Identification of late assembly domains of the human endogenous retrovirus-K(HML-2)

Background: Late assembly (L)-domains are protein interaction motifs, whose dysfunction causes characteristic budding defects in enveloped viruses. Three different amino acid motifs, namely PT/SAP, PPXY and YPXnL have been shown to play a major role in the release of exogenous retroviruses. Although the L-domains of exogenous retroviruses have been studied comprehensively, little is known about these motifs in endogenous human retroviruses. Results: Using a molecular clone of the human endogenous retrovirus K113 that had been engineered to reverse the presumed non-synonymous postinsertional mutations in the major genes, we identified three functional L-domains of the virus, all located in the Gag p15 protein. A consensus PTAP tetrapeptide serves as the core of a main L-domain for the virus and its inactivation reduces virus release in HEK 293T cells by over 80%. Electron microscopy of cells expressing the PTAP mutant revealed predominantly late budding structures and budding chains at the plasma membrane. The fact that this motif determines subcellular colocalization with Tsg101, an ESCRT-I complex protein known to bind to the core tetrapeptide, supports its role as an L-domain. Moreover, two YPXnL motifs providing additional L-domain function were identified in the p15 protein. One is adjacent to the PTAP sequence and the other is in the p15 N-terminus. Mutations in either motif diminishes virus release and induces an L-domain phenotype while inactivation of all three L-domains results in a complete loss of particle release in HEK 293T cells. The flexibility of the virus in the use of L-domains for gaining access to the ESCRT machinery is demonstrated by overexpression of Tsg101 which rescues the release of the YPXnL mutants. Similarly, overexpression of Alix not only enhances release of the PTAP mutant by a factor of four but also the release of a triple mutant, indicating that additional cryptic YPXnL domains with a low affinity for Alix may be present. No L-domain activity is provided by the proline-rich peptides at the Gag C-terminus. Conclusions: Our data demonstrate that HERV-K(HML-2) release is predominantly mediated through a consensus PTAP motif and two auxiliary YPXnL motifs in the p15 protein of the Gag precursor

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Establishment of a technique to combine electron microscopy (EM) with tip- enhanced Raman spectroscopy (TERS) and investigation of the mechanism of infection and ultrastructure of the human endogenous retrovirus K (HERV-K)

Im Hinblick auf die Gefahren von bioterroristischen Angriffen ist es wichtig schnell und auch spezifisch Pathogene im Probenmaterial potentieller Fälle zu identifizieren. Die EM mit ihrer hohen Ortsauflösung und dem breiten diagnostischen Spektrum ist dafür eine sehr gute Methode. Um auf Einzelpartikelniveau zwischen nah verwandten und im EM identischen Erregern zu unterscheiden, wurden im Rahmen dieser Arbeit eine Methode evaluiert, welche eine korrelative Kopplung der TEM mit der TERS erlaubt. In diesem Zusammenhang wurden Schwermetallsalze zur Kontrastierung und weitere Materialien auf ihre Eignung für die korrelative TEM-TERS Technologie getestet. Dabei erwies sich PTA als das am besten geeignete Kontrastmittel. Weiterhin wurden drei verschiedene auf Silizium-basierende Grids (Si-Grids) auf ihre Verwendung im TEM und TERS untersucht. Hierbei erfüllte ein mit Siliziumnitrid beschichtetes Grid (SiN-Grid) die Voraussetzungen, wie Elektronentransparenz, hohe Partikeladsorption und Hitzestabilität, am besten. In Optimierungsversuchen wurde die Partikeladhäsion der Grids gesteigert und erste TERS-Spektren von einzelnen Pocken aufgenommen, die vorher im EM visualisiert wurden. Aufgrund der geringen Partikelstabilität konnten keine Spektren von Retroviren aufgenommen werden. Es gelang jedoch die komplette Rekonstitution eines HERV-K(HML-2) Elements sowie dessen Expression und Darstellung der Virionen in EM-Schnittpräparaten. Trotz vollständiger Rekonstitution ist das Virus nicht in der Lage in humanen und tierischen Zellen zu replizieren. Als eine mögliche Ursache konnte ein Block im Replikationszyklus nach Viruseintritt und vor der Proteinexpression gemessen werden. Dieser Block kann durch die Zugabe von Kompetition-Viren abgesättigt werden. Als einer der inhibierenden Faktoren konnte Trim5α identifiziert werden. Die Identifizierung und Analyse dieser Inhibitoren ist für das Verständnis der Biologie endogener sowie exogener Retroviren von großer Bedeutung und könnte ebenfalls helfen die Beobachtung der HERV-K-Expression in verschiedenen humanen Tumoren besser zu verstehen.Due to the threat of bio-terroristic attacks it is important to be able to rapidly and specifically identify pathogens in environmental specimens. The high resolution of electron microscopy (EM) makes it ideal for this task. Within the framework of this thesis, a correlative combination of TEM and TERS was evaluated for its ability to distinguish between single particles of different species that appear to be identical in standard EM. For this, heavy metal salts for contrast and other materials were tested for their use in the correlative TEM-TERS technology. PTA was found to be most suitable material for contrast staining. Furthermore three different silicon-based grids were validated for their compatibility to TEM and TERS. The silicon nitride covered grids (SiN-Grids) best fulfilled the requirements of electron transparency, high particle adsorption and heat stability. In addition, particle adhesion to the SiN-Grids was further optimised, allowing the first recording of TERS- spectra of pox particles, previously visualised in EM, to be made. However, the low stability of retroviral particles prevented the recording of their spectra. Furthermore, a complete reconstitution of a HERV-K(HML-2) element was achieved and its expression and particle production demonstrated in EM thin sections. Despite complete reconstitution, the virus was unable to replicate in human or animal cells. One possible reason for this could be the demonstrated block in the replicative cycle that occurs after virus entry but before protein expression. This block could be competitively saturated by the addition of excess virus and TRIM5α was identified as one of the inhibitory factors. The characterisation and analysis of this inhibition is important for our understanding of the biology of endogenous and exogenous retroviruses and could lead to a better understanding of the expression of HERV-K that is observed in several human tumours

CMV-Promoter Driven Codon-Optimized Expression Alters the Assembly Type and Morphology of a Reconstituted HERV-K(HML-2)

The HERV-K(HML-2) family contains the most recently integrated and best preserved endogenized proviral sequences in the human genome. All known elements have nevertheless been subjected to mutations or deletions that render expressed particles non-infectious. Moreover, these post-insertional mutations hamper the analysis of the general biological properties of this ancient virus family. The expression of consensus sequences and sequences of elements with reverted post-insertional mutations has therefore been very instrumental in overcoming this limitation. We investigated the particle morphology of a recently reconstituted HERV-K113 element termed oriHERV-K113 using thin-section electron microscopy (EM) and could demonstrate that strong overexpression by substitution of the 5'LTR for a CMV promoter and partial codon optimization altered the virus assembly type and morphology. This included a conversion from the regular C-type to an A-type morphology with a mass of cytoplasmic immature cores tethered to the cell membrane and the membranes of vesicles. Overexpression permitted the release and maturation of virions but reduced the envelope content. A weaker boost of virus expression by Staufen-1 was not sufficient to induce these morphological alterations

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat