Gap junctions establish direct pathways for cells to transfer metabolic and electrical messages. The local lipid environment is known to affect the structure, stability and intercellular channel ...activity of gap junctions; however, the molecular basis for these effects remains unknown. Here, we incorporate native connexin-46/50 (Cx46/50) intercellular channels into a dual lipid nanodisc system, mimicking a native cell-to-cell junction. Structural characterization by CryoEM reveals a lipid-induced stabilization to the channel, resulting in a 3D reconstruction at 1.9 Å resolution. Together with all-atom molecular dynamics simulations, it is shown that Cx46/50 in turn imparts long-range stabilization to the dynamic local lipid environment that is specific to the extracellular lipid leaflet. In addition, ~400 water molecules are resolved in the CryoEM map, localized throughout the intercellular permeation pathway and contributing to the channel architecture. These results illustrate how the aqueous-lipid environment is integrated with the architectural stability, structure and function of gap junction communication channels.
SARS-CoV-2 encodes four structural proteins incorporated into virions, spike (S), envelope (E), nucleocapsid (N), and membrane (M). M plays an essential role in viral assembly by organizing other ...structural proteins through physical interactions and directing them to sites of viral budding. As the most abundant protein in the viral envelope and a target of patient antibodies, M is a compelling target for vaccines and therapeutics. Still, the structure of M and molecular basis for its role in virion formation are unknown. Here, we present the cryo-EM structure of SARS-CoV-2 M in lipid nanodiscs to 3.5 Å resolution. M forms a 50 kDa homodimer that is structurally related to the SARS-CoV-2 ORF3a viroporin, suggesting a shared ancestral origin. Structural comparisons reveal how intersubunit gaps create a small, enclosed pocket in M and large open cavity in ORF3a, consistent with a structural role and ion channel activity, respectively. M displays a strikingly electropositive cytosolic surface that may be important for interactions with N, S, and viral RNA. Molecular dynamics simulations show a high degree of structural rigidity in a simple lipid bilayer and support a role for M homodimers in scaffolding viral assembly. Together, these results provide insight into roles for M in coronavirus assembly and structure.
Translational fidelity, essential for protein and cell function, requires accurate transfer RNA (tRNA) aminoacylation. Purified aminoacyl-tRNA synthetases exhibit a fidelity of one error per 10,000 ...to 100,000 couplings. The accuracy of tRNA aminoacylation in vivo is uncertain, however, and might be considerably lower. Here we show that in mammalian cells, approximately 1% of methionine (Met) residues used in protein synthesis are aminoacylated to non-methionyl-tRNAs. Remarkably, Met-misacylation increases up to tenfold upon exposing cells to live or non-infectious viruses, toll-like receptor ligands or chemically induced oxidative stress. Met is misacylated to specific non-methionyl-tRNA families, and these Met-misacylated tRNAs are used in translation. Met-misacylation is blocked by an inhibitor of cellular oxidases, implicating reactive oxygen species (ROS) as the misacylation trigger. Among six amino acids tested, tRNA misacylation occurs exclusively with Met. As Met residues are known to protect proteins against ROS-mediated damage, we propose that Met-misacylation functions adaptively to increase Met incorporation into proteins to protect cells against oxidative stress. In demonstrating an unexpected conditional aspect of decoding mRNA, our findings illustrate the importance of considering alternative iterations of the genetic code.
Ice-nucleating particles (INPs) are efficiently removed from
clouds through precipitation, a convenience of nature for the study of these
very rare particles that influence multiple climate-relevant ...cloud
properties including ice crystal concentrations, size distributions and
phase-partitioning processes. INPs suspended in precipitation can be used to
estimate in-cloud INP concentrations and to infer their original
composition. Offline droplet assays are commonly used to measure INP
concentrations in precipitation samples. Heat and filtration treatments
are also used to probe INP composition and size ranges. Many previous
studies report storing samples prior to INP analyses, but little is known
about the effects of storage on INP concentration or their sensitivity to
treatments. Here, through a study of 15 precipitation samples collected at a
coastal location in La Jolla, CA, USA, we found INP concentration changes up
to > 1 order of magnitude caused by storage to concentrations of
INPs with warm to moderate freezing temperatures (−7 to
−19 ∘C). We compared four conditions: (1) storage at room
temperature (+21–23 ∘C), (2) storage at +4 ∘C, (3) storage at −20 ∘C and (4) flash-freezing samples with liquid nitrogen prior to storage at −20 ∘C. Results demonstrate that storage can lead to both
enhancements and losses of greater than 1 order of magnitude, with
non-heat-labile INPs being generally less sensitive to storage regime, but
significant losses of INPs smaller than 0.45 µm in all tested storage
protocols. Correlations between total storage time (1–166 d) and changes
in INP concentrations were weak across sampling protocols, with the
exception of INPs with freezing temperatures ≥ −9 ∘C in samples stored at room temperature. We provide the
following recommendations for preservation of precipitation samples from
coastal or marine environments intended for INP analysis: that samples be
stored at −20 ∘C to minimize storage artifacts, that
changes due to storage are likely an additional uncertainty in INP
concentrations, and that filtration treatments be applied only to fresh
samples. At the freezing temperature −11 ∘C, average INP
concentration losses of 51 %, 74 %, 16 % and 41 % were observed for
untreated samples stored using the room temperature, +4, −20 ∘C, and flash-frozen protocols, respectively.
Finally, the estimated uncertainties associated with the four storage protocols
are provided for untreated, heat-treated and filtered samples for INPs
between −9 and −17 ∘C.
A colony of deer mice subspecies ( Peromyscus maniculatus sonoriensis) native to high altitude (HA) has been maintained at sea level for 18-20 generations and remains genetically unchanged. To ...determine if these animals retain responsiveness to hypoxia, one group (9-11 wk old) was acclimated to HA (3,800 m) for 8 wk. Age-matched control animals were acclimated to a lower altitude (LA; 252 m). Maximal O
uptake (V̇o
) was measured at the respective altitudes. On a separate day, lung volume, diffusing capacity for carbon monoxide (DL
), and pulmonary blood flow were measured under anesthesia using a rebreathing technique at two inspired O
tensions. The HA-acclimated deer mice maintained a normal V̇o
relative to LA baseline. Compared with LA control mice, antemortem lung volume was larger in HA mice in a manner dependent on alveolar O
tension. Systemic hematocrit, pulmonary blood flow, and standardized DL
did not differ significantly between groups. HA mice showed a higher postmortem alveolar-capillary hematocrit, larger alveolar ducts, and smaller distal conducting structures. In HA mice, absolute volumes of alveolar type I epithelia and endothelia were higher whereas that of interstitia was lower than in LA mice. These structural changes occurred without a net increase in whole-lung septal tissue-capillary volumes or surface areas. Thus, deer mice bred and raised to adulthood at LA retain phenotypic plasticity and adapt to HA without a decrement in V̇o
via structural (enlarged airspaces, alveolar septal remodeling) and nonstructural (lung expansion under hypoxia) mechanisms and without an increase in systemic hematocrit or compensatory lung growth. NEW & NOTEWORTHY Deer mice ( Peromyscus maniculatus) are robust and very active mammals that are found across the North American continent. They are also highly adaptable to extreme environments. When introduced to high altitude they retain remarkable adaptive ability to the low-oxygen environment via lung expansion and remodeling of existing lung structure, thereby maintaining normal aerobic capacity without generating more red blood cells or additional lung tissue.
Adolescents with type 1 diabetes commonly have poor glycaemic control. We aimed to test the efficacy of a newly developed adaptive behavioral intervention (Flexible Lifestyles Empowering Change; ...FLEX) on metabolic and psychosocial outcomes in adolescents with type 1 diabetes.
Young people (13-16 years, type 1 diabetes duration >1 year, HbA
of 64-119 mmol/mol 8·0-13·0%, and without other serious medical conditions or pregnancy) from two clinical sites (Colorado and Ohio, USA) were eligible for enrolment. One caregiver was required to participate actively in the study. Adolescent participants were randomly assigned to the FLEX intervention, which used motivational interviewing and problem-solving skills training to enhance patients' self-management, or usual care control. Intervention fidelity was assessed by a behavioral psychologist with specific expertise in motivational interviewing and who was not otherwise involved in the study via audiotaped sessions. The primary outcome was measurement of glycated haemoglobin A
(HbA
) at 18 months. Secondary outcomes included motivation and intention, problem solving skills, self-management behaviors, symptoms of depression, health related quality of life, fear of hypoglycemia, diabetes family conflict, risk factors for T1D complications (BMI, blood pressure, and plasma lipids), and hypoglycemia derived from continuous glucose monitoring (percent time below 3·0 and 3·9 mmol/l 54 and 70 mg/dl). Intention-to-treat analyses used mixed effects models, with fixed effects including site, timepoint, intervention group, intervention by timepoint, and baseline level of primary (HbA
) or secondary outcomes (α=0·05). FLEX is registered on clinicaltrials.gov, number NCT01286350.
Young people recruited from May 1, 2014 to April 4, 2016 were randomly assigned to FLEX (n=130) or usual care control (n=128). Mean diabetes duration was 6·4 (SD 3·8) years, and 71% (181 out of 256) of patients used insulin pump therapy. Retention was 93%, with 241 out of 258 completing the 18-month assessment. The intervention fidelity score was 4·40 of 5·00 for motivational interviewing and 97% for session content. At 18 months, HbA
was not significantly different between intervention (83 13 mmol/mol at baseline; 84 19 mmol/mol at follow-up); and control (80 14 mmol/mol at baseline; 82 17 mmol/mol at follow-up); change in intervention versus control was -0·7 mmol/mol (95% CI -4·7 to 3·4, p=0·75). The intervention was associated with improved scores for motivation (p=0·011), problem solving (p=0·024), diabetes self-management profile (p=0·013), youth report of overall quality of life (p=0·0089), selected domains related to fear of hypoglycaemia (p=0·036 for youth's helplessness or worry; p=0·0051 for parent's efforts to maintain high blood glucose), parent report of diabetes family conflict (p=0·0001), total cholesterol (p=0·038), and diastolic blood pressure (p=0·015). A total of 54 serious adverse events were identified; 34 of these were diabetes-related, including low blood glucose requiring assistance (n=3) and high blood glucose with diabetic ketoacidosis and emergency response (n=25).
The FLEX intervention did not significantly change HbA
among these adolescents with elevated HbA
, but did positively affect several psychosocial outcomes over 18 months. Further analyses will provide information regarding drivers of positive response to the intervention and will point to future directions for improvement in the approach.
National Institutes of Health and National Institute of Diabetes Digestive Diseases and Kidney and the Helmsley Charitable Trust.
With oceans covering 71% of the Earth's surface, sea spray aerosol (SSA) particles play an important role in the global radiative budget by acting as cloud condensation nuclei and ice nucleating ...particles (INPs). By acting as INPs, SSA particles affect the structure and properties of mixed‐phase clouds by inducing freezing at warmer temperatures than the homogeneous freezing temperature. Climate models that incorporate marine INPs use the emission of submicron SSA in INP parameterizations because these particles contain a higher fraction of organic mass. Here we show supermicron SSA particles, produced using a natural breaking wave analogue, are the major source of INPs throughout the lifecycle of a phytoplankton bloom. Additionally, supermicron SSA particles are shown to be more efficient INPs than submicron SSA particles, because they carry a greater number of ice active components. Thus, supermicron SSA needs to be incorporated in INP parameterizations for future climate models.
Plain Language Summary
Clouds over high latitude oceans are a major source of uncertainty in global climate models. Sea spray aerosol (SSA) particles are the dominant source of cloud seeds over remote marine locations. Ice nucleating particles (INPs) initiate cloud freezing. Previous climate models approximated INPs released from oceans using the number of submicron SSA. However, field studies show supermicron particles are effective INPs over marine locations. This study was designed to understand the role of particle size for marine INPs in an isolated ocean/atmosphere laboratory setting. Two phytoplankton blooms were induced in seawater and a breaking wave proxy generated SSA. The ice nucleating activities of SSA particles in two size‐segregated populations were measured. Throughout both blooms, supermicron SSA particles were the most ice active population. The efficiency of supermicron particles was still apparent after normalizing to particle surface area. Following normalization to aerosol volume, submicron and total particle populations were comparable, suggesting ice active components in SSA scale with particle volume in the observed temperature range (−26°C to −8°C). These results suggest that climate models use size‐dependent INP activities for SSA. Proper representation of marine INPs will increase model accuracy of ice content in clouds and radiative forcing over high latitude oceans.
Key Points
Supermicron sea spray aerosols have higher ice nucleating particle (INP) concentrations at warm temperatures than submicron sea spray aerosols
The higher concentrations of INPs in supermicron sea spray aerosols occur independent of phytoplankton bloom state
The ice nucleating ability of a sea spray aerosol (SSA) is roughly proportional to the dry particle volume
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