Friedreich’s ataxia (FRDA) is caused primarily
by expanded
GAA repeats in intron 1 of both alleles of the FXN gene, which causes transcriptional silencing and reduced expression
of frataxin mRNA and protein. FRDA is characterized by slowly progressive
ataxia and cardiomyopathy. Symptoms generally appear during adolescence,
and patients slowly progress to wheelchair dependency usually in the
late teens or early twenties with death on average in the 4th decade.
There are two known mature proteoforms of frataxin. Mitochondrial
frataxin (frataxin-M) is a 130-amino acid protein with a molecular
weight of 14,268 Da, and there is an alternatively spliced N-terminally
acetylated 135-amino acid form (frataxin-E) with a molecular weight
of 14,953 Da found in erythrocytes. There is reduced expression of
frataxin in the heart and brain, but frataxin is not secreted into
the systemic circulation, so it cannot be analyzed in serum or plasma.
Blood is a readily accessible biofluid that contains numerous different
cell types that express frataxin. We have found that pig blood can
serve as an excellent surrogate matrix to validate an assay for frataxin
proteoforms because pig frataxin is lost during the immunoprecipitation
step used to isolate human frataxin. Frataxin-M is expressed in blood
cells that contain mitochondria, whereas extra-mitochondrial frataxin-E
is found in erythrocytes. This means that the analysis of frataxin
in whole blood provides information on the concentration of both proteoforms
without having to isolate the individual cell types. In the current
study, we observed that the distributions of frataxin levels for a
sample of 25 healthy controls and 50 FRDA patients were completely
separated from each other, suggesting 100% specificity and 100% sensitivity
for distinguishing healthy controls from FRDA cases, a very unusual
finding for a biomarker assay. Additionally, frataxin levels were
significantly correlated with the GAA repeat length and age of onset
with higher correlations for extra-mitochondrial frataxin-E than those
for mitochondrial frataxin-M. These findings auger well for using
frataxin levels measured by the validated stable isotope dilution
ultrahigh-performance liquid chromatography–multiple reaction
monitoring/mass spectrometry assay to monitor therapeutic interventions
and the natural history of FRDA. Our study also illustrates the utility
of using whole blood for protein disease biomarker discovery and validation.
Background
The COVID-19 pandemic has caused a severe shortage of personal protective equipment (PPE), especially N95 respirators. Efficient, effective and economically feasible methods for large-scale PPE decontamination are urgently needed.
Aims
(1) to develop protocols for effectively decontaminating PPE using vaporized hydrogen peroxide (VHP); (2) to develop novel approaches that decrease set up and take down time while also increasing decontamination capacity (3) to test decontamination efficiency for N95 respirators heavily contaminated by makeup or moisturizers.
Methods
We converted a decommissioned Biosafety Level 3 laboratory into a facility that could be used to decontaminate N95 respirators. N95 respirators were hung on metal racks, stacked in piles, placed in paper bags or covered with makeup or moisturizer. A VHP® VICTORY
TM
unit from STERIS was used to inject VHP into the facility. Biological and chemical indicators were used to validate the decontamination process.
Findings
N95 respirators individually hung on metal racks were successfully decontaminated using VHP. N95 respirators were also successfully decontaminated when placed in closed paper bags or if stacked in piles of up to six. Stacking reduced the time needed to arrange N95 respirators for decontamination by approximately two-thirds while almost tripling facility capacity. Makeup and moisturizer creams did not interfere with the decontamination process.
Conclusions
Respirator stacking can reduce the hands-on time and increase decontamination capacity. When personalization is needed, respirators can be decontaminated in labeled paper bags. Make up or moisturizers do not appear to interfere with VHP decontamination.
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