The nature of the first genetic polymer is the subject of major debate
. Although the 'RNA world' theory suggests that RNA was the first replicable information carrier of the prebiotic era-that is, ...prior to the dawn of life
-other evidence implies that life may have started with a heterogeneous nucleic acid genetic system that included both RNA and DNA
. Such a theory streamlines the eventual 'genetic takeover' of homogeneous DNA from RNA as the principal information-storage molecule, but requires a selective abiotic synthesis of both RNA and DNA building blocks in the same local primordial geochemical scenario. Here we demonstrate a high-yielding, completely stereo-, regio- and furanosyl-selective prebiotic synthesis of the purine deoxyribonucleosides: deoxyadenosine and deoxyinosine. Our synthesis uses key intermediates in the prebiotic synthesis of the canonical pyrimidine ribonucleosides (cytidine and uridine), and we show that, once generated, the pyrimidines persist throughout the synthesis of the purine deoxyribonucleosides, leading to a mixture of deoxyadenosine, deoxyinosine, cytidine and uridine. These results support the notion that purine deoxyribonucleosides and pyrimidine ribonucleosides may have coexisted before the emergence of life
.
Amide hydrogen/deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for analyzing the conformational dynamics of proteins in a solution. Current conventional methods have a measurement ...limit starting from several seconds and are solely reliant on the speed of manual pipetting or a liquid handling robot. Weakly protected regions of polypeptides, such as in short peptides, exposed loops and intrinsically disordered the protein exchange on the millisecond timescale. Typical HDX methods often cannot resolve the structural dynamics and stability in these cases. Numerous academic laboratories have demonstrated the considerable utility of acquiring HDX-MS data in the sub-second regimes. Here, we describe the development of a fully automated HDX-MS apparatus to resolve amide exchange on the millisecond timescale. Like conventional systems, this instrument boasts automated sample injection with software selection of labeling times, online flow mixing and quenching, while being fully integrated with a liquid chromatography–MS system for existing standard “bottom-up” workflows. HDX-MS’s rapid exchange kinetics of several peptides demonstrate the repeatability, reproducibility, back-exchange, and mixing kinetics achieved with the system. Comparably, peptide coverage of 96.4% with 273 peptides was achieved, supporting the equivalence of the system to standard robotics. Additionally, time windows of 50 ms–300 s allowed full kinetic transitions to be observed for many amide groups; especially important are short time points (50–150 ms) for regions that are likely highly dynamic and solvent- exposed. We demonstrate that information on structural dynamics and stability can be measured for stretches of weakly stable polypeptides in small peptides and in local regions of a large enzyme, glycogen phosphorylase.
Abstract
We present the first measurement of the mass function of free-floating planets (FFPs), or very wide orbit planets down to an Earth mass, from the MOA-II microlensing survey in 2006–2014. Six ...events are likely to be due to planets with Einstein radius crossing times
t
E
< 0.5 days, and the shortest has
t
E
= 0.057 ± 0.016 days and an angular Einstein radius of
θ
E
= 0.90 ± 0.14
μ
as. We measure the detection efficiency depending on both
t
E
and
θ
E
with image-level simulations for the first time. These short events are well modeled by a power-law mass function,
dN
4
/
d
log
M
=
(
2.18
−
1.40
+
0.52
)
×
(
M
/
8
M
⊕
)
−
α
4
dex
−1
star
−1
with
α
4
=
0.96
−
0.27
+
0.47
for
M
/
M
⊙
< 0.02. This implies a total of
f
=
21
−
13
+
23
FFPs or very wide orbit planets of mass 0.33 <
M
/
M
⊕
< 6660 per star, with a total mass of
80
−
47
+
73
M
⊕
star
−1
. The number of FFPs is
19
−
13
+
23
times the number of planets in wide orbits (beyond the snow line), while the total masses are of the same order. This suggests that the FFPs have been ejected from bound planetary systems that may have had an initial mass function with a power-law index of
α
∼ 0.9, which would imply a total mass of
171
−
52
+
80
M
⊕
star
−1
. This model predicts that Roman Space Telescope will detect
988
−
566
+
1848
FFPs with masses down to that of Mars (including
575
−
424
+
1733
with 0.1 ≤
M
/
M
⊕
≤ 1). The Sumi et al. large Jupiter-mass FFP population is excluded.
Allostery is a fundamental mechanism of protein activation, yet the precise dynamic changes that underlie functional regulation of allosteric enzymes, such as glycogen phosphorylase (GlyP), remain ...poorly understood. Despite being the first allosteric enzyme described, its structural regulation is still a challenging problem: the key regulatory loops of the GlyP active site (250′ and 280s) are weakly stable and often missing density or have large b-factors in structural models. This led to the longstanding hypothesis that GlyP regulation is achieved through gating of the active site by (dis)order transitions, as first proposed by Barford and Johnson. However, testing this requires a quantitative measurement of weakly stable local structure which, to date, has been technically challenging in such a large protein. Hydrogen–deuterium-exchange mass spectrometry (HDX-MS) is a powerful tool for studying protein dynamics, and millisecond HDX-MS has the ability to measure site-localized stability differences in weakly stable structures, making it particularly valuable for investigating allosteric regulation in GlyP. Here, we used millisecond HDX-MS to measure the local structural perturbations of glycogen phosphorylase b (GlyPb), the phosphorylated active form (GlyPa), and the inhibited glucose-6 phosphate complex (GlyPb:G6P) at near-amino acid resolution. Our results support the Barford and Johnson hypothesis for GlyP regulation by providing insight into the dynamic changes of the key regulatory loops.
Nonalcoholic fatty liver disease (NAFLD) can progress from simple steatosis (i.e., nonalcoholic fatty liver NAFL) to nonalcoholic steatohepatitis (NASH), cirrhosis, and cancer. Currently, the driver ...for this progression is not fully understood; in particular, it is not known how NAFLD and its early progression affects the distribution of lipids in the liver, producing lipotoxicity and inflammation. In this study, we used dietary and genetic mouse models of NAFL and NASH and translated the results to humans by correlating the spatial distribution of lipids in liver tissue with disease progression using advanced mass spectrometry imaging technology. We identified several lipids with distinct zonal distributions in control and NAFL samples and observed partial to complete loss of lipid zonation in NASH. In addition, we found increased hepatic expression of genes associated with remodeling the phospholipid membrane, release of arachidonic acid (AA) from the membrane, and production of eicosanoid species that promote inflammation and cell injury. The results of our immunohistochemistry analyses suggest that the zonal location of remodeling enzyme LPCAT2 plays a role in the change in spatial distribution for AA‐containing lipids. This results in a cycle of AA‐enrichment in pericentral hepatocytes, membrane release of AA, and generation of proinflammatory eicosanoids and may account for increased oxidative damage in pericentral regions in NASH. Conclusion: NAFLD is associated not only with lipid enrichment, but also with zonal changes of specific lipids and their associated metabolic pathways. This may play a role in the heterogeneous development of NAFLD. (Hepatology 2017;65:1165‐1180)
We compare the planet-to-star mass-ratio distribution measured by gravitational microlensing to core accretion theory predictions from population synthesis models. The core accretion theory's runaway ...gas accretion process predicts a dearth of intermediate-mass giant planets that is not seen in the microlensing results. In particular, the models predict ∼10 × fewer planets at mass ratios of than inferred from microlensing observations. This tension implies that gas giant formation may involve processes that have hitherto been overlooked by existing core accretion models or that the planet-forming environment varies considerably as a function of host-star mass. Variation from the usual assumptions for the protoplanetary disk viscosity and thickness could reduce this discrepancy, but such changes might conflict with microlensing results at larger or smaller mass ratios, or with other observations. The resolution of this discrepancy may have important implications for planetary habitability because it has been suggested that the runaway gas accretion process may have triggered the delivery of water to our inner solar system. So, an understanding of giant planet formation may help us to determine the occurrence rate of habitable planets.
Defects in DNA repair and the protection of stalled DNA replication forks are thought to underlie the chemosensitivity of tumors deficient in the hereditary breast cancer genes
and
(BRCA). ...Challenging this assumption are recent findings that indicate chemotherapies, such as cisplatin used to treat BRCA-deficient tumors, do not initially cause DNA double-strand breaks (DSB). Here, we show that ssDNA replication gaps underlie the hypersensitivity of BRCA-deficient cancer and that defects in homologous recombination (HR) or fork protection (FP) do not. In BRCA-deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. Gap suppression by either restoration of fork restraint or gap filling conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored FP and HR could be uncoupled from therapy resistance when gaps were present. Moreover, DSBs were not detected after therapy when apoptosis was inhibited, supporting a framework in which DSBs are not directly induced by genotoxic agents, but rather are induced from cell death nucleases and are not fundamental to the mechanism of action of genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose they are fundamental to the mechanism of action of genotoxic chemotherapies. SIGNIFICANCE: This study suggests that ssDNA replication gaps are fundamental to the toxicity of genotoxic agents and underlie the BRCA-cancer phenotype "BRCAness," yielding promising biomarkers, targets, and opportunities to resensitize refractory disease.
.
Differences in conformational dynamics between two full-length monoclonal antibodies have been probed in detail using Fast Photochemical Oxidation of Proteins (FPOP) followed by proteolysis and ...LC-ESI-MS/MS analyses. FPOP uses hydroxyl radical labeling to probe the surface-accessible regions of proteins and has the advantage that the resulting covalent modifications are irreversible, thus permitting optimal downstream analysis. Despite the two monoclonal antibodies (mAbs) differing by only three amino acids in the heavy chain complementarity determining regions (CDRs), one mAb, MEDI1912-WFL, has been shown to undergo reversible self-association at high concentrations and exhibited poor pharmacokinetic properties in vivo, properties which are markedly improved in the variant, MEDI1912-STT. Identifying the differences in oxidative labeling between the two antibodies at residue level revealed long-range effects which provide a key insight into their conformational differences. Specifically, the amino acid mutations in the CDR region of the heavy chain resulted in significantly different labeling patterns at the interfaces of the CL–CH1 and CH1–CH2 domains, with the nonaggregating variant undergoing up to four times more labeling in this region than the aggregation prone variant, thus suggesting a change in the structure and orientation of the CL–CH1 interface. The wealth of FPOP and LC-MS data obtained enabled the study of the LC elution properties of FPOP-oxidized peptides. Some oxidized amino acids, specifically histidine and lysine, were noted to have unique effects on the retention time of the peptide, offering the promise of using such an analysis as an aid to MS/MS in assigning oxidation sites.
Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of ...coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19 and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from the lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300-mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained threefold above those of convalescent serum at 9 months after AZD7442 administration. About 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19.
Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is ...thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling.
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