Dynamic force microscopy for imaging of viruses under physiological conditions

Dynamic force microscopy (DFM) allows imaging of the structure and the assessment of the function of biological specimens in their physiological environment. In DFM, the cantilever is oscillated at a given frequency and touches the sample only at the end of its downward movement. Accordingly, the problem of lateral forces displacing or even destroying bio-molecules is virtually inexistent as the contact time and friction forces are reduced. Here, we describe the use of DFM in studies of human rhinovirus serotype 2 (HRV2) weakly adhering to mica surfaces. The capsid of HRV2 was reproducibly imaged without any displacement of the virus. Release of the genomic RNA from the virions was initiated by exposure to low pH buffer and snapshots of the extrusion process were obtained. In the following, the technical details of previous DFM investigations of HRV2 are summarized

Insights into Minor Group Rhinovirus Uncoating: The X-ray Structure of the HRV2 Empty Capsid

Upon attachment to their respective receptor, human rhinoviruses (HRVs) are internalized into the host cell via different pathways but undergo similar structural changes. This ultimately results in the delivery of the viral RNA into the cytoplasm for replication. To improve our understanding of the conformational modifications associated with the release of the viral genome, we have determined the X-ray structure at 3.0 Å resolution of the end-stage of HRV2 uncoating, the empty capsid. The structure shows important conformational changes in the capsid protomer. In particular, a hinge movement around the hydrophobic pocket of VP1 allows a coordinated shift of VP2 and VP3. This overall displacement forces a reorganization of the inter-protomer interfaces, resulting in a particle expansion and in the opening of new channels in the capsid core. These new breaches in the capsid, opening one at the base of the canyon and the second at the particle two-fold axes, might act as gates for the externalization of the VP1 N-terminus and the extrusion of the viral RNA, respectively. The structural comparison between native and empty HRV2 particles unveils a number of pH-sensitive amino acid residues, conserved in rhinoviruses, which participate in the structural rearrangements involved in the uncoating process

Uncoating of human rhinovirus serotype 2 from late endosomes

The internalization pathway and mechanism of uncoating of human rhinovirus serotype 2 (HRV2), a minor-group human rhinovirus, were investigated. Kinetic analysis revealed a late endosomal compartment as the site of capsid modification from D to C antigenicity. The conformational change as well as the infection was prevented by the specific V-ATPase inhibitor bafilomycin A1. A requirement for ATP was also demonstrated with purified endosomes in vitro. Capsid modifications occurred at a pH of 5.5 regardless of whether the virus was entrapped in isolated endosomes or free in solution. These findings suggest that the receptor is not directly involved in the structural modification of HRV2. Viral particles found in purified endosomes of infected cells were mostly devoid of RNA. This supports the hypothesis that uncoating of HRV2 occurs in intact endosomes rather than by a mechanism involving endosomal disruption with subsequent release of the RNA into the cytoplasm.</jats:p

Alarming Increase of Ketoacidosis Prevalence at Type 1 Diabetes-Onset in Austria—Results From a Nationwide Registry

ObjectiveWe analyzed the annual prevalence of onset-DKA (diabetic ketoacidosis) from 2012 to 2020 with a sub-analysis for lockdown-periods during the COVID-19 pandemic in 2020.DesignAll newly diagnosed children with type 1 diabetes (T1D) aged &amp;lt;15 years are prospectively registered in the population-based Austrian Diabetes Incidence Study in Austria.Main Outcome MeasuresThe annual DKA prevalence was analyzed using Joinpoint regression. Definition of DKA: pH &amp;lt;7.3, mild DKA: pH 7.3 to ≤ 7.1, severe DKA: pH &amp;lt;7.1. DKA prevalence during the lockdown periods in 2020 and the corresponding periods in 2015–2019 were examined using Fisher's exact test.ResultsIn the years 2012–2020 the mean prevalence for onset-DKA in Austria was 43.6% [95%CI (confidence interval): 41.6, 45.7] and thus above the mean prevalence of previous decades (1989–2011) of 37,1 % (95%CI: 35.6, 38.6). A particularly high prevalence was found among children &amp;lt;2 years of age (72.0% DKA, 32.8% severe DKA). No significant gender difference was found. Prevalence of severe DKA at T1D-onset increased significantly since 2015 (p = 0.023). During the lockdown in 2020, 59.3% of children were diagnosed with DKA at T1D-onset, compared to 42.1% during the previous 5 years (p = 0.022). Moreover, 20% of children had severe DKA at T1D diagnosis, compared to 14% during the comparison period.ConclusionsThe previously already high prevalence of DKA at T1D-onset has further increased over time. The COVID-19 pandemic has exacerbated the problem of a late or delayed diagnosis of diabetes in children resulting in onset-DKA. The alarmingly increased prevalence of DKA in Austrian children with T1D calls for urgent action.</jats:sec

Data_Sheet_1_Alarming Increase of Ketoacidosis Prevalence at Type 1 Diabetes-Onset in Austria—Results From a Nationwide Registry.PDF

ObjectiveWe analyzed the annual prevalence of onset-DKA (diabetic ketoacidosis) from 2012 to 2020 with a sub-analysis for lockdown-periods during the COVID-19 pandemic in 2020.DesignAll newly diagnosed children with type 1 diabetes (T1D) aged Main Outcome MeasuresThe annual DKA prevalence was analyzed using Joinpoint regression. Definition of DKA: pH ResultsIn the years 2012–2020 the mean prevalence for onset-DKA in Austria was 43.6% [95%CI (confidence interval): 41.6, 45.7] and thus above the mean prevalence of previous decades (1989–2011) of 37,1 % (95%CI: 35.6, 38.6). A particularly high prevalence was found among children ConclusionsThe previously already high prevalence of DKA at T1D-onset has further increased over time. The COVID-19 pandemic has exacerbated the problem of a late or delayed diagnosis of diabetes in children resulting in onset-DKA. The alarmingly increased prevalence of DKA in Austrian children with T1D calls for urgent action.</p

Rehabilitation für Kinder und Jugendliche mit Diabetes in Österreich: Konzept der Arbeitsgruppe Pädiatrische Endokrinologie und Diabetologie Österreich/Österreichische Gesellschaft für Kinder- und Jugendheilkunde

ZusammenfassungDiabetes ist eine der häufigsten chronischen Stoffwechselerkrankungen im Kindes- und Jugendalter. Die Zahl der Neuerkrankten hat in der letzten Dekade stark zugenommen.Typ-1-Diabetes ist mit &gt; 90 % die häufigste Form des Diabetes im Kindes- und Jugendalter; deshalb bezieht sich das vorliegende Konzept auf Kinder und Jugendliche mit Typ-1-Diabetes. Seit 2016 gibt es in Österreich die ersten Rehabilitationszentren für Kinder und Jugendliche. Rehabilitation wird dann nötig, wenn sich Probleme mit dem Diabetes in der ambulanten Behandlung nicht lösen lassen oder auch wenn es wegen des Diabetes Schwierigkeiten mit Schule und Ausbildung gibt, und ist deshalb eine wichtiger Bestandteil in der Betreuung von Kindern und Jugendlichen mit Typ-1-Diabetes.Das vorliegende Konzept wurde von Mitgliedern der Arbeitsgruppe Pädiatrische Endokrinologie und Diabetologie Österreich (APEDÖ) in Zusammenarbeit mit den Vorständen der Rehabilitationskliniken und der Österreichischen Gesellschaft für Kinder- und Jugendheilkunde (ÖGKJ) erstellt.Das Konzept beinhaltet Indikationen und Kontraindikationen für eine Diabetes Rehabilitation, geht auf die Ziele der Rehabilitation in den verschiedenen Altersgruppen (Kleinkinder, Schulkinder und Jugendliche) ein und gibt auch einen Überblick über die empfohlenen Anforderungen für das multidisziplinäre Rehabilitationsteam vor Ort.Das Rehabilitationskonzept der APEDÖ/ÖGKJ dient als Basiskonzept, die Rehabilitationspläne bleiben in der Verantwortung des jeweiligen Zentrums und sollen durch Austausch im Rahmen regelmäßiger Treffen zu einer Optimierung und Angleichung der Konzepte führen.</jats:p