A Nup133-dependent NPC-anchored network tethers centrosomes to the nuclear envelope in prophase

Maintenance of stable E-cadherin-dependent adhesion is essential for epithelial function. The small GTPase Rac is activated by initial cadherin clustering, but the precise mechanisms underlying Rac-dependent junction stabilization are not well understood. Ajuba, a LIM domain protein, colocalizes with cadherins, yet Ajuba function at junctions is unknown. We show that, in Ajuba-depleted cells, Rac activation and actin accumulation at cadherin receptors was impaired, and junctions did not sustain mechanical stress. The Rac effector PAK1 was also transiently activated upon cell-cell adhesion and directly phosphorylated Ajuba (Thr172). Interestingly, similar to Ajuba depletion, blocking PAK1 activation perturbed junction maintenance and actin recruitment. Expression of phosphomimetic Ajuba rescued the effects of PAK1 inhibition. Ajuba bound directly to Rac·GDP or Rac·GTP, but phosphorylated Ajuba interacted preferentially with active Rac. Rather than facilitating Rac recruitment to junctions, Ajuba modulated Rac dynamics at contacts depending on its phosphorylation status. Thus, a Rac-PAK1-Ajuba feedback loop integrates spatiotemporal signaling with actin remodeling at cell-cell contacts and stabilizes preassembled cadherin complexes.     

mTOR Signaling Regulates Nucleolar Targeting of the SUMO-Specific Isopeptidase SENP3

Ribosome biogenesis is a multistep cellular pathway that involves more than 200 regulatory components to ultimately generate translation-competent 80S ribosomes. The initial steps of this process, particularly rRNA processing, take place in the nucleolus, while later stages occur in the nucleoplasm and cytoplasm. One critical factor of 28S rRNA maturation is the SUMO-isopeptidase SENP3. SENP3 tightly interacts with the nucleolar scaffold protein NPM1 and is associated with nucleolar 60S preribosomes. A central question is how changes in energy supply feed into the regulation of ribosome maturation. Here, we show that the nutrient-sensing mTOR kinase pathway controls the nucleolar targeting of SENP3 by regulating its interaction with NPM1. We define an N-terminal domain in SENP3 as the critical NPM1 binding region and provide evidence that mTOR-mediated phosphorylation of serine/threonine residues within this region fosters the interaction of SENP3 with NPM1. The inhibition of mTOR triggers the nucleolar release of SENP3, thereby likely compromising its activity in rRNA processing. Since mTOR activity is tightly coupled to nutrient availability, we propose that this pathway contributes to the adaptation of ribosome maturation in response to the cellular energy status.     

Stress-induced changes in gene interactions in human cells

Cells respond to variable environments by changing gene expression and gene interactions. To study how human cells response to stress, we analyzed the expression of >5000 genes in cultured B cells from nearly 100 normal individuals following endoplasmic reticulum stress and exposure to ionizing radiation. We identified thousands of genes that are induced or repressed. Then, we constructed coexpression networks and inferred interactions among genes. We used coexpression and machine learning analyses to study how genes interact with each other in response to stress. The results showed that for most genes, their interactions with each other are the same at baseline and in response to different stresses; however, a small set of genes acquired new interacting partners to engage in stress-specific responses. These genes with altered interacting partners are associated with diseases in which endoplasmic reticulum stress response or sensitivity to radiation has been implicated. Thus, our findings showed that to understand disease-specific pathways, it is important to identify not only genes that change expression levels but also those that alter interactions with other genes.     

Mammalian non-classical major histocompatibility complex I and its receptors: Important contexts of gene,evolution, and immunity

The evolutionary conserved, less-polymorphic, nonclassical major histocompatibility complex (MHC) class I molecules: Qa-1 and its human homologue human leukocyte antigen-E (HLA-E) along with HLA-F, G and H cross-talk with the T-cell receptors and also interact with natural killer T-cells and other lymphocytes. Moreover, these nonclassical MHC molecules are known to interact with CD94/NKG2 heterodimeric receptors to induce immune responses and immune regulations. This dual role of Qa-1/HLA-E in terms of innate and adaptive immunity makes them more interesting. This review highlights the new updates of the mammalian nonclassical MHC-I molecules in terms of their gene organization, evolutionary perspective and their role in immunity.     

Anti-tubercular activity and molecular docking studies of indolizine derivatives targeting mycobacterial InhA enzyme

A series of 1,2,3-trisubstituted indolizines (2a–2f, 3a–3d, and 4a–4c) were screened for in vitro whole-cell anti-tubercular activity against the susceptible H37Rv and multidrug-resistant (MDR) Mycobacterium tuberculosis (MTB) strains. Compounds 2b–2d, 3a–3d, and 4a–4c were active against the H37Rv-MTB strain with minimum inhibitory concentration (MIC) ranging from 4 to 32 µg/mL, whereas the indolizines 4a–4c, with ethyl ester group at the 4-position of the benzoyl ring also exhibited anti-MDR-MTB activity (MIC = 16–64 µg/mL). In silico docking study revealed the enoyl-acyl carrier protein reductase (InhA) and anthranilate phosphoribosyltransferase as potential molecular targets for the indolizines. The X-ray diffraction analysis of the compound 4b was also carried out. Further, a safety study (in silico and in vitro) demonstrated no toxicity for these compounds. Thus, the indolizines warrant further development and may represent a novel promising class of InhA inhibitors and multi-targeting agents to combat drug-sensitive and drug-resistant MTB strains.     

A Murine Inhalation Model to Characterize Pulmonary Exposure to Dry Aspergillus fumigatus Conidia

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×10⁵ twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8⁺ IL17⁺ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.     

A SnapShot of Ubiquitin Chain Elongation: LYSINE 48-TETRA-UBIQUITIN SLOWS DOWN UBIQUITINATION*

We have explored the mechanisms of polyubiquitin chain assembly with reconstituted ubiquitination of IκBα and β-catenin by the Skp1-cullin 1-βTrCP F-box protein (SCF^βTrCP) E3 ubiquitin (Ub) ligase complex. Competition experiments revealed that SCF^βTrCP formed a complex with IκBα and that the Nedd8 modified E3-substrate platform engaged in dynamic interactions with the Cdc34 E2 Ub conjugating enzyme for chain elongation. Using “elongation intermediates” containing β-catenin linked with Ub chains of defined length, it was observed that a Lys-48-Ub chain of a length greater than four, but not its Lys-63 linkage counterparts, slowed the rate of additional Ub conjugation. Thus, the Ub chain length and linkage impact kinetic rates of chain elongation. Given that Lys-48-tetra-Ub is packed into compact conformations due to extensive intrachain interactions between Ub subunits, this topology may limit the accessibility of SCF^βTrCP/Cdc34 to the distal Ub Lys-48 and result in slowed elongation.     

Preclinical evaluation of 89Zr-labeled anti-CD44 monoclonal antibody RG7356 in mice and cynomolgus monkeys: Prelude to Phase 1 clinical studies

RG7356 is a humanized antibody targeting the constant region of CD44. RG7356 was radiolabeled with ⁸⁹Zr for preclinical evaluations in tumor xenograft-bearing mice and normal cynomolgus monkeys to enable study of its biodistribution and the role of CD44 expression on RG7356 uptake. Studies with ⁸⁹Zr-RG7356 were performed in mice bearing tumor xenografts that differ in the level of CD44 expression (CD44⁺ or CD44^-) and RG7356 responsiveness (resp or non-resp): MDA-MB-231 (CD44⁺, resp), PL45 (CD44⁺, non-resp) and HepG2 (CD44^–, non-resp). Immuno-PET whole body biodistribution studies were performed in normal cynomolgus monkeys to determine normal organ uptake after administration of a single dose. At 1, 2, 3, and 6 days after injection, ⁸⁹Zr-RG7356 uptake in MDA-MB-231 (CD44⁺, resp) xenografts was nearly constant and about 9 times higher than in HepG2 (CD44^–, non-resp) xenografts (range 27.44 ± 12.93 to 33.13 ± 7.42% ID/g vs. 3.25 ± 0.38 to 3.90 ± 0.58% ID/g). Uptake of ⁸⁹Zr-RG7356 was similar in MDA-MB-231 (CD44⁺, resp) and PL45 (CD44⁺, non-resp) xenografts. Studies in monkeys revealed antibody uptake in spleen, salivary glands and bone marrow, which might be related to the level of CD44 expression. ⁸⁹Zr-RG7356 uptake in these normal organs decreased with increasing dose levels of unlabeled RG7356.⁸⁹Zr-RG7356 selectively targets CD44⁺ responsive and non-responsive tumors in mice and CD44⁺ tissues in monkeys. These studies indicate the importance of accurate antibody dosing in humans to obtain optimal tumor targeting. Moreover, efficient binding of RG7356 to CD44⁺ tumors may not be sufficient in itself to drive an anti-tumor response.     

The survival of different vibrios in association with a laboratory culture of the red-tide-causing organism Amphidinium carterae

The survival of different vibrios in association with a red-tide-causing organism Amphidinium carterae was studied in the laboratory. Vibrio alginolyticus and V. harveyi could not survive beyond 14 days in an actively growing culture of A. carterae. On the other hand, V. parahaemolyticus could be detected up to 40 days.