24 research outputs found
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Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis
Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR, and SPINK1 variants were associated with pancreatitis risk. We now report two significant genome-wide associations identified and replicated at PRSS1-PRSS2 (1×10-12) and x-linked CLDN2 (p < 1×10-21) through a two-stage genome-wide study (Stage 1, 676 cases and 4507 controls; Stage 2, 910 cases and 4170 controls). The PRSS1 variant affects susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous male) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men – male hemizygous frequency is 0.26, female homozygote is 0.07
The genetic architecture of the human cerebral cortex
The cerebral cortex underlies our complex cognitive capabilities, yet little is known about the specific genetic loci that influence human cortical structure. To identify genetic variants that affect cortical structure, we conducted a genome-wide association meta-analysis of brain magnetic resonance imaging data from 51,665 individuals. We analyzed the surface area and average thickness of the whole cortex and 34 regions with known functional specializations. We identified 199 significant loci and found significant enrichment for loci influencing total surface area within regulatory elements that are active during prenatal cortical development, supporting the radial unit hypothesis. Loci that affect regional surface area cluster near genes in Wnt signaling pathways, which influence progenitor expansion and areal identity. Variation in cortical structure is genetically correlated with cognitive function, Parkinson's disease, insomnia, depression, neuroticism, and attention deficit hyperactivity disorder
Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19
IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19.
Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19.
DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022).
INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days.
MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes.
RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively).
CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes.
TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570
Accurate Determination of Protein Methionine Oxidation by Stable Isotope Labeling and LC-MS Analysis
Methionine
(Met) oxidation is a major modification of proteins,
which converts Met to Met sulfoxide as the common product. It is challenging
to determine the level of Met sulfoxide, because it can be generated
during sample preparation and analysis as an artifact. To determine
the level of Met sulfoxide in proteins accurately, an isotope labeling
and LC-MS peptide mapping method was developed. Met residues in proteins
were fully oxidized using hydrogen peroxide enriched with <sup>18</sup>O atoms before sample preparation. Therefore, it was impossible to
generate Met sulfoxide as an artifact during sample preparation. The
molecular weight difference of 2 Da between Met sulfoxide with the <sup>16</sup>O atom and Met sulfoxide with the <sup>18</sup>O atom was
used to differentiate and calculate the level of Met sulfoxide in
the sample originally. Using a recombinant monoclonal antibody as
a model protein, much lower levels of Met sulfoxide were detected
for the two susceptible Met residues with this new method compared
to a typical peptide mapping procedure. The results demonstrated efficient
elimination of the analytical artifact during LC-MS peptide mapping
for the measurement of Met sulfoxide. This method can thus be used
when accurate determination of the level of Met sulfoxide is critical
Accurate Determination of Protein Methionine Oxidation by Stable Isotope Labeling and LC-MS Analysis
Methionine
(Met) oxidation is a major modification of proteins,
which converts Met to Met sulfoxide as the common product. It is challenging
to determine the level of Met sulfoxide, because it can be generated
during sample preparation and analysis as an artifact. To determine
the level of Met sulfoxide in proteins accurately, an isotope labeling
and LC-MS peptide mapping method was developed. Met residues in proteins
were fully oxidized using hydrogen peroxide enriched with <sup>18</sup>O atoms before sample preparation. Therefore, it was impossible to
generate Met sulfoxide as an artifact during sample preparation. The
molecular weight difference of 2 Da between Met sulfoxide with the <sup>16</sup>O atom and Met sulfoxide with the <sup>18</sup>O atom was
used to differentiate and calculate the level of Met sulfoxide in
the sample originally. Using a recombinant monoclonal antibody as
a model protein, much lower levels of Met sulfoxide were detected
for the two susceptible Met residues with this new method compared
to a typical peptide mapping procedure. The results demonstrated efficient
elimination of the analytical artifact during LC-MS peptide mapping
for the measurement of Met sulfoxide. This method can thus be used
when accurate determination of the level of Met sulfoxide is critical
Characterization of the Acidic Species of a Monoclonal Antibody Using Weak Cation Exchange Chromatography and LC-MS
Charge
variants, especially acidic charge variants, of recombinant
monoclonal antibodies have been challenging to fully characterize
despite the fact that several posttranslational modifications have
already been identified. The acidic species of a recombinant monoclonal
antibody were collected using weak cation exchange (WCX)-10 chromatography
and characterized by LC-MS at multiple levels. In this study, methionine
oxidation and asparagine deamidation are the only two modifications
identified in the acidic species. Incubation of the collected main
chromatographic peak with hydrogen peroxide generated acidic species,
which confirmed that acidic species were enriched in oxidized antibody.
Differences observed between the original acidic species and the oxidization-induced
acidic species indicate that different mechanisms are involved in
the formation of acidic species. Additionally, acidic species were
generated by thermal stress of the collected main peak from the original
sample. Thermal stress of the collected main peak in pH 9 buffer or
ammonium bicarbonate generated chromatograms that are highly similar
to those from the analysis of the original molecule. LC-MS analysis
identified oxidation of the same methionine residue and deamidation
of the same asparagine in the corresponding acidic fractions generated
by thermal stress; however, relatively lower levels of methionine
oxidation and higher levels of asparagine deamdiation were observed.
The results support the use of stressed conditions to generate low
abundance species for detailed characterization of recombinant monoclonal
antibody charge variants, but with caution
Characterization of Recombinant Monoclonal Antibody Charge Variants Using OFFGEL Fractionation, Weak Anion Exchange Chromatography, and Mass Spectrometry
Recombinant
monoclonal antibody charge heterogeneity has been commonly
observed as multiple bands or peaks when analyzed by charge-based
analytical methods such as isoelectric focusing electrophoresis and
cation or anion exchange chromatography. Those charge variants have
been separated by some of the above-mentioned methods and used for
detailed characterization. The utility of a combination of OFFGEL
fractionation and weak anion exchange chromatography to separate the
charge variants of a recombinant monoclonal antibody was demonstrated
in the current study. Charge variants were separated into various
fractions of high purity and then analyzed thoroughly by liquid chromatography
mass spectrometry. Analysis of intact molecular weights identified
the presence of heavy chain leader sequence, C-terminal lysine, and
C-terminal amidation. The identified modifications were further localized
into different regions of the antibody from analysis of antibody fragments
obtained from FabRICATOR digestion. Analysis of tryptic peptides from
various fractions further confirmed the previously identified modifications
in the basic variants. Asparagine deamidation and aspartate isomerization
were identified in acidic fractions from analysis of tryptic peptides.
Basic variants have been fully accounted for by the identified modifications.
However, only a portion of the acidic variants can be explained by
deamidation and isomerization, suggesting that additional modifications
are yet to be identified or acidic variants are an ensemble of molecules
with different structures