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Members of the protein disulfide isomerase (PDI)-C subfamily are chimeric proteins containing the thioredoxin (Trx) domain of PDIs, and the conserved N- and C-terminal Pfam domains of Erv41p/Erv46p-type cargo receptors. They are unique to plants and chromalveolates. The Arabidopsis genome encodes three PDI-C isoforms: PDI7, PDI12 and PDI13. Here we demonstrate that PDI7 is a 65 kDa integral membrane glycoprotein expressed throughout many Arabidopsis tissues. Using a PDI7-specific antibody, we show through immunoelectron microscopy that PDI7 localizes to the endoplasmic reticulum (ER) and Golgi membranes in wild-type root tip cells, and was also detected in vesicles. Tomographic modeling of the Golgi revealed that PDI7 was confined to the cis-Golgi, and accumulated primarily at the cis-most cisterna. Shoot apical meristem cells from transgenic plants overexpressing PDI7 exhibited a dramatic increase in anti-PDI7 labeling at the cis-Golgi. When N- or C-terminal fusions between PDI7 and the green fluorescent protein variant, GFP(S65T), were expressed in mesophyll protoplasts, the fusions co-localized with the ER marker, ER-mCherry. However, when GFP(S65T) was positioned internally within PDI7 (PDI7-GFPint), the fusion strongly co-localized with the cis-Golgi marker, mCherry-SYP31, and faintly labeled the ER. In contrast to the Golgi-resident fusion protein (Man49-mCherry), PDI7-GFPint did not redistribute to the ER after brefeldin A treatment. Protease protection experiments indicated that the Trx domain of PDI7 is located within the ER/Golgi lumen. We propose a model where PDI-C isoforms function as cargo receptors for proteins containing exposed cysteine residues, cycling them from the Golgi back to the ER.

Maize endosperm consists of three distinct types of tissues, including the starchy endosperm (SE), the basal endosperm transfer cell layer (BETL), and the aleurone cell layer (AL). Compartmentalization of these tissues during endosperm differentiation makes the endosperm development an excellent model to study changes in gene expression during development. By utilizing cryo-dissection of developing endosperm, morphologically distinct samples can be obtained for transcriptome and epigenome analysis. Here, we describe methods for the isolation of tissues from developing maize endosperm and for the transcriptome analysis to identify novel long noncoding RNAs. The transcriptome data can be further analyzed to illustrate spatiotemporal changes in both coding and non-coding transcripts during the endosperm development.

In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME., Autophagy mediates the degradation of paternal mitochondria after fertilization in C. elegans to ensure that mitochondria are inherited maternally. Here the authors show that mitochondrial dynamics is critical for the selectivity and kinetics of paternal mitochondrial elimination.

Eliminating paternal mitochondria During fertilization, the oocyte and sperm each bring their mitochondria to the union. Shortly afterward, the paternal mitochondria are degraded, and only the maternal mitochondria are conveyed to the progeny. Zhou et al. observed that the integrity of the inner membrane of paternal mitochondria is compromised, which apparently marks them for degradation (see the Perspective by van der Bliek). Autophagy commences by mitochondrial endonuclease G relocating from the intermembrane space into the matrix and subsequently degrading the paternal mitochondrial DNA. Any delay in this process increases embryonic lethality. Science , this issue p. 394 ; see also p. 351

Two Arabidopsis ABC transporters, ABCG1 and ABCG16, are expressed in the tapetal layer, specifically after postmeiotic microspore release, and play important roles in pollen surface development. The male gametophytic cells of terrestrial plants, the pollen grains, travel far before fertilization, and thus require strong protective layers, which take the form of a pollen coat and a pollen wall. The protective surface structures are generated by the tapetum, the tissue surrounding the developing gametophytes. Many ABC transporters, including Arabidopsis thaliana ABCG1 and ABCG16, have been shown to play essential roles in the development of such protective layers. However, the details of the mechanism of their function remain to be clarified. In this study, we show that ABCG1 and ABCG16 are localized at the plasma membrane of tapetal cells, specifically after postmeiotic microspore release, and play critical roles in the postmeiotic stages of male gametophyte development. Consistent with this stage-specific expression, the abcg1 abcg16 double knockout mutant exhibited defects in pollen development after postmeiotic microspore release; their microspores lacked intact nexine and intine layers, exhibited defects in pollen mitosis I, displayed ectopic deposits of arabinogalactan proteins, failed to complete cytokinesis, and lacked sperm cells. Interestingly, the double mutant exhibited abnormalities in the internal structures of tapetal cells, too; the storage organelles of tapetal cells, tapetosomes and elaioplasts, were morphologically altered. Thus, this work reveals that the lack of ABCG1 and ABCG16 at the tapetal cell membrane causes a broad range of defects in pollen, as well as in tapetal cells themselves. Furthermore, these results suggest that normal pollen surface development is necessary for normal development of the pollen cytoplasm.

Aims Under pressure overload, initial adaptive hypertrophy of the heart is followed by cardiomyocyte elongation, reduced contractile force, and failure. The mechanisms governing the transition to failure are not fully understood. Pressure overload reduced cardiac myosin light chain kinase (cMLCK) by ∼80% within 1 week and persists. Knockdown of cMLCK in cardiomyocytes resulted in reduced cardiac contractility and sarcomere disorganization. Thus, we hypothesized that acute reduction of cMLCK may be causative for reduced contractility and cardiomyocyte remodelling during the transition from compensated to decompensated cardiac hypertrophy. Methods and results To mimic acute cMLCK reduction in adult hearts, the floxed- Mylk3 gene that encodes cMLCK was inducibly ablated in Mylk3flox/flox / merCremer mice ( Mylk3-KO ), and compared with two control mice ( Mylk3flox/flox and Mylk3+/+ / merCremer ) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). In Mylk3-KO mice, reduction of cMLCK protein was evident by 4 days, with a decline to below the level of detection by 6 days. By 7 days, these mice exhibited heart failure, with reduction of fractional shortening compared with those in two control groups (19.8 vs. 28.0% and 27.7%). Severely convoluted cardiomyocytes with sarcomeric disorganization, wavy fibres, and cell death were demonstrated in Mylk3-KO mice. The cardiomyocytes were also unable to thicken adaptively to pressure overload. Conclusion Our results, using a new mouse model mimicking an acute reduction of cMLCK, suggest that cMLCK plays a pivotal role in the transition from compensated to decompensated hypertrophy via sarcomeric disorganization.

Indent ZrTiO4 ceramics are widely used to manufacture electrical and optical devices. This study reveals an approach for the production of micro-porous ceramics consisting of TiO2 and ZrO2 by using a direct foaming method. ZrO2 particles in a colloidal suspension were partially hydrophobized by using propyl gallate as an amphiphile in a suitable pH range. Different mole ratios of the TiO2 suspension were added to the surface-modified ZrO2 suspension. The contact angle was found to be around 54° and the adsorption free energy to be 1.68 × 10−11 ∼ 9.2 × 10−12 J·A Laplace pressure of about 2.0 ∼ 2.2 mPa, corresponding to a wet foam stability of about 80 ∼ 85% was determined. A microstructure analysis was done after the dried samples had been sintered to obtain porous ceramics.

Microalgae are potential sources of energy and high-value materials. To decipher the process of energy metabolism in green algae, we created a mutant pool of strain CC-503 of the model green microalga Chlamydomonas reinhardtii, by random insertion of an antibiotic resistance gene, and screened the pool for lines with altered carbon metabolism. We identified a mutant that harbored the antibiotic resistance gene in CrMEX1, a putative Maltose Exporter-Like protein 1 (Cre12.g486600.t1.2). The mutant had reduced levels of CrMEX1 expression and, similarly to the Arabidopsis mex1 knockout mutant, which cannot export maltose from the chloroplast, it over-accumulated starch granules in the chloroplast. The mutant’s lipid levels were slightly higher than those of the wild type, and its initial growth kinetics were not significantly different from those of the wild type, but the mutant culture did not reach the same high cell density as the wild type in acetate-containing culture medium under continuous light. These results suggest that CrMEX1 encodes a maltose transporter protein, and that export of photoassimilates from chloroplasts is necessary for normal Chlamydomonas growth, even under continuous light with an ample supply of carbon in the form of acetate.

Exogenous metabolites from microbial and dietary origins have profound effects on host metabolism. Here, we report that a sub-population of lipid droplets (LDs), which are conserved organelles for fat storage, is defined by metabolites-driven targeting of theC. elegansseipin ortholog, SEIP-1. Loss of SEIP-1 function reduced the size of a subset of LDs while over-expression of SEIP-1 had the opposite effect. Ultrastructural analysis revealed SEIP-1 enrichment in an endoplasmic reticulum (ER) subdomain, which co-purified with LDs. Analyses ofC. elegansand bacterial genetic mutants indicated a requirement of polyunsaturated fatty acids (PUFAs) and microbial cyclopropane fatty acids (CFAs) for SEIP-1 enrichment, as confirmed by dietary supplementation experiments. In mammalian cells, heterologous expression of SEIP-1 promoted lipid droplet expansion from ER subdomains in a conserved manner. Our results suggest that microbial and polyunsaturated fatty acids serve unexpected roles in regulating cellular fat storage by enforcing LD diversity.

Root border cells lie at the interface between the root cap and the soil, secreting mucilage containing polysaccharides and molecules influencing microbial growth around the root. Border cells are sloughed off from the root surface, and the detachment is associated with secretion of xylogalacturonan (XGA). Recently, we showed that in alfalfa XGA secretion is mediated by large vesicles arising from the trans-Golgi in root cap cells. These vesicles are detected in precursor cells of border cells, but their fusion with the plasma membrane is observed only in border cells. We have now examined XGA secretion from maize border cells and Arabidopsis border-like cells using transmission electron microscopy and immunolabeling. In the root caps of both species, XGA is packaged into vesicles derived from the trans-Golgi, not in the vesicles from the trans-Golgi network as in the alfalfa root cap. Border cell-specific exocytosis of XGA was observed in the maize root suggesting that sorting and secretion of XGA in the root cap are conserved in monocot plants.

Biogenesis of the complex 3D architecture of plant thylakoids remains an unsolved problem. Here, we analyzed this process in chloroplasts of germinating Arabidopsis thaliana cotyledons using 3D electron microscopy and gene expression analyses of chloroplast proteins. Our study identified a linear developmental sequence with five assembly stages: tubulo-vesicular prothylakoids (24 h after imbibition [HAI]), sheet-like pregranal thylakoids that develop from the prothylakoids (36 HAI), proliferation of pro-grana stacks with wide tubular connections to the originating pregrana thylakoids (60 HAI), structural differentiation of pro-grana stacks and expanded stroma thylakoids (84 HAI), and conversion of the pro-grana stacks into mature grana stacks (120 HAI). Development of the planar pregranal thylakoids and the pro-grana membrane stacks coincides with the appearance of thylakoid-bound polysomes and photosystem II complex subunits at 36 HAI. ATP synthase, cytochrome b6f, and light-harvesting complex II proteins are detected at 60 HAI, while PSI proteins and the curvature-inducing CURT1A protein appear at 84 HAI. If stromal ribosome biogenesis is delayed, prothylakoids accumulate until stromal ribosomes are produced, and grana-forming thylakoids develop after polysomes bind to the thylakoid membranes. In fzo-like (fzl) mutants, in which thylakoid organization is perturbed, pro-grana stacks in cotyledons form discrete, spiral membrane compartments instead of organelle-wide membrane networks, suggesting that FZL is involved in fusing membrane compartments together. Our data demonstrate that the assembly of thylakoid protein complexes, CURT1 proteins, and FZL proteins mediate distinct and critical steps in thylakoid biogenesis.

Apicomplexans are a diverse group of obligate parasites occupying different intracellular niches that require modification to meet the needs of the parasite. To efficiently manipulate their environment, apicomplexans translocate numerous parasite proteins into the host cell. Whereas some parasites remain contained within a parasitophorous vacuole membrane (PVM) throughout their developmental cycle, others do not, a difference that affects the machinery needed for protein export. A signal-mediated pathway for protein export into the host cell has been characterized in Plasmodium parasites, which maintain the PVM. Here, we functionally demonstrate an analogous host-targeting pathway involving organellar staging prior to secretion in the related bovine parasite, Babesia bovis, a parasite that destroys the PVM shortly after invasion. Taking into account recent identification of a similar signal-mediated pathway in the coccidian parasite Toxoplasma gondii, we suggest a model in which this conserved pathway has evolved in multiple steps from signal-mediated trafficking to specific secretory organelles for controlled secretion to a complex protein translocation process across the PVM.

Purpose Endocrine-disrupting chemicals interfere with the endocrine system and therefore affect growth and pubertal progression. The study aim was to compare the growth and pubertal progression in wild-type female rats with different bedding types. Methods Twenty 5-week-old female wild-type Sprague Dawley rats were randomly assigned to two groups with different bedding types: one group received wood shaving bedding, while a second group received corncob bedding. We determined crown-rump length and body weight as anthropometric measurements and assessed the serum growth hormone (GH) and estradiol levels. The gh1 mRNA expression levels were compared using quantitative real time transcription polymerase chain reaction. The estrous cycle was evaluated by vaginal smear. Results The anthropometric measurements were not significantly different between the two groups. The mean relative expression of the gh1 gene was lower in the corncob bedding group than that in the wood shaving group (P=0.768). Meanwhile serum GH and estradiol were increased in the wood shaving bedding group; however this difference was not statistically significant. The time to first estrus and the length of the estrous cycle were increased in the corncob bedding group; the proportion of normal estrous cycles was also decreased. These findings indicate irregularities in the estrous cycle. Conclusion Endocrine-disrupting chemicals in corncob bedding might be associated with time to first estrus and length of the estrous cycle. Therefore, the type of bedding should be considered as a factor affecting pubertal progression in rodents.

In order to install the wave power facilities in ocean and coastal area, it is very important to determine the properties of wave data. Discrete wavelet packet transform was applied in this study and was used as a tool to find out the basic properties of waves around Namae coast. Important features of hydrodynamic pressure such as frequency and magnitude were investigated in different observation time. Also the idea of measuring the noise rate was introduced and applied to both stationary and non-stationary time spans for the comparison. These methods would be useful to check the feasibility of wave energy extraction in various types of coasts.

Electron tomography is a powerful tool for visualizing subcellular organelles. With the advances in cryo-fixation techniques, it is now possible to reconstruct complex structures in cells preserved close to their native states in three-dimension (3D) using electron tomography. In order to better visualize these objects, 3D models are made from outlines of organelles in individual tomographic slices, which can be used to display morphological features and quantify structural parameters. While outlines of simple organelles can be drawn by hand fairly quickly, it is possible to accelerate 3D modeling of more complex organelles by means of semiautomatic segmentation. In this chapter, we use the example of reconstructing Golgi cisternae of a plant cell into 3D models using the semiautomatic protocol.

Three-dimensional (3D) printing is an effective tool for preparing tangible 3D models from computer visualizations to assist in scientific research and education. With the recent popularization of 3D printing processes, it is now possible for individual laboratories to convert their scientific data into a physical form suitable for presentation or teaching purposes. Electron tomography is an electron microscopy method by which 3D structures of subcellular organelles or macromolecular complexes are determined at nanometer-level resolutions. Electron tomography analyses have revealed the convoluted membrane architectures of Golgi stacks, chloroplasts, and mitochondria. But the intricacy of their 3D organizations is difficult to grasp from tomographic models illustrated on computer screens. Despite the rapid development of 3D printing technologies, production of organelle models based on experimental data with 3D printing has rarely been documented. In this chapter, we present a simple guide to creating 3D prints of electron tomographic models of plant Golgi stacks using the two most accessible 3D printing technologies.

During cytokinesis in plants, trans-Golgi network-derived vesicles accumulate at the center of dividing cells and undergo various structural changes to give rise to the planar cell plate. However, how this conversion occurs at the molecular level remains elusive. In this study, we report that SH3 Domain-Containing Protein 2 (SH3P2) in Arabidopsis thaliana plays a crucial role in converting vesicles to the planar cell plate. SH3P2 RNAi plants showed cytokinesis-defective phenotypes and produced aggregations of vesicles at the leading edge of the cell plate. SH3P2 localized to the leading edge of the cell plate, particularly the constricted or curved regions of the cell plate. The BAR domain of SH3P2 induced tubulation of vesicles. SH3P2 formed a complex with dynamin-related protein 1A (DRP1A) and affected DRP1A accumulation to the cell plate. Based on these results, we propose that SH3P2 functions together with DRP1A to convert the fused vesicles to tubular structures during cytokinesis.

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three-dimensional structures and macromolecular compositions of these Golgi stacks, we examined high-pressure frozen/freeze-substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans-Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi-associated vesicles. Margins of trans-Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan-I (PGA/RG-I) are detected in the trans-most cisternae and TGN compartments. LVs produced from TGN compartments (TGN-LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN-LVs containing the XG and PGA/RG-I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans-Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans-cisternae accompanying polysaccharide synthesis with a mathematical model.

Autophagy is a conserved pathway for bulk degradation of cytoplasmic material by a double-membrane structure named the autophagosome. The initiation of autophagosome formation requires the recruitment of autophagy-related protein 9 (ATG9) vesicles to the preautophagosomal structure. However, the functional relationship between ATG9 vesicles and the phagophore is controversial in different systems, and the molecular function of ATG9 remains unknown in plants. Here, we demonstrate that ATG9 is essential for endoplasmic reticulum (ER)-derived autophagosome formation in plants. Through a combination of genetic, in vivo imaging and electron tomography approaches, we show that Arabidopsis ATG9 deficiency leads to a drastic accumulation of autophagosome-related tubular structures in direct membrane continuity with the ER upon autophagic induction. Dynamic analyses demonstrate a transient membrane association between ATG9 vesicles and the autophagosomal membrane during autophagy. Furthermore, trafficking of ATG18a is compromised in atg9 mutants during autophagy by forming extended tubules in a phosphatidylinositol 3-phosphate-dependent manner. Taken together, this study provides evidence for a pivotal role of ATG9 in regulating autophagosome progression from the ER membrane in Arabidopsis.

Electron tomography is a powerful tool for visualizing subcellular organelles. With the advances in cryo-fixation techniques, it is now possible to reconstruct complex structures in cells preserved close to their native states in three-dimension (3D) using electron tomography. In order to better visualize these objects, 3D models are made from outlines of organelles in individual tomographic slices, which can be used to display morphological features and quantify structural parameters. While outlines of simple organelles can be drawn by hand fairly quickly, it is possible to accelerate 3D modeling of more complex organelles by means of semiautomatic segmentation. In this chapter, we use the example of reconstructing Golgi cisternae of a plant cell into 3D models using the semiautomatic protocol.

Three-dimensional (3D) printing is an effective tool for preparing tangible 3D models from computer visualizations to assist in scientific research and education. With the recent popularization of 3D printing processes, it is now possible for individual laboratories to convert their scientific data into a physical form suitable for presentation or teaching purposes. Electron tomography is an electron microscopy method by which 3D structures of subcellular organelles or macromolecular complexes are determined at nanometer-level resolutions. Electron tomography analyses have revealed the convoluted membrane architectures of Golgi stacks, chloroplasts, and mitochondria. But the intricacy of their 3D organizations is difficult to grasp from tomographic models illustrated on computer screens. Despite the rapid development of 3D printing technologies, production of organelle models based on experimental data with 3D printing has rarely been documented. In this chapter, we present a simple guide to creating 3D prints of electron tomographic models of plant Golgi stacks using the two most accessible 3D printing technologies.

Unless there are mechanisms to selectively retain membrane proteins in the endoplasmic reticulum (ER) or in the Golgi apparatus, they automatically proceed downstream to the plasma or vacuole membranes. Two types of coat protein complex I (COPI)-interacting motifs in the cytosolic tails of membrane proteins seem to facilitate membrane retention in the early secretory pathway of plants: a dilysine (KKXX) motif (which is typical of p24 proteins) for the ER and a KXE/D motif (which occurs in the Arabidopsis endomembrane protein EMP12) for the Golgi apparatus. The KXE/D motif is highly conserved in all eukaryotic EMPs and is additionally present in hundreds of other proteins of unknown subcellular localization and function. This novel signal may represent a new general mechanism for Golgi targeting and the retention of polytopic integral membrane proteins.

Purpose There were a lot of reports regarding associations of polymorphisms in the estrogen receptor α (ESR1). with many disorders. But, those with constitutional delay of growth and puberty (CDGP) are not known. Our aim is to find out any association between CDGP and ESR1. Methods In a total of 27 subjects, we compared 7 CDGP patients with 20 healthy controls with their heights and sexual maturity rates were within normal range. We selected three single nucleotide polymorphisms from intron 1 of ESR1 (rs3778609, rs12665044, and rs827421) as candidates, respectively. Results In genotype analyses, the frequency of G/G genotype at rs827421 in intron 1 of ESR1 was increased in CDGP boys (P=0.03). Conclusion The genetic variation of ESR1 can be a contributing factor of tempo of growth and puberty.

The cisternal progression/maturation model of Golgi trafficking predicts that cis-Golgi cisternae are formed de novo on the cis-side of the Golgi. Here we describe structural and functional intermediates of the cis cisterna assembly process in high-pressure frozen algae (Scherffelia dubia, Chlamydomonas reinhardtii) and plants (Arabidopsis thaliana, Dionaea muscipula; Venus Flytrap) as determined by electron microscopy, electron tomography and immuno-electron microscopy techniques. Our findings are as follows: (1) The cis-most (C1) Golgi cisternae are generated de novo from cisterna initiators produced by the fusion of 3–5 COPII vesicles in contact with a C2 cis cisterna. (2) COPII vesicles fuel the growth of the initiators, which then merge into a coherent C1 cisterna. (3) When a C1 cisterna nucleates its first cisterna initiator it becomes a C2 cisterna. (4) C2-Cn cis cisternae grow through COPII vesicle fusion. (5) ER-resident proteins are recycled from cis cisternae to the ER via COPIa-type vesicles. (6) In S. dubia the C2 cisternae are capable of mediating the self-assembly of scale protein complexes. (7) In plants, ~90% of native α-mannosidase I localizes to medial Golgi cisternae. (8) Biochemical activation of cis cisternae appears to coincide with their conversion to medial cisternae via recycling of medial cisterna enzymes. We propose how the different cis cisterna assembly intermediates of plants and algae may actually be related to those present in the ERGIC and in the pre-cis Golgi cisterna layer in mammalian cells.

The poxvirus virion contains an inner tubular nucleocapsid structure. The nucleocapsid is apparently labile to conventional electron microscopy fixation procedures and has therefore been largely ignored for decades. Advancements in electron microscopy sample preparation, notably high pressure freezing, better preserve the nucleocapsid structure. Using high pressure freezing and electron microscopy, we have compared the virion structures of wt virus and mutant viruses known to be deficient in packaging of viral transcription enzymes. We show that the mutant viruses lack a defined nucleocapsid. These results support the hypothesis that the nucleocapsid contains the viral DNA genome complexed with viral transcription enzymes and structural proteins. The studies open the door to further investigation of the composition and ultrastructure of the poxvirus nucleocapsid.

Unconventional protein secretion (UPS) describes secretion pathways that bypass one or several of the canonical secretion pit-stops on the way to the plasma membrane, and/or involve the secretion of leaderless proteins. So far, alternatives to conventional secretion were primarily observed and studied in yeast and animal cells. The sessile lifestyle of plants brings with it unique restraints on how they adapt to adverse conditions and environmental challenges. Recently, attention towards unconventional secretion pathways in plant cells has substantially increased, with the large number of leaderless proteins identified through proteomic studies. While UPS pathways in plants are certainly not yet exhaustively researched, an emerging notion is that induction of UPS pathways is correlated with pathogenesis and stress responses. Given the multitude UPS events observed, comprehensively organizing the routes proteins take to the apoplast in defined UPS categories is challenging. With the establishment of a larger collection of studied plant proteins taking these UPS pathways, a clearer picture of endomembrane trafficking as a whole will emerge. There are several novel enabling technologies, such as vesicle proteomics and chemical genomics, with great potential for dissecting secretion pathways, providing information about the cargo that travels along them and the conditions that induce them.

Because of the weak penetrating power of electrons, the signal-to-noise ratio of a transmission electron micrograph (TEM) worsens as section thickness increases. This problem is alleviated by the use of the scanning transmission electron microscopy (STEM). Tomography analyses using STEM of thick sections from yeast and mammalian cells are of higher quality than are bright-field (BF) images. In this study, we compared regular BF tomograms and STEM tomograms from 500-nm thick sections from hypertrophied Golgi stacks of alfalfa root cap cells. Due to their thickness and intense heavy metal staining, BF tomograms of the thick sections suffer from poor contrast and high noise levels. We were able to mitigate these drawbacks by using STEM tomography. When we performed STEM tomography of densely stained chloroplasts of Arabidopsis cotyledon, we observed similar improvements relative to BF tomograms. A longer time is required to collect a STEM tilt series than similar BF TEM images, and dynamic autofocusing required for STEM imaging often fails at high tilt angles. Despite these limitations, STEM tomography is a powerful method for analyzing structures of large or dense organelles of plant cells.

Nuclear transport of immune receptors, signal transducers, and transcription factors is an essential regulatory mechanism for immune activation. Whether and how this process is regulated at the level of the nuclear pore complex (NPC) remains unclear. Here, we report that CPR5, which plays a key inhibitory role in effector-triggered immunity (ETI) and programmed cell death (PCD) in plants, is a novel transmembrane nucleoporin. CPR5 associates with anchors of the NPC selective barrier to constrain nuclear access of signaling cargos and sequesters cyclin-dependent kinase inhibitors (CKIs) involved in ETI signal transduction. Upon activation by immunoreceptors, CPR5 undergoes an oligomer to monomer conformational switch, which coordinates CKI release for ETI signaling and reconfigures the selective barrier to allow significant influx of nuclear signaling cargos through the NPC. Consequently, these coordinated NPC actions result in simultaneous activation of diverse stress-related signaling pathways and constitute an essential regulatory mechanism specific for ETI/PCD induction.

Background In eukaryotes, classical protein disulfide isomerases (PDIs) facilitate the oxidative folding of nascent secretory proteins in the endoplasmic reticulum by catalyzing the formation, breakage, and rearrangement of disulfide bonds. Terrestrial plants encode six structurally distinct subfamilies of PDIs. The novel PDI-B subfamily is unique to terrestrial plants, and in Arabidopsis is represented by a single member, PDI8. Unlike classical PDIs, which lack transmembrane domains (TMDs), PDI8 is unique in that it has a C-terminal TMD and a single N-terminal thioredoxin domain (instead of two). No PDI8 isoforms have been experimentally characterized to date. Here we describe the characterization of the membrane orientation, expression, sub-cellular localization, and biochemical function of this novel member of the PDI family. Results Histochemical staining of plants harboring a PDI8 promoter:β-glucuronidase (GUS) fusion revealed that the PDI8 promoter is highly active in young, expanding leaves, the guard cells of cotyledons, and in the vasculature of several organs, including roots, leaves, cotyledons, and flowers. Immunoelectron microscopy studies using a PDI8-specific antibody on root and shoot apical cells revealed that PDI8 localizes to the endoplasmic reticulum (ER). Transient expression of two PDI8 fusions to green fluorescent protein (spGFP-PDI8 and PDI8-GFP-KKED) in leaf mesophyll protoplasts also resulted in labeling of the ER. Protease-protection immunoblot analysis indicated that PDI8 is a type I membrane protein, with its catalytic domain facing the ER lumen. The lumenal portion of PDI8 was able to functionally complement the loss of the prokaryotic protein foldase, disulfide oxidase (DsbA), as demonstrated by the reconstitution of periplasmic alkaline phosphatase in Escherichia coli. Conclusion The results indicate that PDI8 is a type I transmembrane protein with its catalytic domain facing the lumen of the ER and functions in the oxidation of cysteines to produce disulfide bonds. It likely plays a role in folding newly-synthesized secretory proteins as they translocate across the ER membrane into the lumen. These foundational results open the door to identifying the substrates of PDI8 to enable a more thorough understanding of its function in plants. Electronic supplementary material The online version of this article (doi:10.1186/s12870-016-0869-2) contains supplementary material, which is available to authorized users.

In order to structure the complex nature of the significant wave height time series, Autoregressive Integrated Moving Average model of order (p,1,0) was applied. Each step which is essential for precise modeling is covered comprehensively. First, the stationarity of given time series of SWH with its 1st order difference were checked. Akaike's Information Criteria and Bayesian Information Criteria for each model were calculated thereafter, in order to sort tentative models. And then, Ljung-Box tests were carried out on the residuals of each model to verify whether the residuals show autocorrelation. Through these processes, the optimal models for each time spans were measured. Furthermore, forecasted time series of significant wave height from Monte-Carlo simulations of each model have proven the utility of the proposed method as it mostly complies with the observed time series of significant wave height.

Unconventional protein secretion (UPS) describes secretion pathways that bypass one or several of the canonical secretion pit-stops on the way to the plasma membrane, and/or involve the secretion of leaderless proteins. So far, alternatives to conventional secretion were primarily observed and studied in yeast and animal cells. The sessile lifestyle of plants brings with it unique restraints on how they adapt to adverse conditions and environmental challenges. Recently, attention towards unconventional secretion pathways in plant cells has substantially increased, with the large number of leaderless proteins identified through proteomic studies. While UPS pathways in plants are certainly not yet exhaustively researched, an emerging notion is that induction of UPS pathways is correlated with pathogenesis and stress responses. Given the multitude UPS events observed, comprehensively organizing the routes proteins take to the apoplast in defined UPS categories is challenging. With the establishment of a larger collection of studied plant proteins taking these UPS pathways, a clearer picture of endomembrane trafficking as a whole will emerge. There are several novel enabling technologies, such as vesicle proteomics and chemical genomics, with great potential for dissecting secretion pathways, providing information about the cargo that travels along them and the conditions that induce them.

Because of the weak penetrating power of electrons, the signal-to-noise ratio of a transmission electron micrograph (TEM) worsens as section thickness increases. This problem is alleviated by the use of the scanning transmission electron microscopy (STEM). Tomography analyses using STEM of thick sections from yeast and mammalian cells are of higher quality than are bright-field (BF) images. In this study, we compared regular BF tomograms and STEM tomograms from 500-nm thick sections from hypertrophied Golgi stacks of alfalfa root cap cells. Due to their thickness and intense heavy metal staining, BF tomograms of the thick sections suffer from poor contrast and high noise levels. We were able to mitigate these drawbacks by using STEM tomography. When we performed STEM tomography of densely stained chloroplasts of Arabidopsis cotyledon, we observed similar improvements relative to BF tomograms. A longer time is required to collect a STEM tilt series than similar BF TEM images, and dynamic autofocusing required for STEM imaging often fails at high tilt angles. Despite these limitations, STEM tomography is a powerful method for analyzing structures of large or dense organelles of plant cells.

In plants, a population of non-cell-autonomous proteins (NCAPs), including numerous transcription factors, move cell to cell through plasmodesmata (PD). In many cases, the intercellular trafficking of these NCAPs is regulated by their interaction with specific PD components. To gain further insight into the functions of this NCAP pathway, coimmunoprecipitation experiments were performed on a tobacco (Nicotiana tabacum) plasmodesmal-enriched cell wall protein preparation using as bait the NCAP, pumpkin (Cucurbita maxima) PHLOEM PROTEIN16 (Cm-PP16). A Cm-PP16 interaction partner, Nt-PLASMODESMAL GERMIN-LIKE PROTEIN1 (Nt-PDGLP1) was identified and shown to be a PD-located component. Arabidopsis thaliana putative orthologs, PDGLP1 and PDGLP2, were identified; expression studies indicated that, postgermination, these proteins were preferentially expressed in the root system. The PDGLP1 signal peptide was shown to function in localization to the PD by a novel mechanism involving the endoplasmic reticulum-Golgi secretory pathway. Overexpression of various tagged versions altered root meristem function, leading to reduced primary root but enhanced lateral root growth. This effect on root growth was corrected with an inability of these chimeric proteins to form stable PD-localized complexes. PDGLP1 and PDGLP2 appear to be involved in regulating primary root growth by controlling phloem-mediated allocation of resources between the primary and lateral root meristems.

Huanglongbing (HLB) is a destructive disease of citrus trees caused by phloem-limited bacteria, Candidatus Liberibacter spp. One of the early microscopic manifestations of HLB is excessive starch accumulation in leaf chloroplasts. We hypothesize that the causative bacteria in the phloem may intervene photoassimilate export, causing the starch to over-accumulate. We examined citrus leaf phloem cells by microscopy methods to characterize plant responses to Liberibacter infection and the contribution of these responses to the pathogenicity of HLB. Plasmodesmata pore units (PPUs) connecting companion cells and sieve elements were stained with a callose-specific dye in the Liberibacter-infected leaf phloem cells; callose accumulated around PPUs before starch began to accumulate in the chloroplasts. When examined by transmission electron microscopy, PPUs with abnormally large callose deposits were more abundant in the Liberibacter-infected samples than in the uninfected samples. We demonstrated an impairment of symplastic dye movement into the vascular tissue and delayed photoassimilate export in the Liberibacter-infected leaves. Liberibacter infection was also linked to callose deposition in the sieve plates, which effectively reduced the sizes of sieve pores. Our results indicate that Liberibacter infection is accompanied by callose deposition in PPUs and sieve pores of the sieve tubes and suggest that the phloem plugging by callose inhibits phloem transport, contributing to the development of HLB symptoms.

Endosperm in a developing seed of maize is a major storage tissue for starch and proteins, and is of immense economic significance for its role in food, feed, and biofuel production. Of the total four cell types in developing endosperm, basal endosperm transfer cells (BETCs) are known to perform a major role in solute acquisition from post-phloem maternal tissues of developing seeds of the plant. To identify all transcripts expressed in the BETCs, we employed here a simplified microdissection method to isolate the BETCs from developing kernels at 12 days after pollination. Total RNA was extracted from such BETCs and used for cDNA synthesis. The 5,790 reads of cDNAs with an average length of 218 nucleotides were generated through 454 sequencing of a partial (1/16) run of a picotiter plate. After trimming, a total of 4,505 reads were used for clustering and assembly that led to 682 contigs and 1,791 singletons. A select set of genes showing high abundance in the above transcript reads were tested and validated for the BETC-specific expression by qPCR and by in situ hybridization. Positive results from these tests and the undetectability of expression of the marker genes relating to the embryo surrounding region located adjacent to the BETCs suggested that our microdissection method was successful in harvesting the BETC-enriched cells. Functional annotation and categorization of the 2,473 unique sequences showed high abundance of transcripts related to mitochondrial activity, stress stimulus, alkaloid biosynthesis related to defense genes and various signaling functions essential for seed development.

The trans Golgi network (TGN) of plant cells sorts and packages Golgi products into secretory (SV) and clathrin-coated (CCV) vesicles. We have analyzed of TGN cisternae in Arabidopsis root meristem cells by cell fractionation and electron microscopy/tomography to establish reliable criteria for identifying TGN cisternae in plant cells, and to define their functional attributes. Transformation of a trans Golgi cisterna into a Golgi-associated TGN cisterna begins with cisternal peeling, the formation of SV buds outside the plane of the cisterna and a 30-35% reduction in cisternal membrane area. Free TGN compartments are defined as cisternae that have detached from the Golgi to become independent organelles. Golgi-associated and free TGN compartments, but not trans Golgi cisternae, bind anti-RabA4b and anti-phosphatidylinositol-4 kinase (PI-4K) antibodies. RabA4b and PI-4Kβ1 localize to budding SVs in the TGN and to SVs en route to the cell surface. SV and CCV release occurs simultaneously via cisternal fragmentation, which typically yields ∼30 vesicles and one to four residual cisternal fragments. Early endosomal markers, VHA-a1-green fluorescent protein (GFP) and SYP61-cyan fluorescent protein (CFP), colocalized with RabA4b in TGN cisternae, suggesting that the secretory and endocytic pathways converge at the TGN. pi4k1/pi4k2 knockout mutant plants produce SVs with highly variable sizes indicating that PI-4Kβ1/2 regulates SV size.

Summary Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER–PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.

In plants, the mechanisms that regulate the transit of vacuolar soluble proteins containing C-terminal and N-terminal vacuolar sorting determinants (VSDs) to the vacuole are largely unknown. In a screen for Arabidopsis thaliana mutants affected in the trafficking of C-terminal VSD containing proteins, we isolated the ribosomal biogenesis mutant rpl4a characterized by its partial secretion of vacuolar targeted proteins and a plethora of developmental phenotypes derived from its aberrant auxin responses. In this study, we show that ribosomal biogenesis can be directly regulated by auxins and that the exogenous application of auxins to wild-type plants results in vacuolar trafficking defects similar to those observed in rpl4a mutants. We propose that the influence of auxin on ribosomal biogenesis acts as a regulatory mechanism for auxin-mediated developmental processes, and we demonstrate the involvement of this regulatory mechanism in the sorting of vacuolar targeted proteins in Arabidopsis.

The miniature1 (mn1) seed phenotype in maize (Zea mays) is due to a loss-of-function mutation at the Mn1 locus that encodes a cell wall invertase (INCW2) that localizes exclusively to the basal endosperm transfer cells (BETCs) of developing seeds. A common feature of all transfer cells is the labyrinth-like wall-in-growth (WIG) that increases the plasma membrane area, thereby enhancing transport capacity in these cells. To better understand WIG formation and roles of INCW2 in the BETC development, we examined wild-type and mn1 mutant developing kernels by cryofixation and electron microscopy. In Mn1 seeds, WIGs developed uniformly in the BETC layer during 7 to 17 d after pollination, and the secretory/endocytic organelles proliferated in the BETCs. Mitochondria accumulated in the vicinity of WIGs, suggesting a functional link between them. In the mn1 BETCs, WIGs were stunted and their endoplasmic reticulum was swollen; Golgi density in the mutant BETCs was 51% of the Mn1 Golgi density. However, the polarized distribution of mitochondria was not affected. INCW2-specific immunogold particles were detected in WIGs, the endoplasmic reticulum, Golgi stacks, and the trans-Golgi network in the Mn1 BETCs, while immunogold particles were extremely rare in the mutant BETCs. Levels of WIG development in the empty pericarp4 mutant was heterogeneous among BETCs, and INCW2 immunogold particles were approximately four times more abundant in the larger WIGs than in the stunted WIGs. These results indicate that polarized secretion is activated during WIG formation and that INCW2 is required for normal development of WIGs to which INCW2 is localized.

The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3, or the profission gene drp-1, are not required for apoptosis activation in C. elegans. However minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9, and downstream of the CED-3 caspase, to promote elimination of mitochondria in dying cells, an event that could facilitate cell death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1’s proapoptotic function but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell death execution downstream of caspase activation.

Modern architecture is guided by the axiom “form follows function,” which emphasizes the need for the shape of a building or an object to reflect its intended function or purpose. Biologists tend to prefer the phrase “form begets function,” because in living organisms, form not only reflects

Coat protein I (COPI) vesicles arise from Golgi cisternae and mediate the recycling of proteins from the Golgi back to the endoplasmic reticulum (ER) and the transport of Golgi resident proteins between cisternae. In vitro studies have produced evidence for two distinct types of COPI vesicles, but the in vivo sites of operation of these vesicles remain to be established. We have used a combination of electron tomography and immunolabeling techniques to examine Golgi stacks and associated vesicles in the cells of the scale-producing alga Scherffelia dubia and Arabidopsis preserved by high-pressure freezing/freeze-substitution methods. Five structurally distinct types of vesicles were distinguished. In Arabidopsis , COPI and COPII vesicle coat proteins as well as vesicle cargo molecules (mannosidase I and sialyltransferase–yellow fluorescent protein) were identified by immunogold labeling. In both organisms, the COPI-type vesicles were further characterized by a combination of six structural criteria: coat architecture, coat thickness, membrane structure, cargo staining, cisternal origin, and spatial distribution. Using this multiparameter structural approach, we can distinguish two types of COPI vesicles, COPIa and COPIb. COPIa vesicles bud exclusively from cis cisternae and occupy the space between cis cisternae and ER export sites, whereas the COPIb vesicles bud exclusively from medial- and trans-Golgi cisternae and are confined to the space around these latter cisternae. We conclude that COPIa vesicle-mediated recycling to the ER occurs only from cis cisternae, that retrograde transport of Golgi resident proteins by COPIb vesicles is limited to medial and trans cisternae, and that diffusion of periGolgi vesicles is restricted.

The BURP domain is a plant-specific protein domain that has been identified in secretory proteins, and some of these are involved in cell wall remodeling. Among Arabidopsis BURP domain proteins, three proteins exhibit strong amino acid similarities with the tomato polygalacturonase 1 beta (PG1β) protein that interacts with a pectin-digesting enzyme. To investigate biological roles of the Arabidopsis PG1β-like proteins (AtPGLs), we generated Arabidopsis lines in which expression of AtPGLs is altered. Among the three AtPGLs, AtPGL3 exhibited highest transcriptional activity throughout all developmental stages. When tissue-specific expression pattern of AtPGL3 was examined, the gene was observed to be active in epidermal cell layers of rosette leaves and in the trichomes. AtPGL triple mutant plants were smaller than wild type plants because cells were smaller in the mutant plants. Interestingly, when we overexpressed AtPGL3 using a 35S promoter, cells in transgenic plants grew larger than those of the wild type, suggesting that AtPGL3 plays a role in cell expansion. A C-terminal GFP fusion protein of AtPGL3 complemented phenotypes of the triple mutant plants and localized to the cell wall. A truncated AtPGL3-GFP fusion protein that lacks the BURP domain failed to rescue the mutant phenotypes even though the GFP protein was targeted to the cell wall, indicating that the BURP domain is required for its effect on cell expansion. Quantitative RT-PCR and immunoblot analyses indicated that 2 α-expansin genes are down-regulated and up-regulated in the triple mutant and overexpressor lines, respectively. Taken together, AtPGL3 is a cell wall protein required for normal cell expansion and the coexpression results suggest that AtPGLs regulate cell wall loosening, in conjunction with α-expansins, to promote cell growth.

Method Twenty female Sprague Dawley rat (SD rat) were randomly divided into two groups according to their bedding types. The 1 group was raised in wood shaving bedding as a control and 2 one was in corncob bedding as an endocrine disrupting agent. Each group was checked daily for the first day of vaginal opening, and their vaginal smears were collected to determine estrous cyclicity after vaginal opening. The interval between vaginal opening and the first normal estrous cycle was recorded for determining sexual maturation. The phases of normal estrous cycles were as follows, proestrus, estrus, metestrus and diestrus. We compared the proportion of normal estrous cycles in each group.

Genetic networks that determine rates of organ initiation and organ size are key regulators of plant architecture. Whereas several genes that influence the timing of lateral organ initiation have been identified, the regulatory pathways in which these genes operate are poorly understood. Here, we identify a class of genes implicated in regulation of the lateral organ initiation rate. Loss-of-function mutations in the MATE transporter encoded by maize (Zea mays) Big embryo 1 (Bige1) cause accelerated leaf and root initiation as well as enlargement of the embryo scutellum. BIGE1 is localized to trans-Golgi, indicating a possible role in secretion of a signaling molecule. Interestingly, phenotypes of bige1 bear striking similarity to cyp78a mutants identified in diverse plant species. We show that a CYP78A gene is upregulated in bige1 mutant embryos, suggesting a role for BIGE1 in feedback regulation of a CYP78A pathway. We demonstrate that accelerated leaf formation and early flowering phenotypes conditioned by mutants of Arabidopsis thaliana BIGE1 orthologs are complemented by maize Bige1, showing that the BIGE1 transporter has a conserved function in regulation of lateral organ initiation in plants. We propose that BIGE1 is required for transport of an intermediate or product associated with the CYP78A pathway.

Introduction: Under sustained pressure overload, myocytes that initially remodel to become short and thick, eventually become elongated and maladaptive with little force for contraction. Mechanisms underlying this transition are largely unknown. We previously demonstrated that 1 week of transverse aortic constriction in wild-type mice reduced cardiac myosin light chain kinase (cMLCK) levels by ~85%. In cultured cardiomyocytes, cMLCK knockdown led to sarcomere disorganization. Germline Mylk3 (encoding cMLCK) knockout mice demonstrated moderate heart failure. Hypothesis: Acute cMLCK reduction is causative for reduced contractility and remodeling of cardiomyocytes during the transition from compensated to decompensated hypertrophy. Methods: To mimic acute cMLCK reduction, adult inducible Mylk3 gene knockout mice, Mylk3flox/flox/merCremer, were generated and compared to 2 control mice (Mylk3flox/flox and Mylk3+/+/merCremer) following tamoxifen injection (50 mg/kg/day, 2 consecutive days). We assessed cardiac function (MRI, echocardiogram) in addition to histopathologic (tissue sectioning, immunostaining, and EM), cellular (cell size and cell shortening and intracellular free Ca2+) and molecular analyses. Results: Reduction of cMLCK proteins was evident at day 4 following tamoxifen-injection in inducible Mylk3 knockout mice. At day 7, inducible Mylk3 knockout but not control mice demonstrated heart failure [%FS, 28.0% in flox/flox (n=6), 27.7% in +/+/merCremer (n=6), 19.8% in flox/flox/merCremer (n=8)]. Analysis of inducible Mylk3 knockout mice revealed unique, severely convoluted cell morphology; isolated cardiomyocytes were elongated and thinner with disorganized sarcomere and reduced contractility (%FS in sarcomere length, control 5.1% vs. inducible knockout 2.4%). Adenovirus encoding cMLCK enhanced organized sarcomere structures; this was not prevented by co-infection of adenovirus unphosphorylatable MLC2v(Ser14/15Ala) mutants incorporated into sarcomere. Conclusion: Results of acute cMLCK reduction in a novel mouse model suggest that cMLCK plays a pivotal role in transition from compensated to decompensated hypertrophy via sarcomere disorganization likely independent of MLC2 phosphorylation.

The delay-insensitive Null Convention Logic (NCL) as one of innovative asynchronous logic design methodologies has many advantages of inherent robustness, power consumption, and easy design reuses. However, transistor-level topologies of conventional NCL gates have weakness of logic speed, area overhead or wire complexity. Therefore, this paper proposes a new NCL gates designed at transistor level for high-speed, low area overhead. A 4×4 multiplier using the proposed NCL gates has been compared to the multiplier using conventional NCL gates in terms of delay, area and energy consumption.