10 research outputs found
Near infrared (NIR)-spectroscopy and in-vitro dissolution absorption system 2 (IDAS2) can help detect changes in the quality of generic drugs
While Health authorities in Panama strive to increase generic drug use to contain the rising costs of medicines, there is still hesitation to embrace generic drugs. Thus, regulators and drug companies need to ensure the quality, safety and efficacy of generic drugs. One prevailing concern is the absence of control over lot-to-lot changes, which may impact consistent therapeutic performance. The objective of this work was to determine whether near-infrared spectroscopy (NIR) could detect product changes. Calibration models were built using reference (standard) tablets of two products: ViraxÂź (200 mg acyclovir) and AmlopinÂź
(5 mg amlodipine). Then, to assess the sensitivity of NIR to product changes we compared reference versus deliberately-modified formulations of these products. Comparisons were made using principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) of NIR spectra. Several modified lots were different from reference lots, and 3D score plots showed greater discrimination by PLS-DA than PCA. The Kth nearest neighbour scores (KNN) of
the modified batches were used to classify formulations as identical or not identical versus the reference. In addition, the differences detected by NIR were correlated with different in vitro dissolution and/or permeation in the in vitro dissolution absorption system 2 (IDAS2): NIR and IDAS2 yielded the same difference rank-order of difference for the modified lots tested. This study suggests that NIR and IDAS2 can help detect lots of generic drugs that differ from the reference lots. This strategy may help regulatory agencies in developing countries to safeguard patients against changes in generic products
Cytoplasmic chromatin triggers inflammation in senescence and cancer
Chromatin is traditionally viewed as a nuclear entity that regulates gene expression and silencing. However, we recently discovered the presence of cytoplasmic chromatin fragments that pinch off from intact nuclei of primary cells during senescence, a form of terminal cell-cycle arrest associated with pro-inflammatory responses. The functional significance of chromatin in the cytoplasm is unclear. Here we show that cytoplasmic chromatin activates the innate immunity cytosolic DNA-sensing cGAS-STING (cyclic GMP-AMP synthase linked to stimulator of interferon genes) pathway, leading both to short-term inflammation to restrain activated oncogenes and to chronic inflammation that associates with tissue destruction and cancer. The cytoplasmic chromatin-cGAS-STING pathway promotes the senescence-associated secretory phenotype in primary human cells and in mice. Mice deficient in STING show impaired immuno-surveillance of oncogenic RAS and reduced tissue inflammation upon ionizing radiation. Furthermore, this pathway is activated in cancer cells, and correlates with pro-inflammatory gene expression in human cancers. Overall, our findings indicate that genomic DNA serves as a reservoir to initiate a pro-inflammatory pathway in the cytoplasm in senescence and cancer. Targeting the cytoplasmic chromatin-mediated pathway may hold promise in treating inflammation-related disorders
Low Resolution Data-Independent Acquisition in an LTQ-Orbitrap Allows for Simplified and Fully Untargeted Analysis of Histone Modifications
Label-free
peptide quantification in liquid chromatographyâmass
spectrometry (LCâMS) proteomics analyses is complicated by
the presence of isobaric coeluting peptides, as they generate the
same extracted ion chromatogram corresponding to the sum of their
intensities. Histone proteins are especially prone to this, as they
are heavily modified by post-translational modifications (PTMs). Their
proteolytic digestion leads to a large number of peptides sharing
the same mass, while carrying PTMs on different amino acid residues.
We present an application of MS data-independent acquisition (DIA)
to confidently determine and quantify modified histone peptides. By
introducing the use of low-resolution MS/MS DIA, we demonstrate that
the signals of 111 histone peptides could easily be extracted from
LCâMS runs due to the relatively low sample complexity. By
exploiting an LTQ-Orbitrap mass spectrometer, we parallelized MS and
MS/MS scan events using the Orbitrap and the linear ion trap, respectively,
decreasing the total scan time. This, in combination with large windows
for MS/MS fragmentation (50 <i>m</i>/<i>z</i>)
and multiple full scan events within a DIA duty cycle, led to a MS
scan cycle speed of âŒ45 full MS per minute, improving the definition
of extracted LCâMS chromatogram profiles. By using such acquisition
method, we achieved highly comparable results to our optimized acquisition
method for histone peptide analysis (<i>R</i><sup>2</sup> correlation > 0.98), which combines data-dependent acquisition
(DDA)
and targeted MS/MS scans, the latter targeting isobaric peptides.
By using DIA, we could also remine our data set and quantify 16 additional
isobaric peptides commonly not targeted during DDA experiments. Finally,
we demonstrated that by performing the full MS scan in the linear
ion trap, we achieve highly comparable results as when adopting high-resolution
MS scans (<i>R</i><sup>2</sup> correlation 0.97). Taken
together, results confirmed that histone peptide analysis can be performed
using DIA and low-resolution MS with high accuracy and precision of
peptide quantification. Moreover, DIA intrinsically enables data remining
to later identify and quantify isobaric peptides unknown at the time
of the LCâMS experiment. These methods will open up epigenetics
analyses to the proteomics community who do not have routine access
to the newer generation high-resolution MS/MS generating instruments
EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data
Epigenetics has become a fundamental
scientific discipline with
various implications for biology and medicine. Epigenetic marks, mostly
DNA methylation and histone post-translational modifications (PTMs),
play important roles in chromatin structure and function. Accurate
quantification of these marks is an ongoing challenge due to the variety
of modifications and their wide dynamic range of abundance. Here we
present EpiProfile 2.0, an extended version of our 2015 software (v1.0),
for accurate quantification of histone peptides based on liquid chromatographyâtandem
mass spectrometry (LCâMS/MS) analysis. EpiProfile 2.0 is now
optimized for data-independent acquisition through the use of precursor
and fragment extracted ion chromatography to accurately determine
the chromatographic profile and to discriminate isobaric forms of
peptides. The software uses an intelligent retention time prediction
trained on the analyzed samples to enable accurate peak detection.
EpiProfile 2.0 supports label-free and isotopic labeling, different
organisms, known sequence mutations in diseases, different derivatization
strategies, and unusual PTMs (such as acyl-derived modifications).
In summary, EpiProfile 2.0 is a universal and accurate platform for
the quantification of histone marks via LCâMS/MS. Being the
first software of its kind, we anticipate that EpiProfile 2.0 will
play a fundamental role in epigenetic studies relevant to biology
and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family
EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data
Epigenetics has become a fundamental
scientific discipline with
various implications for biology and medicine. Epigenetic marks, mostly
DNA methylation and histone post-translational modifications (PTMs),
play important roles in chromatin structure and function. Accurate
quantification of these marks is an ongoing challenge due to the variety
of modifications and their wide dynamic range of abundance. Here we
present EpiProfile 2.0, an extended version of our 2015 software (v1.0),
for accurate quantification of histone peptides based on liquid chromatographyâtandem
mass spectrometry (LCâMS/MS) analysis. EpiProfile 2.0 is now
optimized for data-independent acquisition through the use of precursor
and fragment extracted ion chromatography to accurately determine
the chromatographic profile and to discriminate isobaric forms of
peptides. The software uses an intelligent retention time prediction
trained on the analyzed samples to enable accurate peak detection.
EpiProfile 2.0 supports label-free and isotopic labeling, different
organisms, known sequence mutations in diseases, different derivatization
strategies, and unusual PTMs (such as acyl-derived modifications).
In summary, EpiProfile 2.0 is a universal and accurate platform for
the quantification of histone marks via LCâMS/MS. Being the
first software of its kind, we anticipate that EpiProfile 2.0 will
play a fundamental role in epigenetic studies relevant to biology
and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family
EpiProfile 2.0: A Computational Platform for Processing Epi-Proteomics Mass Spectrometry Data
Epigenetics has become a fundamental
scientific discipline with
various implications for biology and medicine. Epigenetic marks, mostly
DNA methylation and histone post-translational modifications (PTMs),
play important roles in chromatin structure and function. Accurate
quantification of these marks is an ongoing challenge due to the variety
of modifications and their wide dynamic range of abundance. Here we
present EpiProfile 2.0, an extended version of our 2015 software (v1.0),
for accurate quantification of histone peptides based on liquid chromatographyâtandem
mass spectrometry (LCâMS/MS) analysis. EpiProfile 2.0 is now
optimized for data-independent acquisition through the use of precursor
and fragment extracted ion chromatography to accurately determine
the chromatographic profile and to discriminate isobaric forms of
peptides. The software uses an intelligent retention time prediction
trained on the analyzed samples to enable accurate peak detection.
EpiProfile 2.0 supports label-free and isotopic labeling, different
organisms, known sequence mutations in diseases, different derivatization
strategies, and unusual PTMs (such as acyl-derived modifications).
In summary, EpiProfile 2.0 is a universal and accurate platform for
the quantification of histone marks via LCâMS/MS. Being the
first software of its kind, we anticipate that EpiProfile 2.0 will
play a fundamental role in epigenetic studies relevant to biology
and translational medicine. EpiProfile is freely available at https://github.com/zfyuan/EpiProfile2.0_Family
Slow-Binding Inhibition of <i>Mycobacterium tuberculosis</i> Shikimate Kinase by Manzamine Alkaloids
Tuberculosis represents a significant
public health crisis. There
is an urgent need for novel molecular scaffolds against this pathogen.
We screened a small library of marine-derived compounds against shikimate
kinase from <i>Mycobacterium tuberculosis</i> (<i>Mt</i>SK), a promising target for antitubercular drug development. Six
manzamines previously shown to be active against <i>M. tuberculosis</i> were characterized as <i>Mt</i>SK inhibitors: manzamine
A (<b>1</b>), 8-hydroxymanzamine A (<b>2</b>), manzamine
E (<b>3</b>), manzamine F (<b>4</b>), 6-deoxymanzamine
X (<b>5</b>), and 6-cyclohexamidomanzamine A (<b>6</b>). All six showed mixed noncompetitive inhibition of <i>Mt</i>SK. The lowest <i>K</i><sub>I</sub> values were obtained
for <b>6</b> across all <i>Mt</i>SKâsubstrate
complexes. Time-dependent analyses revealed two-step, slow-binding
inhibition. The behavior of <b>1</b> was typical; initial formation
of an enzymeâinhibitor complex (EI) obeyed an apparent <i>K</i><sub>I</sub> of âŒ30 ÎŒM with forward (<i>k</i><sub>5</sub>) and reverse (<i>k</i><sub>6</sub>) rate constants for isomerization to an EI* complex of 0.18 and
0.08 min<sup>â1</sup>, respectively. In contrast, <b>6</b> showed a lower <i>K</i><sub>I</sub> for the initial encounter
complex (âŒ1.5 ÎŒM), substantially faster isomerization
to EI* (<i>k</i><sub>5</sub> = 0.91 min<sup>â1</sup>), and slower back conversion of EI* to EI (<i>k</i><sub>6</sub> = 0.04 min<sup>â1</sup>). Thus, the overall inhibition
constants, <i>K</i><sub>I</sub>*, for <b>1</b> and <b>6</b> were 10 and 0.06 ÎŒM, respectively. These findings
were consistent with docking predictions of a favorable binding mode
and a second, less tightly bound pose for <b>6</b> at <i>Mt</i>SK. Our results suggest that manzamines, in particular <b>6</b>, constitute a new scaffold from which drug candidates with
novel mechanisms of action could be designed for the treatment of
tuberculosis by targeting <i>Mt</i>SK