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Edman Degradation Reveals Unequivocal Analysis of the Disulfide Connectivity in Peptides and Proteins
No full textDisulfide bridges in peptides and proteins play an essential
role
in maintaining their conformation, structural integrity, and consequently
function. Despite ongoing efforts, it is still not possible to detect
disulfide bonds and the connectivity of multiply bridged peptides
directly through a simple and sufficiently validated protein sequencing
or peptide mapping method. Partial or complete reduction and chemical
cysteine modification are required as initial steps, followed by the
application of a proper detection method. Edman degradation (ED) has
been used for primary sequence determination but is largely neglected
since the establishment of mass spectrometry (MS)-based protein sequencing.
Here, we evaluated and thoroughly characterized the phenyl thiohydantoin
(PTH) cysteine derivatives PTH-S-methyl cysteine and PTH-S-carbamidomethyl
cysteine as bioanalytical standards for cysteine detection and quantification
as well as for the elucidation of the disulfide connectivity in peptides
by ED. Validation of the established derivatives was performed according
to the guidelines of the International Committee of Harmonization
on bioanalytical method validation, and their analytical properties
were confirmed as reference standards. A series of model peptides
was sequenced to test the usability of the PTH-Cys-derivatives as
standards, whereas the native disulfide-bonded peptides CCAP-vil,
μ-conotoxin KIIIA, and human insulin were used as case studies
to determine their disulfide bond connectivity completely independent
of MS analysis
Beyond CSF and Neuroimaging Assessment: Evaluating Plasma miR-145-5p as a Potential Biomarker for Mild Cognitive Impairment and Alzheimer’s Disease
No full textAlzheimer’s
disease (AD) is the most common cause
of dementia.
New strategies for the early detection of MCI and sporadic AD are
crucial for developing effective treatment options. Current techniques
used for diagnosis of AD are invasive and/or expensive, so they are
not suitable for population screening. Cerebrospinal fluid (CSF) biomarkers
such as amyloid β1–42 (Aβ1–42), total tau
(T-tau), and phosphorylated tau181 (P-tau181) levels are core biomarkers
for early diagnosis of AD. Several studies have proposed the use of
blood-circulating microRNAs (miRNAs) as potential novel early biomarkers
for AD. We therefore applied a novel approach to identify blood-circulating
miRNAs associated with CSF biomarkers and explored the potential of
these miRNAs as biomarkers of AD. In total, 112 subjects consisting
of 28 dementia due to AD cases, 63 MCI due to AD cases, and 21 cognitively
healthy controls were included. We identified seven Aβ1–42-associated
plasma miRNAs, six P-tau181-associated plasma miRNAs, and nine Aβ1–42-associated
serum miRNAs. These miRNAs were involved in AD-relevant biological
processes, such as PI3K/AKT signaling. Based on this signaling pathway,
we constructed an miRNA-gene target network, wherein miR-145-5p has
been identified as a hub. Furthermore, we showed that miR-145-5p performs
best in the prediction of both AD and MCI. Moreover, miR-145-5p also
improved the prediction performance of the mini-mental state examination
(MMSE) score. The performance of this miRNA was validated using different
datasets including an RT-qPCR dataset from plasma samples of 23 MCI
cases and 30 age-matched controls. These findings indicate that blood-circulating
miRNAs that are associated with CSF biomarkers levels and specifically
plasma miR-145-5p alone or combined with the MMSE score can potentially
be used as noninvasive biomarkers for AD or MCI screening in the general
population, although studies in other AD cohorts are necessary for
further validation
Strong, Antifatigue, and Ionically Conductive Organogels for High-Performance Strain Sensors
No full textConductive organohydrogels with flexibility and biocompatibility
have attracted extensive attention in bioelectronic devices. However,
poor mechanical properties and crack propagation resistance have severely
limited their applications. Herein, strong, tough, and ionically conductive
organogels (ICOs) with outstanding fatigue resistance are prepared
based on simultaneous construction of dense cross-linked polymer network
with numerous crystalline domains and ionically conductive network
during the solvent exchange. ICOs show excellent mechanical properties
with tensile strength and elongation at break as high as 16.7 ±
0.9 MPa and 1112.4 ± 120.3%, respectively. Moreover, the fracture
energy and fatigue threshold can reach 34.0 ± 4.7 KJ/m2 and 561.3 ± 59.6 J/m2, respectively, exhibiting
outstanding crack resistant properties. ICOs with antifreezing performance
are used for strain sensing with a linear working strain up to 80%
and superior cycling stability, and the ICO strain sensor can monitor
various body motions. The mechanically strong and antifatigue organogels
show promising applications in flexible and smart electronics even
in extreme environments
Table_4_Gene-edited Mtsoc1 triple mutant Medicago plants do not flower.xlsx
No full textOptimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. SOC1-like genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model Arabidopsis thaliana (Arabidopsis), where SOC1 promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. Medicago truncatula (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one SOC1 gene, multiple gene duplication events have given rise to three MtSOC1 paralogs within the Medicago genus in legumes: one Fabaceae group A SOC1 gene, MtSOC1a, and two tandemly repeated Fabaceae group B SOC1 genes, MtSOC1b and MtSOC1c. Previously, we showed that MtSOC1a has unique functions in floral promotion in Medicago. The Mtsoc1a Tnt1 retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, Mtsoc1b Tnt1 single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of Mtsoc1a Mtsoc1b Mtsoc1c triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three MtSOC1 genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single Mtsoc1a mutant and the Arabidopsis soc1 mutant; implicating multiple MtSOC1 genes in critical overlapping roles in the transition to flowering in Medicago.</p
FIGURE 2 from Inhibition of Aurora Kinase Induces Endogenous Retroelements to Induce a Type I/III IFN Response via RIG-I
No full textActivation of IFN is mediated through inhibition of Aurora B. A, Western blot analysis of the HCT116-IFI27 reporter line treated with either DMSO, 1,000 U/mL IFNα, 1 µmol/L alisertib, or 100 nmol/L decitabine for the indicated amount of time. A total of 20 µg of whole cell lysates per lane were blotted with the indicated antibodies, with vinculin blotted as a loading control. B, qPCR analysis showing induction of IFN and target genes IFNβ, IFNλ1, CXCL10, IFI27, IFIT1, IFIT3 after 5 days treatment of the HCT116-IFI27 reporter line with DMSO, 100 nmol/L decitabine or 1 µmol/L alisertib. Significance is shown versus the DMSO control for each gene. C, Dose–response titration of Aurora A, Aurora B selective drugs on the IFI27 reporter suggests that IFN induction occurs preferentially after inhibition of Aurora B. The Aurora A selective drugs TC-7010 and MK8745, the weakly Aurora A selective drug alisertib, and the Aurora B selective drugs barasertib and GSK1070916 were dosed at 12, 37, 110, 330, 1,000, or 2,500 nmol/L on the HCT116-IFI27 reporter line for 5 days, then luciferase activity measured. Significance is shown for each drug versus the DMSO control at each timepoint. D, Western blot analysis of p-S10-H3 after treatment with alisertib, barasertib, or the Aurora A selective inhibitor MK8745 suggest that induction of the IFI27 reporter coincides with inhibition of P-S10-H3, a biomarker of Aurora B activity. E, Ruxolitinib inhibition of IFI27 reporter induction by Aurora kinase inhibitors demonstrates dependence of IFN induction by Aurora inhibition on JAK signaling. Cells were treated with DMSO, 1 µmol/L alisertib, 830 nmol/L TC-7010, 280 nmol/L barasertib, or 280 nmol/L GSK1070916, without or with 10 µmol/L ruxolitinib for 5 days, then luciferase activity measured. Significance shown for each treatment compared with DMSO alone. F, siRNA against Aurora A versus Aurora B shows that Aurora B inhibition is critical for induction of the IFN response. HCT116-IFI27 reporter cells were transfected with siRNAs to PPIB, or pooled siRNAs (4 per target) to Aurora A or Aurora B, and luciferase activity was measured 5 days later. Significance is shown for each Aurora siRNA versus the PPIB siRNA condition for each dose. G, GFP fluorescent and phase imaging of cells shown in F, with characteristic morphologic changes accompanying siRNA transfection of either Aurora A or Aurora B, but induction of the reporter only with knockdown of Aurora B. H, Western blot analysis showing phospho-Aurora A/B and total Aurora A and B after siRNA knockdown. HCT116-IFI27 reporter cells were transfected with the indicated siRNAs and 48 hours later additionally treated with nocodazole, then 24 hours later whole cell lysates prepared and blotted with Aurora A, Aurora B, or p-T288-Aurora A/T232-B antibodies. Vinculin blotting was also performed as a loading control. I, Individual siRNAs against Aurora A and Aurora B show that siRNAs against Aurora B activate IFI27 reporter. HCT116-IFI27 reporter cells were transfected with indicated siRNAs and reporter activity measured 72 hours later. Significance shown is relative to the siPPIB transfection.</p
π‑Conjugated Lewis Base for Efficient Tin Halide Perovskite Solar Cells with Retarded Sn<sup>2+</sup> Oxidation
No full textLead-free
tin halide perovskite solar cells (TPSCs) have driven
much research attention for their environmental friendliness. However,
the low efficiency and large open-circuit voltage (Voc) deficit limit their further development. Here, we
introduced a π-conjugated Lewis base, 1H-pyrrolo[2,3-b]pyridin-6-ylaamine (1H6An), a N donor for the unique electron-donating
role of pyridine and pyrrole N with unpaired lone pair electrons to
interact with the tin halide perovskites and thus retard the oxidation
of Sn2+ during the aging process of precursors. Meanwhile,
the interaction stabilized the perovskite lattice and decreased the
microstrain of deposited films. The carrier kinetics further revealed
a notably enhanced carrier extraction and transport as well as decreased
nonradiative recombination with 1H6An. Consequently, this approach
delivered an efficiency of 13.28% with a remarkable Voc enhancement from 853 to 907 mV. Meanwhile, the 1H6An
device exhibited an extended lifespan of over 2500 h with around 90%
retention of its initial value in a N2 atmosphere
New Multitarget Molecules Derived from Caffeine as Potentiators of the Cholinergic System
No full textCholinergic deficit is a characteristic
factor of several
pathologies,
such as myasthenia gravis, some types of congenital myasthenic syndromes,
and Alzheimer’s Disease. Two molecular targets for its treatment
are acetylcholinesterase (AChE) and nicotinic acetylcholine receptor
(nAChR). In previous studies, we found that caffeine behaves as a
partial nAChR agonist and confirmed that it inhibits AChE. Here, we
present new bifunctional caffeine derivatives consisting of a theophylline
ring connected to amino groups by different linkers. All of them were
more potent AChE inhibitors than caffeine. Furthermore, although some
of them also activated muscle nAChR as partial agonists, not all of
them stabilized nAChR in its desensitized conformation. To understand
the molecular mechanism underlying these results, we performed docking
studies on AChE and nAChR. The nAChR agonist behavior of the compounds
depends on their accessory group, whereas their ability to stabilize
the receptor in a desensitized state depends on the interactions of
the linker at the binding site. Our results show that the new compounds
can inhibit AChE and activate nAChR with greater potency than caffeine
and provide further information on the modulation mechanisms of pharmacological
targets for the design of novel therapeutic interventions in cholinergic
deficit