Abstract: Plant intracellular nucleotide-binding domain, leucine-rich repeat-containing receptors (NLRs) activate a robust immune response upon detection of pathogen effectors. How NLRs induce downstream immune defense genes remains poorly understood. The Mediator complex plays a central role in transducing signals from gene-specific transcription factors to the transcription machinery for gene transcription/activation. In this study, we demonstrate that MED10b and MED7 of the Mediator complex mediate jasmonate-dependent transcription repression, and coiled-coil NLRs (CNLs) in Solanaceae modulate MED10b/MED7 to activate immunity. Using the tomato CNL Sw-5b , which confers resistance to tospovirus , as a model, we found that the CC domain of Sw-5b directly interacts with MED10b. Knockout/down of MED10b and other subunits including MED7 of the middle module of Mediator activates plant defense against tospovirus . MED10b was found to directly interact with MED7, and MED7 directly interacts with JAZ proteins, which function as transcriptional repressors of jasmonic acid (JA) signaling. MED10b–MED7–JAZ together can strongly repress the expression of JA-responsive genes. The activated Sw-5b CC interferes with the interaction between MED10b and MED7, leading to the activation of JA-dependent defense signaling against tospovirus . Furthermore, we found that CC domains of various other CNLs including helper NLR NRCs from Solanaceae modulate MED10b/MED7 to activate defense against different pathogens. Together, our findings reveal that MED10b/MED7 serve as a previously unknown repressor of jasmonate-dependent transcription repression and are modulated by diverse CNLs in Solanaceae to activate the JA-specific defense pathways.

Abstract: Collapsed replication forks, which are a major source of DNA double-strand breaks (DSBs), are repaired by sister chromatid recombination (SCR). The Mre11–Rad50–Nbs1 (MRN) protein complex, assisted by CtIP/Sae2/Ctp1, initiates SCR by nucleolytically resecting the single-ended DSB (seDSB) at the collapsed fork. The molecular architecture of the MRN intercomplex, in which zinc hooks at the apices of long Rad50 coiled-coils connect two Mre11 2 –Rad50 2 complexes, suggests that MRN also structurally assists SCR. Here, Rad50 ChIP assays in Schizosaccharomyces pombe show that MRN sequentially localizes with the seDSB and sister chromatid at a collapsed replication fork. Ctp1, which has multivalent DNA-binding and DNA-bridging activities, has the same DNA interaction pattern. Provision of an intrachromosomal repair template alleviates the nonnucleolytic requirement for MRN to repair the broken fork. Mutations of zinc-coordinating cysteines in the Rad50 hook severely impair SCR. These data suggest that the MRN complex facilitates SCR by linking the seDSB and sister chromatid.

Abstract: The nuclear envelope (NE) separates genomic DNA from the cytoplasm and regulates transport between the cytosol and the nucleus in eukaryotes. Nuclear stiffening enables the cell nucleus to protect itself from extensive deformation, loss of NE integrity, and genome instability. It is known that the reorganization of actin, lamin, and chromatin can contribute to nuclear stiffening. In this work, we show that structural alteration of NE also contributes to instantaneous nuclear stiffening under indentation. In situ mechanical characterization of cell nuclei in intact cells shows that nuclear stiffening and unfolding of NE wrinkles occur simultaneously at the indentation site. A positive correlation between the initial state of NE wrinkles, the unfolding of NE wrinkles, and the stiffening ratio (stiffness fold-change) is found. Additionally, NE wrinkles unfold throughout the nucleus outside the indentation site. Finite element simulation, which involves the purely passive process of structural unfolding, shows that unfolding of NE wrinkles alone can lead to an increase in nuclear stiffness and a reduction in stress and strain levels. Together, these results provide a perspective on how cell nucleus adapts to mechanical stimuli through structural alteration of the NE.

Abstract: Patients with type 2 diabetes (T2D) have disease-associated changes in B-cell function, but the role these changes play in disease pathogenesis is not well established. Data herein show B cells from obese mice produce a proinflammatory cytokine profile compared with B cells from lean mice. Complementary in vivo studies show that obese B cell–null mice have decreased systemic inflammation, inflammatory B- and T-cell cytokines, adipose tissue inflammation, and insulin resistance (IR) compared with obese WT mice. Reduced inflammation in obese/insulin resistant B cell–null mice associates with an increased percentage of anti-inflammatory regulatory T cells (Tregs). This increase contrasts with the sharply decreased percentage of Tregs in obese compared with lean WT mice and suggests that B cells may be critical regulators of T-cell functions previously shown to play important roles in IR. We demonstrate that B cells from T2D (but not non-T2D) subjects support proinflammatory T-cell function in obesity/T2D through contact-dependent mechanisms. In contrast, human monocytes increase proinflammatory T-cell cytokines in both T2D and non-T2D analyses. These data support the conclusion that B cells are critical regulators of inflammation in T2D due to their direct ability to promote proinflammatory T-cell function and secrete a proinflammatory cytokine profile. Thus, B cells are potential therapeutic targets for T2D.

Abstract: The ER-to-Golgi transport pathway is required by all cells. Our findings here reveal a surprising tissue-specific variation in dependence on SEC23B for maintenance of the secretion pathway, and suggest that murine SEC23B is particularly important for the COPII-mediated ER exit of highly abundant cargo in many tissues that specialize in protein secretion. The contrast between the species may be explained by a potential phenotypic difference between complete deficiency in mice and the residual activity observed in humans, or, alternatively, by evolutionary differences between the relative contributions of SEC23A and SEC23B across tissues. Apoptosis induced by deficiency in protein export from the ER could play a previously unappreciated role in the pathogenesis of pancreatitis. In the pancreas of SEC23B-deficient mice, defects occur in exocrine tissues that secrete digestive enzymes, as well as in endocrine tissues that secrete protein hormones, including insulin and glucagon. Endocrine and exocrine progenitor cells in the early SEC23B-deficient embryonic pancreas are normal, suggesting that abnormalities resulting in the pancreatic phenotype arise after cell fate determination. In normal exocrine pancreatic cells, digestive enzymes are stored in numerous zymogen granules after they move out of the Golgi. However, in SEC23B-deficient pancreas, exocrine cells are completely devoid of zymogen granules, and the ER becomes severely distended as a result of accumulation of proteins in the lumen ( Fig. P1 B ). Similar ultrastructural changes are also observed in other affected tissues such as the salivary glands, but not in unaffected tissues, such as the liver. Accumulation of proteins in the ER lumen activates the proapoptotic pathway of the unfolded protein response, suggesting a central role for apoptosis in the degeneration of these tissues in SEC23B-deficient embryos. The affected tissues all express relatively high levels of SEC23B compared with unaffected tissues and to the levels of SEC23A. Why do mutations in different paralogues of the COPII components result in these distinct but limited phenotypes? Such differences could result from distinct functional activities exerted by each paralogue, different temporal and/or spatial expression patterns, both of these, or unique features of the specific human mutations. Only missense mutations have been identified in CLSD, and all reported patients with CDAII retain at least one missense allele, suggesting that complete deficiencies of SEC23A or SEC23B may result in more severe phenotypes. We found that SEC23B-deficient mice survive to term, but die shortly after birth, suggesting that SEC23A alone can support major biological functions during mouse embryogenesis. In contrast to humans with CDAII, mice completely deficient for SEC23B are born with no apparent anemia phenotype, but show degeneration of specific secretory tissues, including the pancreas and the salivary, gastric, intestinal, and nasal glands ( Fig. P1 A ). Two distinct human genetic disorders have been associated with defects in SEC23 paralogues. Craniolenticulosutural dysplasia (CLSD) is characterized by craniofacial and skeletal malformation caused by homozygosity for one of two missense mutations in SEC23A ( 2 ). One mutation has been shown to interfere with the recruitment of SEC13–SEC31 to the site of vesicle formation, blocking COPII coat assembly and resulting in accumulation of cargo proteins in the ER lumen ( 3 ). Skin fibroblasts from patients with CLSD exhibit defects in collagen secretion. In contrast, mutations in SEC23B cause congenital dyserythropoietic anemia type II (CDAII) ( 4 , 5 ). CDAII patients exhibit moderate anemia due to ineffective erythropoiesis. A portion of erythroblasts in the bone marrow of patients with CDAII contains multiple nuclei, and aberrant glycosylation of specific red blood cell membrane proteins is observed. How SEC23B deficiency leads to these selective red blood cell defects in humans is unknown. The two basic functions of the COPII complex are capturing cargo into vesicles and mediating vesicle budding from the ER ( 1 ). Cargo recognition appears to be mediated primarily by the SEC24 subunit, which recognizes divergent export signals located in the cytosolic domain of cargo proteins. The GTP-bound form of SAR1 initiates vesicle budding by binding to the ER membrane and recruits the SEC23–SEC24 heterodimer, which in turn, recruits the outer coat, composed of SEC13–SEC31 heterotetramers, to complete the COPII coat structure. SEC23 is a GTPase-activating protein that activates the SAR1 GTPase. Conversion of SAR1-GTP to SAR1-GDP results in its dissociation from the COPII coat. After SAR1 release, SEC23 interacts with a tethering complex that is thought to facilitate the fusion of COPII vesicles with the target membrane. In eukaryotic cells, 20% to 30% of all synthesized proteins require export to the extracellular space or transport to the plasma membrane and internal organelles to exert their functions. Nearly all these cargo proteins are first synthesized in the endoplasmic reticulum (ER), processed by the ER quality-control machinery, and then transported to the Golgi complex. From the Golgi, cargo proteins are further sorted and moved to their final destinations. Proteins move from one organelle to the next in the secretion pathway in small membrane-enclosed packets called vesicles. Vesicle movement from the ER to the Golgi is controlled by coat protein complex II (COPII). COPII is composed of five core proteins: the small GTPase SAR1 and the two cytosolic protein complexes SEC23–SEC24 and SEC13–SEC31. Mammals express multiple paralogues of COPII proteins, including two paralogues each for SAR1, SEC23, and SEC31, and four paralogues for SEC24. The complexity of mammalian COPII proteins may be an evolutionary response to accommodate the heterogeneity of cargo molecules in different cell types and at different stages of development. Here, we studied the functions of the COPII proteins SEC23A and SEC23B by using mice genetically deficient for SEC23B expression. We found that SEC23A alone supports major biological functions during embryogenesis, and that mice completely deficient for SEC23B, unlike humans with SEC23B gene mutations, are not anemic but have degeneration of multiple secretory tissues.

Abstract: Nonvolatile phase-change memory has been successfully commercialized, but further density scaling below 10 nanometers requires compositionally and structurally homogeneous materials for both the memory cell and the associated vertically stacked two-terminal access switch. The selector switches are mostly amorphous-chalcogenide Ovonic threshold switches (OTSs), operating with a nonlinear current response above a threshold voltage in the amorphous state. However, they currently suffer from the chemical complexity introduced by the quaternary or even more diverse chalcogenide compositions used. We present a single-element tellurium (Te) volatile switch with a large (≥11 megaamperes per square centimeter) drive current density, ~10 3 ON/OFF current ratio, and faster than 20 nanosecond switching speed. The low OFF current arises from the existence of a ~0.95–electron volt Schottky barrier at the Te–electrode interface, whereas a transient, voltage pulse–induced crystal-liquid melting transition of the pure Te leads to a high ON current. Our discovery of a single-element electrical switch may help realize denser memory chips.

Abstract: The incidence of tuberculosis has been increasing substantially on a worldwide basis over the past decade, but no tuberculosis-specific drugs have been discovered in 40 years. We identified a diarylquinoline, R207910, that potently inhibits both drug-sensitive and drug-resistant Mycobacterium tuberculosis in vitro (minimum inhibitory concentration 0.06 μg/ml). In mice, R207910 exceeded the bactericidal activities of isoniazid and rifampin by at least 1 log unit. Substitution of drugs included in the World Health Organization's first-line tuberculosis treatment regimen (rifampin, isoniazid, and pyrazinamide) with R207910 accelerated bactericidal activity, leading to complete culture conversion after 2 months of treatment in some combinations. A single dose of R207910 inhibited mycobacterial growth for 1 week. Plasma levels associated with efficacy in mice were well tolerated in healthy human volunteers. Mutants selected in vitro suggest that the drug targets the proton pump of adenosine triphosphate (ATP) synthase.