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Molecular Mechanisms of Bortezomib Action: Novel Evidence for the miRNA−mRNA Interaction Involvement
(2020)
Bortezomib is an anti-tumor agent, which inhibits 26S proteasome degrading ubiquitinated
proteins. While apoptotic transcription-associated activation in response to bortezomib has been
suggested, mechanisms related to its influence on post-transcriptional gene silencing mediated
regulation by non-coding RNAs remain not fully elucidated. In the present study, we examined
changes in global gene and miRNA expression and analyzed the identified miRNA–mRNA interactions
after bortezomib exposure in human neuroblastoma cells to define pathways affected by this agent in
this type of cells. Cell viability assays were performed to assess cytotoxicity of bortezomib. Global gene
and miRNA expression profiles of neuroblastoma cells after 24-h incubation with bortezomib were
determined using genome-wide RNA and miRNA microarray technology. Obtained results were
then confirmed by qRT-PCR and Western blot. Further bioinformatical analysis was performed
to identify affected biological processes and pathways. In total, 719 genes and 28 miRNAs were
downregulated, and 319 genes and 61 miRNAs were upregulated in neuroblastoma cells treated with
bortezomib. Possible interactions between dysregulated miRNA/mRNA, which could be linked to
bortezomib-induced neurotoxicity, affect neurogenesis, cellular calcium transport, and neuron death.
Bortezomib might exert toxic effects on neuroblastoma cells and regulate miRNA–mRNA interactions
influencing vital cellular functions. Further studies on the role of specific miRNA–mRNA interactions
are needed to elucidate mechanisms of bortezomib action.
Chemerin ist ein Adipokin, welches unter anderem vom Fettgewebe exprimiert wird und wichtige Funktionen im Rahmen des Fettstoffwechsels übernimmt. Verschiedene klinische Studien konnten Korrelationen zwischen der Chemerinkonzentration im Blut und diversen metabolischen, renalen sowie kardiovaskulären Phänotypen identifizieren. Aufgrund der Verwendung von sehr unterschiedlichen Patientenpopulationen mit häufig kleinen Kollektiven ist die derzeitige Befundlage insgesamt allerdings noch recht widersprüchlich. Das Ziel der vorliegenden Arbeit war daher, den Zusammenhang zwischen Chemerin und einem breiten Spektrum von inflammatorischen, metabolischen, renalen sowie kardiovaskulären Parametern in einer großen, gut charakterisierten, populationsbasierten Studie zu analysieren und dabei den Einfluss der Fettmasse und anderer Störfaktoren angemessen zu berücksichtigen.
Unter Zuhilfenahme von Methoden der multivariablen linearen und logistischen Regressionsanalyse wurden in dieser Arbeit Daten von 4420 Probanden der SHIP-TREND-Population ausgewertet. Die Analysen zeigten positive Assoziationen zwischen Chemerin und den Inflammationsparametern hsCRP und Fibrinogen. Darüber hinaus konnten positive Zusammenhänge mit dem HbA1c-Wert, dem Nüchterninsulinwert und dem HOMA-IR identifiziert werden. Für den Glukosespiegel ließ sich dagegen kein Zusammenhang mit Chemerin nachweisen. Die Ergebnisse zeigten weiterhin, dass ein hoher Chemerinspiegel mit einem unvorteilhaften Lipid-Profil assoziiert ist. Während eine glockenförmige Assoziation zwischen Chemerin und dem diastolischen Blutdruck vorlag, wurde kein Zusammenhang mit dem systolischen Blutdruck beobachtet. Alle beschriebenen Zusammenhänge waren unabhängig von Unterschieden der Probanden in der individuellen Fettmasse sichtbar. Die Analysen zeigten darüber hinaus, dass ein hoher Chemerinspiegel signifikant mit einer schlechteren Nierenfunktion einhergeht und dass sich dieser Effekt mit zunehmender Fettmasse noch verstärkt. Bezüglich der verschiedenen subklinischen Parameter der Atherosklerose konnte nach vollständiger Modelladjustierung eine inverse Assoziation von Chemerin mit dem ABI sowie eine positive Assoziation mit dem Vorliegen einer Karotisstenose beobachtet werden.
Zusammenfassend zeigen die dargestellten Ergebnisse, dass Chemerin unabhängig von individuellen Unterschieden in der Fettmasse mit einer ganzen Reihe von verschiedenen inflammatorischen, metabolischen, renalen und kardiovaskulären Parametern assoziiert ist. Dementsprechend kann vermutet werden, dass Chemerin auch abseits seiner Funktionen im Fettgewebe wichtige pathophysiologische Prozesse im Körper steuert. Existierende experimentelle Arbeiten liefern erste Hinweise auf die zugrundeliegenden Mechanismen. Das gesamte Funktionsspektrum von Chemerin ist zum jetzigen Zeitpunkt allerdings nicht vollständig geklärt. Die vorliegende Arbeit liefert einen umfassenden Überblick über die epidemiologischen Zusammenhänge der genannten Parameter in einer allgemeinen Bevölkerung und stellt damit eine wichtige Grundlage für weitere Forschungsarbeiten dar. Langfristig ist denkbar, dass Chemerin als diagnostischer Biomarker oder Therapeutikum im klinischen Alltag eingesetzt wird.
Abstract
Background
Duchenne muscular dystrophy (DMD) is a progressive muscle‐wasting disease caused by mutations in the dystrophin gene, which leads to structural instability of the dystrophin–glycoprotein‐complex with subsequent muscle degeneration. In addition, muscle inflammation has been implicated in disease progression and therapeutically addressed with glucocorticosteroids. These have numerous adverse effects. Treatment with human immunoglobulin G (IgG) improved clinical and para‐clinical parameters in the early disease phase in the well‐established mdx mouse model. The aim of the present study was to confirm the efficacy of IgG in a long‐term pre‐clinical study in mdx mice.
Methods
IgG (2 g/kg body weight) or NaCl solution as control was administered monthly over 18 months by intraperitoneal injection in mdx mice beginning at 3 weeks of age. Several clinical outcome measures including endurance, muscle strength, and echocardiography were assessed. After 18 months, the animals were sacrificed, blood was collected for analysis, and muscle samples were obtained for ex vivo muscle contraction tests, quantitative PCR, and histology.
Results
IgG significantly improved the daily voluntary running performance (1.9 m more total daily running distance, P < 0.0001) and slowed the decrease in grip strength by 0.1 mN, (P = 0.018). IgG reduced fatigability of the diaphragm (improved ratio to maximum force by 0.09 ± 0.04, P = 0.044), but specific tetanic force remained unchanged in the ex vivo muscle contraction test. Cardiac function was significantly better after IgG, especially fractional area shortening (P = 0.012). These results were accompanied by a reduction in cardiac fibrosis and the infiltration of T cells (P = 0.0002) and macrophages (P = 0.0027). In addition, treatment with IgG resulted in a significant reduction of the infiltration of T cells (P ≤ 0.036) in the diaphragm, gastrocnemius, quadriceps, and a similar trend in tibialis anterior and macrophages (P ≤ 0.045) in gastrocnemius, quadriceps, tibialis anterior, and a similar trend in the diaphragm, as well as a decrease in myopathic changes as reflected by a reduced central nuclear index in the diaphragm, tibialis anterior, and quadriceps (P ≤ 0.002 in all).
Conclusions
The present study underscores the importance of an inflammatory contribution to the disease progression of DMD. The data demonstrate the long‐term efficacy of IgG in the mdx mouse. IgG is well tolerated by humans and could preferentially complement gene therapy in DMD. The data call for a clinical trial with IgG in DMD.
Dengue virus (DV) is a positive-strand RNA virus of the Flavivirus genus. It is one of the most prevalent mosquito-borne viruses, infecting globally 390 million individuals per year. The clinical spectrum of DV infection ranges from an asymptomatic course to severe complications such as dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), the latter because of severe plasma leakage. Given that the outcome of infection is likely determined by the kinetics of viral replication and the antiviral host cell immune response (HIR) it is of importance to understand the interaction between these two parameters. In this study, we use mathematical modeling to characterize and understand the complex interplay between intracellular DV replication and the host cells' defense mechanisms. We first measured viral RNA, viral protein, and virus particle production in Huh7 cells, which exhibit a notoriously weak intrinsic antiviral response. Based on these measurements, we developed a detailed intracellular DV replication model. We then measured replication in IFN competent A549 cells and used this data to couple the replication model with a model describing IFN activation and production of IFN stimulated genes (ISGs), as well as their interplay with DV replication. By comparing the cell line specific DV replication, we found that host factors involved in replication complex formation and virus particle production are crucial for replication efficiency. Regarding possible modes of action of the HIR, our model fits suggest that the HIR mainly affects DV RNA translation initiation, cytosolic DV RNA degradation, and naïve cell infection. We further analyzed the potential of direct acting antiviral drugs targeting different processes of the DV lifecycle in silico and found that targeting RNA synthesis and virus assembly and release are the most promising anti-DV drug targets.
Over the past 10 years, the crisis of sepsis has remained a great challenge. According to data from 2016, the sepsis-related mortality rate remains high. In addition, sepsis consumes extensive medical resources in intensive care units, and anti-inflammatory agents fail to improve sepsis-associated hyperinflammation and symptoms of immunosuppression. The specific immune mechanism of sepsis remains to be elucidated. Reactive oxygen species (ROS) are triggered by energy metabolism and respiratory dysfunction in sepsis, which not only cause oxidative damage to tissues and organelles, but also directly and indirectly promote NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) inflammasome activation. NLRP3 inflammasomes enlarge the inflammatory response and trigger apoptosis of immune cells to exacerbate sepsis progression. Inhibiting the negative effects of ROS and NLRP3 inflammasomes therefore provides the possibility of reversing the excessive inflammation during sepsis. In this review, we describe the interaction of ROS and NLRP3 inflammasomes during sepsis, provide prevention strategies, and identify fields that need further study.
Bats belong to the most gregarious and diverse mammals with highly complex social behaviors. Despite extensive research on their ecology and social behavior in some bat species, gained insights are restricted to only few of the more than 1300 species. In the recent past, bats have also become a central topic of a different branch of research: Since the 1990s bats came to the fore of virologists and immunologists due to the bats’ apparent importance as reservoir hosts and vectors of several (mostly tropical) diseases. While this research is focused mainly on emerging infectious diseases linked to bats, and their zoonotic potential, little has been invested regarding the link between disease transmission and bat social systems.
In my work, I aim at filling this gap by merging automated daily roosting observations, social network analysis, and a virological screening in Natterer’s bats (Myotis nattereri). In a collaborative approach, my co-workers and I analyzed the social structure of individually marked Natterer’s bats, their astrovirus detection rate and transmission pathways within their colony, as well as roosting interactions between different co-occurring con- and heterospecific bat colonies.
We discovered Natterer’s bats to display a very divergent social network structure that contradicts the findings of previous studies on large fission-fusion groups. Contrary to the modular social network structure found in e.g. primates or other bats species, the social network of Natterer’s bats consists of only one highly interconnected community. Moreover, although the close proximity between bat hosts in the colony should strongly promote direct transmission, we found indications that astrovirus infections follow at least partly an indirect transmission pathway via contaminated roost use. Lastly, our results prove that co-occurring con- and heterospecific bat colonies, e.g. as in this study Natterer’s bats, brown long-eared bats and Bechstein’s bats, can influence each other in their roost use by avoiding conspecific roosts and by being attracted towards those of heterospecifics. This holds implication for the transmission of parasites and pathogens within and between different colonies with opportunities for spillovers. To conclude, this multidisciplinary study led to valuable insights in the hitherto hidden mechanisms within and among bat colonies.
Abstract
Ecosystems with highly pulsed water supply must be better understood as climate change may increase frequency and severity of intense storms, droughts and floods. Here we collected data over 3 years (2016–2018) in the episodic wetland outflow channel (Aluize), Banhine National Park, in which the system state changed from dry to wet to dry. Field sampling included vegetation records, small‐scale vegetation zoning, the seed bank and water and soil quality. The same main plant species were found in both dry and wet conditions across the riverbed of the outflow channel. We found only very few diaspores of plants in the soil after prolonged drought. In the subsequent flooded state, we examined very dense vegetation on the water surface, which was dominated by the gramineous species Paspalidium obtusifolium. This species formed a compact floating mat that was rooted to the riverbed. The Cyperaceae Bolboschoenus glaucus showed high clonal growth in the form of root tubers, which likely serve as important food reservoir during drought. Soil and water analyses do not indicate a limitation by nutrients. We outline how resident people may change the plant community structure with an increasing practice of setting fire to the meadows in the dried‐up riverbed to facilitate plant regrowth as food for their livestock.
Hintergrund: Die Infektion mit dem Humanen Immundefizienz-Virus (HIV) ist keine tödliche Krankheit mehr [1]. Mit dem Aufkommen der HAART (highly active antiretroviral therapy) im Jahre 1996 und ihrer stetigen Weiterentwicklung hin zur cART (combined antiretroviral therapy) stieg der Behandlungserfolg bei HIV-infizierten Personen drastisch an. Eine Kombination aus unterschiedlichen Wirkstoffklassen machte die Therapie effizienter und verbesserte die Lebensqualität von HIV-Patienten [92]. Doch obwohl sich die Medikation als gut und wirksam erwiesen hat, ist sie nicht frei von Nachteilen. Die Adhärenz bei der Medikamenteneinnahme ist bei vielen Patienten nicht ausreichend, was unter anderem Nebenwirkungen, sozialen Faktoren, Stigmatisierung und Komorbiditäten geschuldet ist. Unterschreitet die Medikamenteneinnahme 95 % der verordneten Dosis, besteht die Gefahr von Resistenzbildung, die mit hohen Kosten für das Gesundheitssystem und gesundheitlichen Risiken für die infizierte Person verbunden ist [4, 10]. Die kontinuierliche Weiterentwicklung der antiretroviralen Therapie hat zu einer deutlichen Veränderung der Ansprüche an die Qualität der Versorgung geführt. Während in den Anfangstagen der HIV-Behandlung das Hauptaugenmerk auf die Verhinderung von opportunistischen Infektionen (OI) gelegt wurde, sind inzwischen Aspekte wie die langfristige Virussuppression und die stabile Rekonstitution des Immunsystems die Mindestanforderungen der Therapie. Trotz dieser allgemeingültigen Ziele gibt es regional unterschiedliche Herausforderungen. Zur unterschiedlichen Versorgungsqualität in ländlichen und städtischen Regionen existieren nur rudimentäre Daten. Ziel dieser Arbeit war, diese Daten prototypisch für Berlin und Greifswald zu erheben.
Methoden: Im Rahmen der Qualitätssicherungsvereinbarung zur strukturierten Patientenversorgung nach § 135 Abs. 2 SGB V wurden gemäß EBM seit 2009 in Berlin sowie in Greifswald Daten HIV-positiver Patienten im Rahmen der Routineversorgung erhoben. Von 43 Patienten in Greifswald konnten 41 mit einer HIV-Erkrankung in unsere Studie eingeschlossen werden. In Berlin umfasste die Ursprungskohorte einer Schwerpunktpraxis 1669 Patienten. Eingeschlossen wurden aufgrund der von uns angelegten Kriterien (Sampling der Berliner Patientendaten nach Alter und Geschlecht) jedoch nur 187 Patienten. Die wichtigsten Ausschlusskriterien waren eine fehlende Einwilligung zur Studienteilnahme sowie ein Lost to follow-up (LTFU) über mehr als drei Quartale. Die Auswertung der bizentrischen, nicht interventionellen Korrelationsstudie erfolgte retrospektiv. Der Beobachtungszeitraum betrug fünf Jahre (01/2009 – 01/2014). Hauptaugenmerk der Datenauswertung wurde auf die Erfassung, den Vergleich und die Herausarbeitung von Gemeinsamkeiten und Unterschieden der demografischen, immunologischen, virologischen, klinischen und therapeutischen Daten der beiden Kohorten gelegt. Die Auswertung erfolgte mithilfe des Statistikprogrammes R (Version 3.2.1). Statistische Signifikanz wurde bei p ≤ 0,05 angenommen.
Ergebnisse: Vor Durchführung des Samplings bestanden deutliche Unterschiede hinsichtlich der demografischen Daten in beiden Kohorten. Bei gleichem Altersdurchschnitt (Median Berlin (B): 44,4 Jahre; Greifswald (G): 44,5 Jahre; p = 0,94) war der Anteil HIV-infizierten Frauen in Greifswald deutlich höher (B: 9,4%; G: 22%; p = 0,01), was sich im ländlichen Raum auch in einer vergleichsweise geringen Transmissionsrate der HIV-Infektion über Männer, die Sex mit Männern haben (MSM) niederschlug (B: 141/187 [75,4%]; G: 17/41 [41,46%]; p = 5,384e-05). Zu Therapiebeginn zeigte sich eine im Median signifikant geringere CD4+ T-Lymphozytenzellzahl in Greifswald als in Berlin (B: 516/µl; G: 266/µl; p < 0,001). Weiterhin hatten in Greifswald mehr Patienten einen Ausgangswert von <200 CD4+ T-Zellen je µl Blut (B: 12/187 [6,41%]; G: 13/41 [31,7%]; p < 0,001). Ungeachtet dieser unterschiedlichen Ausgangssituationen glichen sich die CD4+ T-Zellzahlen unter cART nach drei Quartalen auf ein einheitlich hohes Niveau an (B: 564/µl; G: 416/µl; p = 0,095). Die Viruslast (VL) lag in Greifswald zu Therapiebeginn nicht signifikant über der von Berlin (p = 0,17). Ein Abfall der VL unter cART auf ein Level von < 50 Kopien/ml erfolgte in beiden Kohorten ähnlich schnell (B: 128d; G: 137d; p= 0,8). Von allen 184 bzw. 41 diagnostizierten Patienten wurden in beiden Kohorten > 80 % mit cART versorgt (B: 153/184 [83,15%]; G: 36/41 [87,8%]; p = 0,12). Ein Absenken der VL unter die Nachweisgrenze (NG) gelang in beiden Kohorten bei mehr als der Hälfte der Patienten (B: 115/184 [62,5%]; G: 23/41 [56,09%]; p = 0,085). Die Firstline-Therapie (FL) wurde in Berlin im Median 1127 d und in Greifswald 809 d eingenommen (p = 0,09). Eingesetzte Therapieregime in FL sowie in Secondline (SL) waren in beiden Kohorten weitgehend übereinstimmend (FL: p = 0,48; SL: p = 0,08). Therapiewechsel fanden etwa in gleicher Häufigkeit statt (B: 33,5%; G: 32,1%; p = 0,87), während die Gründe für einen Therapiewechsel voneinander abwichen (p = 0,0076). Therapieumstellungen fanden in Berlin am häufigsten aufgrund einer Therapievereinfachung (Umstellung auf Single-tablet regimen (STR)) oder auf Patientenwunsch statt, während in Greifswald medikamentenassoziierte Probleme wie Resistenzbildung und das Auftreten von Nebenwirkungen am häufigsten als Ursache für einen Therapiewechsel benannt wurden. Koinfektionen wie Hepatitis C (HCV) und Hepatitis B (HBV) traten in beiden Kohorten mit gleicher Häufigkeit auf (p = 1), auch die Anzahl an durchgeführten HBV-Impfungen differierte nicht (p = 0,68). Ein Rückgang der OI war nach Beginn mit cART in beiden Kohorten gleichermaßen zu verzeichnen (p = 0,87).
Diskussion: Die Auswertungen unserer Studie zeigen insgesamt, dass die Versorgung von HIV-infizierten Patienten sowohl in städtischen als auch in ländlichen Regionen leitlinienkonform durchgeführt wird und die Qualität sehr hoch ist. Dieses Fazit kann gezogen werden, obgleich sich die Patienten aus Greifswald mit ihrer Erkrankung bei Therapiebeginn in einem deutlich weiter fortgeschrittenen Stadium befanden. Die Zahl der CD4+ T-Lymphozyten bei Erstdiagnose bzw. bei Therapiebeginn hat sich dabei als wichtigster Vorhersagewert etabliert, da niedrige Werte in direkter Verbindung mit dem Auftreten von Komorbiditäten stehen. Ein später Beginn mit cART ist weiterhin direkt mit einer erhöhten Morbidität und Mortalität assoziiert [48, 49], besonders dann, wenn die CD4+ T-Lymphozyten bei Beginn mit cART bereits unter 200/µl abgesunken sind [50,51]. Trotzdem gelingt durch eine leitlinienorientierte und stringente Therapie eine Angleichung der Werte innerhalb kurzer Zeit, was für ein hohes Versorgungsniveau auch im ländlichen Raum spricht. Zwischen dem Leben im ländlichen Raum und einem späten Beginn mit cART scheint des Weiteren ein Zusammenhang zu bestehen [52]. Als mögliche Ursachen hierfür werden verminderte Risikowahrnehmung, größere Stigmatisierung, weniger Diskretion und Anonymität, ein geringerer Bildungsgrad sowie ein schlechterer Zugang zu Aufklärungskampagnen und Screening-Maßnahmen angegeben [52, 54, 55]. Ob diese Gründe auch für Greifswald Gültigkeit besitzen, erfordert weiteren Nachforschungen. Mitverantwortlich für die unterschiedlichen Ausgangssituationen könnte des Weiteren sein, dass in Greifswald die Versorgung von HIV-positiven Patienten nicht durch eine Fachabteilung, sondern - aus historischen Gründen - durch verschiedene Ambulanzen bzw. Klinken mitgetragen wurde. Ein Umstand, der Ende 2014 durch die Betreuungsübernahme aller HIV-Patienten in die Klinik und Poliklinik für Hautkrankheiten der Universitätsmedizin Greifswald bereits geändert wurde und dessen Effekte es im Nachgang zu evaluieren gilt.
Die cART wurde in beiden Kohorten wirkstoffgleich verordnet, während die Gründe für Therapiewechsel voneinander abwichen. Am ehesten erklärbar ist die häufige Umstellung der cART auf Patientenwunsch in Berlin dabei durch viele HIV-Schwerpunktpraxen, HIV-Selbsthilfegruppen und AIDS-Treffpunkte in der Hauptstadt, die einen regeren Austausch über neue Therapieregime und Möglichkeiten zur Verbesserung bzw. zur Vereinfachung der Therapie ermöglichen.
Abstract
Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.