Two mitotic cyclin types, cyclin A and B, exist in higher eukaryotes, but their specialised functions in mitosis are incompletely understood. Using degron tags for rapid inducible protein removal, we ...analyse how acute depletion of these proteins affects mitosis. Loss of cyclin A in G2‐phase prevents mitotic entry. Cells lacking cyclin B can enter mitosis and phosphorylate most mitotic proteins, because of parallel PP2A:B55 phosphatase inactivation by Greatwall kinase. The final barrier to mitotic establishment corresponds to nuclear envelope breakdown, which requires a decisive shift in the balance of cyclin‐dependent kinase Cdk1 and PP2A:B55 activity. Beyond this point, cyclin B/Cdk1 is essential for phosphorylation of a distinct subset of mitotic Cdk1 substrates that are essential to complete cell division. Our results identify how cyclin A, cyclin B and Greatwall kinase coordinate mitotic progression by increasing levels of Cdk1‐dependent substrate phosphorylation.
Synopsis
The specific functions of mitotic CDK1‐activating cyclins A and B in higher eukaryotes have remained unclear. Acute depletion studies combined with phosphoproteomics and computation modeling now define their exact roles and interplay with other pathways during initiation, progression and completion of mammalian cell division.
A novel degron‐tagging strategy in RPE‐1 cells reveals how acute loss of either cyclin A or B affects mitosis.
Cyclin A is critical to trigger the feedback loops involved in initial Cdk1 activation.
Cyclin B is not required for mitotic entry, but essential for phosphorylation of specific Cdk1 substrates, and for sister chromatid segregation and cytokinesis.
Mitotic cells buffer loss of Cyclin B by inhibition of PP2A:B55 phosphatase via the Greatwall kinase pathway.
Acute degron‐mediated depletion defines the exact roles of mitotic cyclins and related CDK1 substrate phosphorylation during initiation, progression and completion of mammalian cell division.
Extensive ecosystem restoration is increasingly seen as being central to conserving biodiversity
and stabilizing the climate of the Earth
. Although ambitious national and global targets have been ...set, global priority areas that account for spatial variation in benefits and costs have yet to be identified. Here we develop and apply a multicriteria optimization approach that identifies priority areas for restoration across all terrestrial biomes, and estimates their benefits and costs. We find that restoring 15% of converted lands in priority areas could avoid 60% of expected extinctions while sequestering 299 gigatonnes of CO
-30% of the total CO
increase in the atmosphere since the Industrial Revolution. The inclusion of several biomes is key to achieving multiple benefits. Cost effectiveness can increase up to 13-fold when spatial allocation is optimized using our multicriteria approach, which highlights the importance of spatial planning. Our results confirm the vast potential contributions of restoration to addressing global challenges, while underscoring the necessity of pursuing these goals synergistically.
We report new polarimetric and photometric maps of the massive star-forming region OMC-1 using the HAWC+ instrument on the Stratospheric Observatory for Infrared Astronomy. We present continuum ...polarimetric and photometric measurements of this region at 53, 89, 154, and 214 m at angular resolutions of 5″, 8″, 14″, and 19″ for the four bands, respectively. The photometric maps enable the computation of improved spectral energy distributions for the region. We find that at the longer wavelengths, the inferred magnetic field configuration matches the "hourglass" configuration seen in previous studies, indicating magnetically regulated star formation. The field morphology differs at the shorter wavelengths. The magnetic field inferred at these wavelengths traces the bipolar structure of the explosive Becklin-Neugebauer/Kleinman-Low outflow emerging from OMC-1 behind the Orion Nebula. Using statistical methods to estimate the field strength in the region, we find that the explosion dominates the magnetic field near the center of the feature. Farther out, the magnetic field is close to energetic equilibrium with the ejecta and may be providing confinement to the explosion. The correlation between polarization fraction and the local polarization angle dispersion indicates that the depolarization as a function of unpolarized intensity is a result of intrinsic field geometry as opposed to decreases in grain alignment efficiency in denser regions.
Herpes simplex virus is a common causative agent of oral and genital diseases. Novel vaccines and therapeutics are needed to combat herpes infections especially after the failure of subunit vaccines ...in human clinical trials. We have shown that the live-attenuated HSV-1 VC2 vaccine strain is unable to establish latency in vaccinated animals and produces a robust immune response capable of completely protecting mice against lethal vaginal HSV-1 or HSV-2 infections. The guinea pig represents the best small animal model of genital HSV-2 disease. Reported here, twenty-one female Hartley guinea pigs received intramuscular injection with either the VC2 vaccine, or equal volume of conditioned tissue culture media. Animals received 2 booster vaccinations at 21 day intervals following the initial vaccination. After vaccination, animals were challenged with the highly virulent HSV-2 (G) strain. Histologically, VC2 vaccinated animals had little to no apparent inflammation/disease following challenge. Unvaccinated animals developed moderate to severe erosive and ulcerative vaginitis. Quantitative reverse-transcriptase PCR analysis in VC2 vaccinated and challenged animals identified transcriptional signatures of Th17 and regulatory Tr1 cells associated with the inflammatory response primed by VC2 vaccination. Treatment of cultured human vaginal epithelial cells (VK2 cells) with a combination of IL-17A and IL-22 resulted in the significant induction of beta-defensin 3 expression. Further, treatment of VK2 cells with IL-17A, IL-22, IL-36 or beta-defensin 3 resulted in diminished HSV-2 replication. Overall, these results suggest that intramuscular vaccination with the live-attenuated vaccine VC2 primes a mucosal immune response predisposing the adaptive expression of transcripts associated with a Th17 response to challenge and these responses contribute to antiviral immunity.
Antibodies targeting Receptor Binding Domain (RBD) of SARS-CoV-2 have been suggested to account for the majority of neutralizing activity in COVID-19 convalescent sera and several neutralizing ...antibodies (nAbs) have been isolated, characterized and proposed as emergency therapeutics in the form of monoclonal antibodies (mAbs). However, SARS-CoV-2 variants are rapidly spreading worldwide from the sites of initial identification. The variants of concern (VOC) B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.167.2 (Delta) showed mutations in the SARS-CoV-2 spike protein potentially able to cause escape from nAb responses with a consequent reduction of efficacy of vaccines and mAbs-based therapy. We produced the recombinant RBD (rRBD) of SARS-CoV-2 spike glycoprotein from the Wuhan-Hu 1 reference sequence in a mammalian system, for mice immunization to isolate new mAbs with neutralizing activity. Here we describe four mAbs that were able to bind the rRBD in Enzyme-Linked Immunosorbent Assay and the transmembrane full-length spike protein expressed in HEK293T cells by flow cytometry assay. Moreover, the mAbs recognized the RBD in supernatants of SARS-CoV-2 infected VERO E6 cells by Western Blot under non-reducing condition or in supernatants of cells infected with lentivirus pseudotyped for spike protein, by immunoprecipitation assay. Three out of four mAbs lost their binding efficiency to completely N-deglycosylated rRBD and none was able to bind the same recombinant protein expressed in
, suggesting that the epitopes recognized by three mAbs are generated by the conformational structure of the glycosylated native protein. Of particular relevance, three mAbs were able to inhibit Wuhan SARS-CoV-2 infection of VERO E6 cells in a plaque-reduction neutralization test and the Wuhan SARS-CoV-2 as well as the Alpha, Beta, Gamma and Delta VOC in a pseudoviruses-based neutralization test. These mAbs represent important additional tools for diagnosis and therapy of COVID-19 and may contribute to the understanding of the functional structure of SARS-CoV-2 RBD.
Oncolytic virotherapy (OVT) is now understood to be an immunotherapy that uses viral infection to liberate tumor antigens in an immunogenic context to promote the development of antitumor immune ...responses. The only currently FDA-approved oncolytic virotherapy, T-Vec, is a modified type 1 herpes simplex virus (HSV-1). While T-Vec is associated with limited response rates, its modest efficacy supports the continued development of novel OVT viruses. Herein, we test the efficacy of a recombinant HSV-1, VC2, as an OVT in a syngeneic B16F10-derived mouse model of melanoma. VC2 possesses mutations that block its ability to enter neurons via axonal termini. This greatly enhances its safety profile by precluding the ability of the virus to establish latent infection. VC2 has been shown to be a safe, effective vaccine against both HSV-1 and HSV-2 infection in mice, guinea pigs, and nonhuman primates. We found that VC2 slows tumor growth rates and that VC2 treatment significantly enhances survival of tumor-engrafted, VC2-treated mice over control treatments. VC2-treated mice that survived initial tumor engraftment were resistant to a second engraftment as well as colonization of lungs by intravenous introduction of tumor cells. We found that VC2 treatment induced substantial increases in intratumoral T cells and a decrease in immunosuppressive regulatory T cells. This immunity was critically dependent on CD8
T cells and less dependent on CD4
T cells. Our data provide significant support for the continued development of VC2 as an OVT for the treatment of human and animal cancers.
Current oncolytic virotherapies possess limited response rates. However, when certain patient selection criteria are used, oncolytic virotherapy response rates have been shown to increase. This, in addition to the increased response rates of oncolytic virotherapy in combination with other immunotherapies, suggests that oncolytic viruses possess significant therapeutic potential for the treatment of cancer. As such, it is important to continue to develop novel oncolytic viruses as well as support basic research into their mechanisms of efficacy. Our data demonstrate significant clinical potential for VC2, a novel type 1 oncolytic herpes simplex virus. Additionally, due to the high rates of survival and the dependence on CD8
T cells for efficacy, our model will enable study of the immunological correlates of protection for VC2 oncolytic virotherapy and oncolytic virotherapy in general. Understanding the mechanisms of efficacious oncolytic virotherapy will inform the rational design of improved oncolytic virotherapies.
Stratospheric Observatory for Infrared Astronomy High-resolution Airborne Wideband Camera Plus polarimetry at 154 m is reported for the face-on galaxy M51 and the edge-on galaxy NGC 891. For M51, the ...polarization vectors generally follow the spiral pattern defined by the molecular gas distribution, the far-infrared (FIR) intensity contours, and other tracers of star formation. The fractional polarization is much lower in the FIR-bright central regions than in the outer regions, and we rule out loss of grain alignment and variations in magnetic field strength as causes. When compared with existing synchrotron observations, which sample different regions with different weighting, we find the net position angles are strongly correlated, the fractional polarizations are moderately correlated, but the polarized intensities are uncorrelated. We argue that the low fractional polarization in the central regions must be due to significant numbers of highly turbulent segments across the beam and along lines of sight in the beam in the central 3 kpc of M51. For NGC 891, the FIR polarization vectors within an intensity contour of 1500 are oriented very close to the plane of the galaxy. The FIR polarimetry is probably sampling the magnetic field geometry in NGC 891 much deeper into the disk than is possible with NIR polarimetry and radio synchrotron measurements. In some locations in NGC 891, the FIR polarization is very low, suggesting we are preferentially viewing the magnetic field mostly along the line of sight, down the length of embedded spiral arms. There is tentative evidence for a vertical field in the polarized emission off the plane of the disk.
We present far-infrared polarimetry observations of M82 at 53 and 154 m and NGC 253 at 89 m, which were taken with High-resolution Airborne Wideband Camera-plus (HAWC+) in polarimetry mode on the ...Stratospheric Observatory for Infrared Astronomy. The polarization of M82 at 53 m clearly shows a magnetic field geometry perpendicular to the disk in the hot dust emission. For M82 the polarization at 154 m shows a combination of field geometry perpendicular to the disk in the nuclear region, but closer to parallel to the disk away from the nucleus. The fractional polarization at 53 m (154 m) ranges from 7% (3%) off nucleus to 0.5% (0.3%) near the nucleus. A simple interpretation of the observations of M82 invokes a massive polar outflow, dragging the field along, from a region ∼700 pc in diameter that has entrained some of the gas and dust, creating a vertical field geometry seen mostly in the hotter (53 m) dust emission. This outflow sits within a larger disk with a more typical planar geometry that more strongly contributes to the cooler (154 m) dust emission. For NGC 253, the polarization at 89 m is dominated by a planar geometry in the tilted disk, with weak indication of a vertical geometry above and below the plane from the nucleus. The polarization observations of NGC 253 at 53 m were of a insufficient signal-to-noise ratio for a detailed analysis.
SARS-CoV-2 vaccination is known to induce antibodies that recognize also variants of concerns (VoCs) of the virus. However, epidemiological and laboratory evidences indicate that these antibodies ...have a reduced neutralization ability against VoCs. We studied binding and neutralizing antibodies against the Spike protein domains and subunits of the Wuhan-Hu-1 virus and its alpha, beta, delta VoCs and of seasonal betacoronaviruses (HKU1 and OC43) in a cohort of 31 health care workers prospectively followed post-vaccination with BNT162b2-Comirnaty. The study of sequential samples collected up to 64 days post-vaccination showed that serological assays measuring IgG against Wuhan-Hu-1 antigens were a poor proxy for VoC neutralization. In addition, in subjects who had asymptomatic or mild COVID-19 prior to vaccination, the loss of nAbs following disease could be rapid and accompanied by post-vaccination antibody levels similar to those of naïve vaccinees. Interestingly, in health care workers naïve for SARS-CoV-2 infection, vaccination induced a rapid and transient reactivation of pre-existing seasonal coronaviruses IgG responses that was associated with a subsequent reduced ability to neutralize alpha and beta VoCs.
PDF
