Multiple Controls Regulate Nucleostemin Partitioning Between Nucleolus and Nucleoplasm

Nucleostemin plays an essential role in maintaining the continuous proliferation of stem cells and cancer cells. The movement of nucleostemin between the nucleolus and the nucleoplasm provides a dynamic way to partition the nucleostemin protein between these two compartments. Here, we showed that nucleostemin contained two nucleolus-targeting regions, the basic and the GTP-binding domains, which exhibited a short and a long nucleolar retention time, respectively. In a GTP-unbound state, the nucleolus-targeting activity of nucleostemin was blocked by a mechanism that trapped its intermediate domain in the nucleoplasm. A nucleostemin-interacting protein, RSL1D1, was identified that contained a ribosomal L1-domain, co-resided with nucleostemin in the same subnucleolar compartment non-identical to the B23 and fibrillarin distributions, and displayed a longer nucleolar residence time than nucleostemin. RSL1D1 interacted with both the basic and the GTP-binding domains of nucleostemin through a non-nucleolus-targeting region. Overexpression of the nucleolus-targeting domain of RSL1D1 alone dispersed the nucleolar nucleostemin. Loss of RSL1D1 expression reduced the compartmental size and amount of nucleostemin in the nucleolus. This work reveals that the partitioning of nucleostemin employs complex mechanisms involving both nucleolar and nucleoplasmic components, and provides insight into the post-translational regulation of its activity

Fast Estimation of Leakage Area in a Multizone Test Facility

The effects of building leakage on heat and mass transfer in buildings are difficult to model due to the difficulty in knowing crack size, shape, position, and number. Nevertheless, interest remains high in the estimation of crack areas in buildings because of the important effects cracks have on infiltration, indoor air quality, building energy performance, and because of interest in exploring mitigation/ventilation strategies. We show how a steady-state model is developed that can provide fast estimation of the crack areas for a test facility. The facility is then modeled in CONTAM to perform simulations and make prediction of overpressure values using the set of estimated crack areas determined from the above model. Comparisons are made between the CONTAM model predictions and experimental results in terms of overpressure, and good agreement is achieved. The experimental overpressure ranges from 3 Pa to 155 Pa, covering the normal range of overpressure in commercial and residential buildings. The method outlined in the present work can be extended to estimate crack areas of the openings in multizone pressurization tests. Further, this method holds promise in filling the knowledge gap of interzonal leakage in multizone buildings

On real-time multi-stage speech enhancement systems

Recently, multi-stage systems have stood out among deep learning-based speech enhancement methods. However, these systems are always high in complexity, requiring millions of parameters and powerful computational resources, which limits their application for real-time processing in low-power devices. Besides, the contribution of various influencing factors to the success of multi-stage systems remains unclear, which presents challenges to reduce the size of these systems. In this paper, we extensively investigate a lightweight two-stage network with only 560k total parameters. It consists of a Mel-scale magnitude masking model in the first stage and a complex spectrum mapping model in the second stage. We first provide a consolidated view of the roles of gain power factor, post-filter, and training labels for the Mel-scale masking model. Then, we explore several training schemes for the two-stage network and provide some insights into the superiority of the two-stage network. We show that the proposed two-stage network trained by an optimal scheme achieves a performance similar to a four times larger open source model DeepFilterNet2.Comment: To appear at ICASSP 202

Nucleostemin inhibits TRF1 dimerization and shortens its dynamic association with the telomere

TRF1 is a key component of the telomere-capping complex and binds double-strand telomeric DNA as homodimers. So far, it is not clear whether TRF1 dimerization coincides with its telomere binding or is actively controlled before it binds the telomere, and in the latter case, how this event might affect its telomere association. We previously found that TRF1 dimerization and its telomere binding can be increased by GNL3L, which is the vertebrate paralogue of nucleostemin (NS). Here, we show that NS and GNL3L bind TRF1 directly but competitively through two separate domains of TRF1. In contrast to GNL3L, NS prevents TRF1 dimerization through a mechanism not determined by its ability to displace TRF1-bound GNL3L. Furthermore, NS is capable of shortening the dynamic association of TRF1 with the telomere in normal and TRF2(ΔBΔM)-induced telomere-damaged cells without affecting the amount of telomere-bound TRF1 proteins in vivo. Importantly, NS displays a protective function against the formation of telomere-dysfunction-induced foci. This work demonstrates that TRF1 dimerization is actively and oppositely regulated by NS and GNL3L extrachromosomally. Changing the relative amount of TRF1 monomers versus dimers in the nucleoplasm might affect the dynamic association of TRF1 with the telomere and the repair of damaged telomeres

Nucleostemin deletion reveals an essential mechanism that maintains the genomic stability of stem and progenitor cells

Stem and progenitor cells maintain a robust DNA replication program during the tissue expansion phase of embryogenesis. The unique mechanism that protects them from the increased risk of replication-induced DNA damage, and hence permits self-renewal, remains unclear. To determine whether the genome integrity of stem/progenitor cells is safeguarded by mechanisms involving molecules beyond the core DNA repair machinery, we created a nucleostemin (a stem and cancer cell-enriched protein) conditional-null allele and showed that neural-specific knockout of nucleostemin predisposes embryos to spontaneous DNA damage that leads to severe brain defects in vivo. In cultured neural stem cells, depletion of nucleostemin triggers replication-dependent DNA damage and perturbs self-renewal, whereas overexpression of nucleostemin shows a protective effect against hydroxyurea-induced DNA damage. Mechanistic studies performed in mouse embryonic fibroblast cells showed that loss of nucleostemin triggers DNA damage and growth arrest independently of the p53 status or rRNA synthesis. Instead, nucleostemin is directly recruited to DNA damage sites and regulates the recruitment of the core repair protein, RAD51, to hydroxyurea-induced foci. This work establishes the primary function of nucleostemin in maintaining the genomic stability of actively dividing stem/progenitor cells by promoting the recruitment of RAD51 to stalled replication-induced DNA damage foci

Nucleostemin and GNL3L exercise distinct functions in genome protection and ribosome synthesis, respectively

The mammalian nucleolar proteins nucleostemin (NS) and GNL3L (for GNL3-like) are encoded by paralogous genes that arose from an invertebrate ancestral gene, GNL3. Invertebrate GNL3 has been implicated in ribosome biosynthesis as has its mammalian descendent GNL3L, whereas the paralogous mammalian NS gene has instead been implicated in cell renewal. Here we found that NS depletion in a human breast carcinoma cell line triggered a prompt and significant effect of DNA damage in S-phase cells without perturbing the initial step of rRNA synthesis and only mildly affected the total ribosome production. In contrast, GNL3L depletion markedly impaired ribosome production without inducing appreciable DNA damage. These results indicate that during vertebrate evolution GNL3L retained the role of the ancestral gene in ribosome biosynthesis while the paralogous NS acquired a novel genome-protective function. Our results provide a coherent explanation for what had seemed to be contradictory findings about the functions of the invertebrate vs. vertebrate genes, and also speak to how the nucleolus was fine-tuned for a role in genome protection and cell cycle control as the vertebrates evolved.</jats:p

GNL3L stabilizes the TRF1 complex and promotes mitotic transition

Telomeric repeat binding factor 1 (TRF1) is a component of the multiprotein complex “shelterin,” which organizes the telomere into a high-order structure. TRF1 knockout embryos suffer from severe growth defects without apparent telomere dysfunction, suggesting an obligatory role for TRF1 in cell cycle control. To date, the mechanism regulating the mitotic increase in TRF1 protein expression and its function in mitosis remains unclear. Here, we identify guanine nucleotide-binding protein-like 3 (GNL3L), a GTP-binding protein most similar to nucleostemin, as a novel TRF1-interacting protein in vivo. GNL3L binds TRF1 in the nucleoplasm and is capable of promoting the homodimerization and telomeric association of TRF1, preventing promyelocytic leukemia body recruitment of telomere-bound TRF1, and stabilizing TRF1 protein by inhibiting its ubiquitylation and binding to FBX4, an E3 ubiquitin ligase for TRF1. Most importantly, the TRF1 protein-stabilizing activity of GNL3L mediates the mitotic increase of TRF1 protein and promotes the metaphase-to-anaphase transition. This work reveals novel aspects of TRF1 modulation by GNL3L

Modeling contaminant spread and mitigation in the indoor environment

Experimental and modeling efforts, using a pilot-scale testbed and multizone modeling, are undertaken to develop filtration and ventilation strategies aimed at improving indoor air quality (IAQ). As part of this effort, a model is developed to effectively estimate crack areas of the multizone testbed. The model is divided into two sub-approaches: one approach is to assume the same crack area for the same type of opening and determine them by minimizing the sum of the squares of relative error between the calculated and experimental ventilation rates for the whole facility; the other is to assume that the crack areas are independent of each other and a similar least-squares minimization is applied to determine these crack areas zone by zone. A comparison of the two approaches shows that both can provide satisfactory results, and the latter approach is preferred, because it provides more flexibility and detail. Ventilation systems are explored using multizone simulations. The model results suggest a distributed unbalanced ventilation system is preferred for maintaining IAQ, because 1) it can provide positive pressure difference across the building envelope to prevent exterior contaminant infiltration; and 2) some contaminated indoor zones can be “isolated” from adjacent ones by adjusting the relative pressure differences. Realistic particle distributions typical to a particular contamination threat of interest are considered, and an acoustically enhanced impaction (AEI) filtration device is investigated together with other filters. The protection factor (PF, a ratio of concentration integrated over time in the ambient to that indoors) is chosen as a performance metric. A PF-oriented evaluation framework has been established such that ventilation system/strategy (or filter) comparison in terms of IAQ enhancement is straightforward. For instance, 16 filtration schemes are compared to identify preferred ventilation and filtration strategies. For the indoor environment, a highly efficient outside air (OA) filter is recommended, but a recirculated air (RA) filter is relatively much less effective. For vestibule protection, a stand-alone balanced system with 100% RA filtration is recommended. The AEI device can be an alternative to a HEPA filter when the ambient contamination level is low to moderate. Extension of an existing analytical steady-state PF model is undertaken to demonstrate the advantages of pressurization protection of buildings over non-pressurization protection. The analytical PF model can be used to determine the ventilation flow rate and filter efficiency at a specific PF level and guide the vestibule door operation. It is found that the minimum closing period of the vestibule interior door typically should be 20 minutes to protect the room

Nucleolar Trafficking of Nucleostemin Family Proteins: Common versus Protein-Specific Mechanisms▿ §

The nucleolus has begun to emerge as a subnuclear organelle capable of modulating the activities of nuclear proteins in a dynamic and cell type-dependent manner. It remains unclear whether one can extrapolate a rule that predicts the nucleolar localization of multiple proteins based on protein sequence. Here, we address this issue by determining the shared and unique mechanisms that regulate the static and dynamic distributions of a family of nucleolar GTP-binding proteins, consisting of nucleostemin (NS), guanine nucleotide binding protein-like 3 (GNL3L), and Ngp1. The nucleolar residence of GNL3L is short and primarily controlled by its basic-coiled-coil domain, whereas the nucleolar residence of NS and Ngp1 is long and requires the basic and the GTP-binding domains, the latter of which functions as a retention signal. All three proteins contain a nucleoplasmic localization signal (NpLS) that prevents their nucleolar accumulation. Unlike that of the basic domain, the activity of NpLS is dynamically controlled by the GTP-binding domain. The nucleolar retention and the NpLS-regulating functions of the G domain involve specific residues that cannot be predicted by overall protein homology. This work reveals common and protein-specific mechanisms underlying the nucleolar movement of NS family proteins