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Abstract The eye, which is under constant exposure to environmental pathogens, has evolved various anatomic and immunological barriers critical to the protection of tissues lacking regenerative capacity, and the maintenance of a clear optic pathway essential to vision. By bypassing the ocular barriers, intravitreal (IVT) injection has become the mainstay for the delivery of drugs to treat conditions that affect the back of the eye. Both small molecules and biotherapeutics have been successfully administered intravitreally, and several drugs have been approved for the treatment of (wet) age‐related macular degeneration and diabetic macular edema. However, IVT injection is an invasive procedure, which requires sufficient technical expertise from the healthcare professional administering the drug. Potential side effects include bleeding, retinal tear, cataracts, infection, uveitis, loss of vision, and increased ocular pressure. Pharmaceutical companies often differ in their drug development plan, including drug administration techniques, collection of ocular tissues and fluids, ophthalmology monitoring, and overall conduct of nonclinical and clinical studies. The present effort, under the aegis of the Innovation & Quality Ophthalmic Working Group, aims at understanding these differences, identifying pros and cons of the various approaches, determining the gaps in knowledge, and suggesting feasible good practices for nonclinical and early clinical IVT drug development.

Summary: Single-stranded DNA-binding proteins (SSBs) have been regarded as indispensable replication factors. Herein, we report that the genes encoding the canonical SSB (SisSSB) and the non-canonical SSB (SisDBP) in Saccharolobus islandicus REY15A are not essential for cell viability. Interestingly, at a lower temperature (55°C), the protein level of SisSSB increases and the growth of ΔSisssb and ΔSisssbΔSisdbp is retarded. SisSSB exhibits melting activity on dsRNA and DNA/RNA hybrid in vitro and is able to melt RNA hairpin in Escherichia coli. Furthermore, the core SisSSB domain is able to complement the absence of cold-shock proteins in E. coli. Importantly, these activities are conserved in the canonical SSBs from Crenarchaeota species that lack bacterial Csp homologs. Overall, our study has clarified the function of the archaeal canonical SSBs which do not function as a DNA-processing factor, but play a role in the processes requiring melting of dsRNA or DNA/RNA hybrid.

Abstract Kinase, putative Endopeptidase, and Other Proteins of Small size (KEOPS) is a multisubunit protein complex conserved in eukaryotes and archaea. It is composed of Pcc1, Kae1, Bud32, Cgi121, and Gon7 in eukaryotes and is primarily involved in N6‐threonylcarbamoyl adenosine (t6A) modification of transfer RNAs (tRNAs). Recently, it was reported that KEOPS participates in homologous recombination (HR) repair in yeast. To characterize the KEOPS in archaea (aKEOPS), we conducted genetic and biochemical analyses of its encoding genes in the hyperthermophilic archaeon Saccharolobus islandicus. We show that aKEOPS also possesses five subunits, Pcc1, Kae1, Bud32, Cgi121, and Pcc1‐like (or Gon7‐like), just like eukaryotic KEOPS. Pcc1‐like has physical interactions with Kae1 and Pcc1 and can mediate the monomerization of the dimeric subcomplex (Kae1‐Pcc1‐Pcc1‐Kae1), suggesting that Pcc1‐like is a functional homolog of the eukaryotic Gon7 subunit. Strikingly, none of the genes encoding aKEOPS subunits, including Pcc1 and Pcc1‐like, can be deleted in the wild type and in a t6A modification complementary strain named TsaKI, implying that the aKEOPS complex is essential for an additional cellular process in this archaeon. Knock‐down of the Cgi121 subunit leads to severe growth retardance in the wild type that is partially rescued in TsaKI. These results suggest that aKEOPS plays an essential role independent of the cellular t6A modification level. In addition, archaeal Cgi121 possesses dsDNA‐binding activity that relies on its tRNA 3ʹ CCA tail binding module. Our study clarifies the subunit organization of archaeal KEOPS and suggests an origin of eukaryotic Gon7. The study also reveals a possible link between the function in t6A modification and the additional function, presumably HR.

ABSTRACT Forkhead-associated (FHA) domain proteins specifically recognize phosphorylated threonine via the FHA domain and are involved in signal transduction in various processes especially DNA damage response (DDR) and cell cycle regulation in eukaryotes. Although FHA domain proteins are found in prokaryotes, archaea, and bacteria, their functions are far less clear as compared to the eukaryotic counterparts, and it has not been studied whether archaeal FHA proteins play a role in DDR. Here, we have characterized an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) by genetic, biochemical, and transcriptomic approaches. We find that ΔSisarnA exhibits higher resistance to DNA damage agent 4-nitroquinoline 1-oxide (NQO). The transcription of ups genes, encoding the proteins for pili-mediated cell aggregation and cell survival after DDR, is elevated in ΔSisarnA. The interactions of SisArnA with two predicted partners, SisvWA1 (SisArnB) and SisvWA2 (designated as SisArnE), were enhanced by phosphorylation in vitro. ΔSisarnB displays higher resistance to NQO than the wild type. In addition, the interaction between SisArnA and SisArnB, which is reduced in the NQO-treated cells, is indispensable for DNA binding in vitro. These indicate that SisArnA and SisArnB work together to inhibit the expression of ups genes in vivo. Interestingly, ΔSisarnE is more sensitive to NQO than the wild type, and the interaction between SisArnA and SisArnE is strengthened after NQO treatment, suggesting a positive role of SisArnE in DDR. Finally, transcriptomic analysis reveals that SisArnA represses a number of genes, implying that archaea apply the FHA/phospho-peptide recognition module for extensive transcriptional regulation. IMPORTANCE Cellular adaption to diverse environmental stresses requires a signal sensor and transducer for cell survival. Protein phosphorylation and its recognition by forkhead-associated (FHA) domain proteins are widely used for signal transduction in eukaryotes. Although FHA proteins exist in archaea and bacteria, investigation of their functions, especially those in DNA damage response (DDR), is limited. Therefore, the evolution and functional conservation of FHA proteins in the three domains of life is still a mystery. Here, we find that an FHA protein from the hyperthermophilic Crenarchaeon Saccharolobus islandicus (SisArnA) represses the transcription of pili genes together with its phosphorylated partner SisArnB. SisArnA derepression facilitates DNA exchange and repair in the presence of DNA damage. The fact that more genes including a dozen of those involved in DDR are found to be regulated by SisArnA implies that the FHA/phosphorylation module may serve as an important signal transduction pathway for transcriptional regulation in archaeal DDR.

Abstract Distant metastasis is the main cause of mortality in breast cancer patients. Using the breast cancer genomic data from The Cancer Genome Atlas (TCGA), we identified brain specific Cav2.2 as a critical regulator of metastasis. Cav2.2 expression is significantly upregulated in breast cancer and its higher expression is inversely correlated with survival suggesting a previously unappreciated role of Cav2.2 in breast cancer. Cav2.2 is required for breast cancer migration, invasion, and metastasis. Interestingly, Cav2.2 promotes invadopodia formation and extracellular matrix (ECM) degradation through the stabilization of invadopodia component cortactin in a proteosome-dependent manner. Moreover, deubiquitinating enzyme USP43 mediated the functions of Cav2.2 in cortactin stabilization, invadopodia formation, ECM degradation, and metastasis. Interestingly, Cav2.2 upregulates USP43 expression through NFAT2 dephosphorylation and nuclear localization. Our study uncovered a novel pathway that regulates cortactin expression and invadopodia formation in breast cancer metastasis.

Abstract BRAF V600E-mutant colorectal cancer (CRC) is a rare subtype of colorectal cancer with poor prognosis. Compelling evidence indicates that the heparanase (HPSE) gene has multiple functions in cancer, however, its role in BRAF V600E-mutant CRC remains elusive. Differentially expressed genes between BRAF V600E-mutant and wild-type patients were explored by analyzing public data from The Cancer Genome Atlas and the Gene Expression Omnibus. Clinical samples of 172 patients with BRAF V600E-mutant CRC diagnosed at Zhongshan Hospital Fudan University were collected. Overall survival was analyzed using Kaplan–Meier curves and Cox regression models. Cell models and xenografts were utilized to investigate the effect of HPSE on tumor proliferation. HPSE was significantly highly expressed in the BRAF V600E-mutant group. High HPSE expression level was independently associated with inferior survival in the BRAF V600E-mutant cohort. HPSE knockdown impeded tumor proliferation of BRAF V600E-mutant CRC cells in vitro and in vivo. Mechanistically, HPSE silencing arrested cell cycle in G0/G1 phase by downregulating Cyclin E2 expression via the AKT/p27Kip1 pathway. These findings support a role for HPSE in promoting BRAF V600E-mutant CRC progression, which suggests it holds great promise as a prognostic biomarker and a potential therapeutic target for the aggressive CRC subtype.

The emergent universe provides a possible method to avoid the Big Bang singularity by considering that the universe stems from a stable Einstein static universe rather than the singularity. Since the Einstein static universe exists before inflation, it may leave some relics in the CMB power spectrum. In this paper, we analyze the stability condition for the Einstein static universe in general relativity with k-essence against both the scalar and tensor perturbations. Furthermore, we find the emergent universe can be successfully realized by constructing a scalar potential and an equation of state parameter. Solving the curved Mukhanov–Sasaki equation, we obtain the analytical approximation for the primordial power spectrum, and then depict the TT-spectrum of the emergent universe. The results show that both the primordial power spectrum and CMB TT-spectrum are suppressed on large scales.

BackgroundIntravenous lidocaine has been shown to reduce opioid consumption and is associated with favourable outcomes after surgery. In this study, we explored whether intraoperative lidocaine reduces intraoperative opioid use and length of stay (LOS) and improves long-term survival after primary debulking surgery for ovarian cancer and explored the correlation between SCN9A expression and ovarian cancer prognosis.MethodsThis retrospective study included patients who underwent primary debulking surgery(PDS) for ovarian cancer from January 2015 to December 2018. The patients were divided into non-lidocaine and lidocaine [bolus injection of 1.5 mg/kg lidocaine at the induction of anaesthesia followed by a continuous infusion of 2 mg/(kg∙h) intraoperatively] groups. Intraoperative opioid consumption, the verbal numeric rating scale (VNRS) at rest and LOS were recorded. Propensity score matching was used to minimize bias, and disease-free survival (DFS) and overall survival (OS) were compared between the two groups.ResultsAfter propensity score matching(PSM), the demographics were not significantly different between the groups. The intraoperative sufentanil consumption in the lidocaine group was significantly lower than that in the non-lidocaine group (Mean: 35.6 μg vs. 43.2 μg, P=0.035). LOS was similar between the groups (12.0 days vs. 12.4 days, P=0.386). There was a significant difference in DFS between the groups (32.3% vs. 21.6%, P=0.015), and OS rates were significantly higher in the lidocaine group than in the non-lidocaine group (35.2% vs. 25.6%, P=0.042). Multivariate analysis indicated that intraoperative lidocaine infusion was associated with prolonged OS and DFS.ConclusionIntraoperative intravenous lidocaine infusion appears to be associated with improved OS and DFS in patients undergoing primary debulking surgery for ovarian cancer. Our study has the limitations of a retrospective review. Hence, our results should be confirmed by a prospective randomized controlled trial.

In this paper, using the latest Pantheon+ sample of Type Ia supernovae, baryon acoustic oscillation measurements, and observational Hubble data, we carry out a joint constraint on the Hubble constant H _0 , the spatial curvature Ω _K , and the sound horizon at the end of the drag epoch r _d . To be model-independent, four cosmography models—i.e., the Taylor series in terms of redshift y _1 = z /(1 + z ), ${y}_{2}=\arctan (z)$ , ${y}_{3}=\mathrm{ln}(1+z)$ , and the Padé approximants—are used without the assumption of a flat Universe. The results show that H _0 is anticorrelated with Ω _K and r _d , indicating that smaller Ω _K or r _d would be helpful in alleviating the Hubble tension. The values of H _0 and r _d are consistent with the estimate derived from the Planck cosmic microwave background data based on the flat ΛCDM model, but H _0 is in 2.3 ∼ 3.0 σ tension with that obtained by Riess et al. in all these cosmographic approaches. Meanwhile, a flat Universe is preferred by the present observations under all approximations except the third order of y _1 and y _2 of the Taylor series. Furthermore, according to the values of the Bayesian evidence, we found that the flat ΛCDM remains to be the most favored model by the joint data sets, and the Padé approximant of order (2,2), the third order of y _3 and y _1 are the top three cosmographic expansions that fit the data sets best, while the Taylor series in terms of y _2 are essentially ruled out.

Imaging through the wavy water–air interface is challenging since the random fluctuations of water will cause complex geometric distortion and motion blur in the images, seriously affecting the effective identification of the monitored object. Considering the problems of image recovery accuracy and computational efficiency, an efficient reconstruction scheme that combines lucky-patch search and image registration technologies was proposed in this paper. Firstly, a high-quality reference frame is rebuilt using a lucky-patch search strategy. Then an iterative registration algorithm is employed to remove severe geometric distortions by registering warped frames to the reference frame. During the registration process, we integrate JADE and LBFGS algorithms as an optimization strategy to expedite the control parameter optimization process. Finally, the registered frames are refined using PCA and the lucky-patch search algorithm to remove residual distortions and random noise. Experimental results demonstrate that the proposed method significantly outperforms the state-of-the-art methods in terms of sharpness and contrast.

Orc1-2 is a non-initiator ortholog of archaeal/eukaryotic Orc1 proteins, which functions as a global regulator in DNA damage-responsive (DDR) expression. As for Orc1 initiators, the DDR regulator harbors an AAA+ ATPase domain, an Initiator-Specific Motif (ISM) and a winged-helix (wH) DNA-binding domain, which are also organized in a similar fashion. To investigate how Orc1-2 mediates the DDR regulation, the orc1-2 mutants inactivating each of these functional domains were constructed with Saccharolobus islandicus and genetically characterized. We found that disruption of each functional domain completely abolished the DDR regulation in these orc1-2 mutants. Strikingly, inactivation of ATP hydrolysis of Orc1-2 rendered an inviable mutant. However, the cell lethality can be suppressed by the deficiency of the DNA binding in the same protein, and it occurs independent of any DNA damage signal. Mutant Orc1-2 proteins were then obtained and investigated for DNA-binding in vitro. This revealed that both the AAA+ ATPase and the wH domains are involved in DNA-binding, where ISM and R381R383 in wH are responsible for specific DNA binding. We further show that Orc1-2 regulation occurs in two distinct steps: (a) eliciting cell division inhibition at a low Orc1-2 content, and this regulation is switched on by ATP binding and turned off by ATP hydrolysis; any failure in turning off the regulation leads to growth inhibition and cell death; (b) activation of the expression of DDR gene encoding DNA repair proteins at an elevated level of Orc1-2.

Using the Barrow entropy and considering the timescale as IR cutoff, a new holographic dark energy model named Barrow agegraphic dark energy (BADE) was proposed. We use phase space analysis method to discuss the evolution of the universe in three different mode of BADE (Q=0; Q=3αH(ρm+ρD); Q=H(αρm+βρD)). We find the attractor which represents the dark energy-dominated era exists in all cases. In the case Q=0 and Q=H(αρm+βρD) with β=0, the attractor can behave as the cosmological constant, and these models can used to mimic the cosmological constant.