly reduced the fine particle fraction, while roughening of the surface led to a remarkable increase. The latter could be ascribed to decreased adhesive forces. In the course of this thesis, the impact of the dimensioning of important inhaler parts of a dry powder inhaler was investigated as well. Results showed that air-ducting parts like the air inlet or the mouthpiece should be narrowed to increase the air stream velocity and consequently kinetic energy and level of turbulence. In addition, a small grid proved to be beneficial, as it led to a slow and controlled discharge of the deagglomeration chamber.Zur Therapie von Erkrankungen der Atemwege werden vornehmlich Medikamente eingesetzt, die pulmonal verabreicht werden. Hierbei wird der Arzneistoff in der Regel zunächst mit einem groben Träger zu einer interaktiven Pulvermischung verarbeitet, um so unter anderem die Dosiergenauigkeit zu erhöhen. Die zwischen Arzneistoff und Träger vorherrschenden interpartikulären Wechselwirkungen bestimmen maßgeblich das aerodynamische Verhalten der Pulvermischung und damit das Ausmaß der Deposition des Arzneistoffs in der Lunge. Diese Wechselwirkungen werden durch eine Reihe verschiedener Faktoren bestimmt, wie die Oberflächenchemie und –topographie des Trägers. Um deren Einfluss genauer zu untersuchen wurden Glaskugeln als Modellträgersystem verwendet. Diese wurden mit speziellen Modifizierungstechniken wie Silanisierung, Inkubation mit Flusssäure und Mahlung mit Wolframcarbid verändert. Die chemischen Eigenschaften zeigten unterschiedliche Effekte auf die pulmonale Deposition abhängig von dem verwendeten Arzneistoff. Hingegen zeigte die Modifizierung der Oberflächentopographie den gleichen Effekt auf das aerodynamische Verhalten der Arzneistoffe. Eingebrachte Kavitäten im Mikrometermaßstab führten zu einer deutlichen Verschlechterung des aerodynamischen Verhaltens, während ein Anstieg der Feinpartikelfraktion durch das Aufrauen der Oberfläche bei der Wolframcarbidmahlung erreicht werden konnte. Dies wurde einer Abnahme der adhäsiven Kräfte zwischen Arzneistoff und Träger zugeschrieben. Des Weiteren wurde in dieser Arbeit der Einfluss der Dimensionierung wichtiger Bauteile eines Pulverinhalators untersucht. Es wurde gezeigt, dass luftführende Bauteile möglichst klein dimensioniert werden sollten, um eine hohe Luftgeschwindigkeit im Inhalator zu generieren. Dies führt zu erhöhten kinetischen Energie und Turbulenzgrad. Auch ein kleines Gitter zeigte einen vorteilhaften Effekt, da hierdurch eine kontrollierte Entleerung der Deagglomerationskammer erzielt wird

Hepatocellular carcinoma progression during bridging before liver transplantation

Background Recipient selection for liver transplantation in hepatocellular carcinoma (HCC) is based primarily on criteria affecting the chance of long-term success. Here, the relationship between pretransplant bridging therapy and long-term survival was investigated in a subgroup analysis of the SiLVER Study. Methods Response to bridging, as defined by comparison of imaging at the time of listing and post-transplant pathology report, was categorized into controlled versus progressive disease (more than 20 per cent tumour growth or development of new lesions). Results Of 525 patients with HCC who had liver transplantation, 350 recipients underwent pretransplant bridging therapy. Tumour progression despite bridging was an independent risk factor affecting overall survival (hazard ratio 1.80; P = 0.005). For patients within the Milan criteria (MC) at listing, mean overall survival was longer for those with controlled versus progressive disease (6.8 versus 5.8 years; P < 0.001). Importantly, patients with HCCs outside the MC that were downsized to within the MC before liver transplantation had poor outcomes compared with patients who never exceeded the MC (mean overall survival 6.2 versus 6.6 years respectively; P = 0.030). Conclusion Patients with HCCs within the MC that did not show tumour progression under locoregional therapy had the best outcomes after liver transplantation. Downstaging into the limits of the MC did not improve the probability of survival. Prognostic factors determining the long-term success of liver transplantation in patients with hepatocellular carcinoma are still under discussion. A subgroup analysis of the SiLVER trial showed that disease control under bridging therapy is strongly associated with improved prognosis in terms of overall survival. However, in tumours exceeding the limits of the Milan criteria, downstaging did not restore the probability of survival compared with that of patients within the Milan criteria

DWI - histology: a possible means of determining degree of liver fibrosis?

Objectives: The aim of this study was to determine the diagnostic value of diffusion-weighted MRI of the liver at 3T to classify liver fibrosis/cirrhosis. Methods: 62 patients who underwent both histopathological examination and diffusion-weighted imaging of the liver via 3T MRI within a period of 3 months were included in the study. The Ishak score (1-6) was used to determine the degree of fibrosis: No liver fibrosis (NLF; Ishak 0, n = 16), mild liver fibrosis (MLF; Ishak 1-2, n = 23), advanced liver fibrosis (ALF; Ishak 3-5, n = 12), and liver cirrhosis (LC; Ishak 6, n = 11). Results: The corresponding ADC values for the individual patient groups were as follows: NLF: 1123 (SD 95.8); MLF: 1032 (SD 77.6); ALF: 962 (SD 68.8); LC: 1015 (SD 60.2) mm2/s. There is a significant difference between NLF and MLF (p = 0.004) and between MLF and ALF (p = 0.022). A significant difference between patients with ALF and LC (p = 0.117) could not be found. Conclusion: Liver fibrosis/cirrhosis lowers the ADC values of the liver parenchyma in 3T MRI. However, the degree of fibrosis can only be conditionally determined on the basis of ADC values

Similar complication rates for irreversible electroporation and thermal ablation in patients with hepatocellular tumors

Background. To compare the frequency of adverse events of thermal microwave (MWA) and radiofrequency ablation (RFA) with non-thermal irreversible electroporation (IRE) in percutaneous ablation of hepatocellular carcinoma (HCC). Patients and methods. We retrospectively analyzed 117 MWA/RFA and 47 IRE procedures (one tumor treated per procedure; 144 men and 20 women; median age, 66 years) regarding adverse events, duration of hospital and intensive care unit (ICU) stays and occurrence of a post-ablation syndrome. Complications were classified according to the Clavien & Dindo classification system. Results. 70.1% of the RFA/MWA and 63.8% of the IRE procedures were performed without complications. Grade I and II complications (any deviation from the normal postinterventional course, e.g., analgesics) occurred in 26.5% (31/117) of MWA/RFA and 34.0% (16/47) of IRE procedures. Grade III and IV (major) complications occurred in 2.6% (3/117) of MWA/RFA and 2.1% (1/47) of IRE procedures. There was no significant difference in the frequency of complications (p = 0.864), duration of hospital and ICU stay and the occurrence of a post-ablation syndrome between the two groups. Conclusions. Our results suggest that thermal (MWA and RFA) and non-thermal IRE ablation of malignant liver tumors have comparable complication rates despite the higher number of punctures and the lack of track cauterization in IRE

Severe T cell hyporeactivity in ventilated COVID-19 patients correlates with prolonged virus persistence and poor outcomes

Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity.Coronavirus disease 2019 (COVID-19) can lead to pneumonia and hyperinflammation. Here we show a sensitive method to measure polyclonal T cell activation by downstream effects on responder cells like basophils, plasmacytoid dendritic cells, monocytes and neutrophils in whole blood. We report a clear T cell hyporeactivity in hospitalized COVID-19 patients that is pronounced in ventilated patients, associated with prolonged virus persistence and reversible with clinical recovery. COVID-19-induced T cell hyporeactivity is T cell extrinsic and caused by plasma components, independent of occasional immunosuppressive medication of the patients. Monocytes respond stronger in males than females and IL-2 partially restores T cell activation. Downstream markers of T cell hyporeactivity are also visible in fresh blood samples of ventilated patients. Based on our data we developed a score to predict fatal outcomes and identify patients that may benefit from strategies to overcome T cell hyporeactivity

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery

Exploring the impact of fossil constraints on the divergence time estimates of derived liverworts

In this study, we evaluate the impact of fossil assignments and different models of calibration on divergence time estimates carried out as Bayesian analyses. Estimated ages from preceding studies and liverwort inclusions from Baltic amber are used as constraints on a molecular phylogeny of Cephaloziineae (Jungermanniopsida) obtained from sequences of two chloroplast coding regions: rbcL and psbA. In total, the comparison of 12 different analyses demonstrates that an increased reliability of the chronograms is linked to the number of fossils assigned and to the accuracy of their assignments. Inclusion of fossil constraints leads to older ages of most crown groups, but has no influence on lineage through time plots suggesting a nearly constant accumulation of diversity since the origin of Cephaloziineae in the early to Middle Jurassic. Our results provide a note of caution regarding the interpretation of chronograms derived from DNA sequence variation of extant species based on a single calibration point and/or low accuracy of the assignment of fossils to nodes in the phylogeny

Biomechanics of Ankle Ligament Reconstruction: A Cadaveric Study to Compare Stability of Reconstruction Techniques Using 1 or 2 Fibular Tunnels

Background Anatomic lateral ankle ligament reconstruction has been proposed for patients with chronic ankle instability. A reliable approach is a reconstruction technique using an allograft and 2 fibular tunnels. A recently introduced approach that entails 1-fibular tunnel reconstruction might reduce the risk of intraoperative complications and ultimately improve patient outcome. Hypothesis We hypothesized that both reconstruction techniques show similar ankle stability (joint laxity and stiffness) and are similar to the intact joint condition. Study Design Controlled laboratory study. Methods A total of 10 Thiel-conserved cadaveric ankles were divided into 2 groups and tested in 3 stages-intact, transected, and reconstructed lateral ankle ligaments-using either the 1- or the 2-fibular tunnel technique. To quantify stability in each stage, anterior drawer and talar tilt tests were performed in 0°, 10°, and 20° of plantarflexion (anterior drawer test) or dorsiflexion (talar tilt test). Bone displacements were measured using motion capture, from which laxity and stiffness were calculated together with applied forces. Finally, reconstructed ligaments were tested to failure in neutral position with a maximal applicable torque in inversion. A mixed linear model was used to describe and compare the outcomes. Results When ankle stability of intact and reconstructed ligaments was compared, no significant difference was found between reconstruction techniques for any flexion angle. Also, no significant difference was found when the maximal applicable torque of the 1-tunnel technique (9.1 ± 4.4 N·m) was compared with the 2-tunnel technique (8.9 ± 4.8 N·m). Conclusion Lateral ankle ligament reconstruction with an allograft using 1 fibular tunnel demonstrated similar biomechanical stability to the 2-tunnel approach. Clinical Relevance Demonstrating similar stability in a cadaveric study and given the potential to reduce intraoperative complications, the 1-fibular tunnel approach should be considered a viable option for the surgical therapy of chronic ankle instability. Clinical randomized prospective trials are needed to determine the clinical outcome of the 1-tunnel approach