Retinoic acid-driven Hox1 is required in the epidermis for forming the otic/atrial placodes during ascidian metamorphosis

Retinoic acid (RA)-mediated expression of the homeobox gene Hox1 is a hallmark of the chordate central nervous system (CNS). It has been suggested that the RA-Hox1 network also functions in the epidermal ectoderm of chordates. Here, we show that in the urochordate ascidian Ciona intestinalis, RA-Hox1 in the epidermal ectoderm is necessary for formation of the atrial siphon placode (ASP), a structure homologous to the vertebrate otic placode. Loss of Hox1 function resulted in loss of the ASP, which could be rescued by expressing Hox1 in the epidermis. As previous studies showed that RA directly upregulates Hox1 in the epidermis of Ciona larvae, we also examined the role of RA in ASP formation. We showed that abolishment of RA resulted in loss of the ASP, which could be rescued by forced expression of Hox1 in the epidermis. Our results suggest that RA-Hox1 in the epidermal ectoderm played a key role in the acquisition of the otic placode during chordate evolution.     

Genus-specific and phase-dependent effects of nitrate on a sulfate-reducing bacterial community as revealed by dsrB-based DGGE analyses of wastewater reactors

The biogenic production of hydrogen sulfide is a serious problem associated with wastewater treatment. The aim of this study was to investigate the inhibitory effect of nitrate on the dynamics of sulfate-reducing bacteria (SRB) community in a laboratory-scale wastewater reactor, originating from a denitrifying plant using activated sludge. For this purpose, denaturing gradient gel electrophoresis (DGGE) analysis targeting the dsrB (dissimilatory sulfite reductase) gene was used in combination with chemical analyses and measurement of oxidation and reduction potential (ORP). The reactors were initially dosed with 1.0 and 4.0 g/L potassium nitrate and anaerobically incubated for 490 h. Addition of 4.0 g/L nitrate to the reactor was associated with a prolonged inhibition (over 300 h, i.e., 12.5 days) of sulfate reduction and this was consistent with a rapid decrease in ORP associated with nitrate depletion. The DGGE analysis revealed that nitrate addition remarkably attenuated a distinct group of dsrB related to Desulfovibrio, whereas other dsrB groups were not influenced. Furthermore, another sulfate reduction by Syntrophobacter in the later stages of the incubation period occurred in both reactors (regardless of the nitrate concentration), suggesting that different SRB groups are associated with sulfate reduction at different stages of the wastewater treatment process.     

ZG16p, an animal homolog of β-prism fold plant lectins, interacts with heparan sulfate proteoglycans in pancreatic zymogen granules

ZG16p is a soluble 16?kDa pancreatic protein having structural similarities with plant β-prism fold lectins such as the banana lectin BanLec and the jackfruit lectin jacalin. ZG16p is postulated to be involved in the formation of zymogen granules by interacting with proteoglycans (PGs) localized in pancreatic exocrine granule membranes, but direct evidence was lacking. We characterized the structural properties of rat pancreatic zymogen granule PGs and examined their interaction with ZG16p. Structural analysis of the glycosaminoglycans (GAGs) showed that rat pancreatic zymogen granule PGs have heparan sulfate chains with a unique property, a high degree of sulfation (ΔUA-GlcNAc:ΔUA-GlcNS:ΔUA-GlcNAc6S:ΔUA-GlcNS6S:ΔUA2S-GlcNS:ΔUA2S-GlcNS6S, 27.9:16.6:5.7:22.5:6.2:21.1). After heparin lyase II digestion, the core proteins derived from the PGs were detected at molecular weights of 66,000 and 35,000-40,000. An overlay binding assay revealed that ZG16p binds specifically to heparan sulfate PGs by recognizing their GAG chains. Affinity chromatography demonstrated that ZG16p binds most strongly to heparin among the zymogen granule proteins. Site-directed mutational analysis revealed that the basic amino acid residues located in two putative carbohydrate-binding sites (CBSs) of ZG16p, which were found in association with the crystal structure of BanLec, are responsible for the recognition of heparin. These observations suggest that ZG16p is the primary binding partner of the granule heparan sulfate PGs. ZG16p may cross-link the granule heparan sulfate chains via two CBSs and facilitate the formation of a submembranous matrix, a sorting platform for enzyme proteins on the luminal side of the zymogen granule membrane.     

The wavy growth 3 E3 ligase family controls the gravitropic response in Arabidopsis roots

Regulation of the root growth pattern is an important control mechanism during plant growth and propagation. To better understand alterations in root growth direction in response to environmental stimuli, we have characterized an Arabidopsis thaliana mutant, wavy growth 3 (wav3), whose roots show a short‐pitch pattern of wavy growth on inclined agar medium. The wav3 mutant shows a greater curvature of root bending in response to gravity, but a smaller curvature in response to light, suggesting that it is a root gravitropism‐enhancing mutation. This wav3 phenotype also suggests that enhancement of the gravitropic response in roots strengthens root tip impedance after contact with the agar surface and/or causes an increase in subsequent root bending in response to obstacle‐touching stimulus in these mutants. WAV3 encodes a protein with a RING finger domain, and is mainly expressed in root tips. RING‐containing proteins often function as an E3 ubiquitin ligase, and the WAV3 protein shows such activity in vitro. There are three genes homologous to WAV3 in the Arabidopsis genome [EMBRYO SAC DEVELOPMENT ARREST 40 (EDA40), WAVH1 and WAVH2 ], and wav3 wavh1 wavh2 triple mutants show marked root gravitropism abnormalities. This genetic study indicates that WAV3 functions positively rather than negatively in root gravitropism, and that enhancement of the gravitropic response in wav3 roots is dependent upon the function of WAVH2 in the absence of WAV3. Hence, our results demonstrate that the WAV3 family of proteins are E3 ligases that are required for root gravitropism in Arabidopsis.     

Design and synthesis of estrogen receptor degradation inducer based on a protein knockdown strategy

We designed and synthesized estrogen receptor (ER) degradation inducers 5, 6, and 7, which crosslink the ER and the cellular inhibitor of apoptosis protein 1 (cIAP1). Compounds 5, 6, and 7 induced cIAP1-mediated ubiquitylation of ERα resulting in its proteasomal degradation.     

Skin TRM mediates distributed border patrol

Tissue-resident memory T cells (T_RM) are a new subset of memory cells that have been associated with enhanced protective immunity for their tissue of residence. A recent study by Jiang et al. sheds light on the migration behavior of T_RM in both infected and unifected skin, and their ability to provide protection against re-infection even within a previously uninfected skin tissue.T cell memory is a hallmark of adaptive immunity. Two broad subsets, effector memory (T_EM) and central memory (T_CM) have been defined largely by their migratory capacities¹. CD62L^lo CCR7^lo T_EM circulate through peripheral tissues, while CD62L^hi CCR7^hi T_CM home to secondary lymphoid tissues^1,2. More recently, another distinct subset, tissue-resident memory T cells (T_RM) have been described. T_RM have been found in several tissues, including the skin, gut, lung and brain^3,4,5,6. Once established, T_RM are maintained independently of circulating T_EM and T_CM and their presence correlates with superior protection against local viral challenge^3,5,7. However, the exact role of T_RM vs. recruited circulating memory T cells in controlling viral replication is unclear. Furthermore, due to the confined patrolling area of T_RM, the capacity of T_RM to mediate protection against infection at distal sites is unknown.A recent paper by Xiaodong Jiang et al.⁸ investigated these issues in a skin infection model using adoptively transferred TCR transgenic CD8 T cells that recognize the ovalbumin protein (OT-I) and vaccinia virus expressing ovalbumin (VACV). They first characterized the requirements necessary for establishment of T_RM in the skin, and found that unlike other epithelial tissues such as the female genital tract⁹, entry of CD8 T cells into the skin was independent of CD4 T cells and IFNγ. However, T cell expression of ligands for E- and P-selectin, two adhesion molecules that are expressed constitutively and are upregulated on inflamed endothelial cells of skin blood vessels, was necessary for seeding of the skin with OT-I T cells. These data parallel the requirement for circulating T_EM CD4 T cell entry into inflamed skin¹⁰. Furthermore, in agreement with previous studies using a different virus, herpes simplex virus type 1 (HSV-1)¹⁰, the authors found that after VACV infection, while CD8 T cells were retained within the skin, CD4 T cells were not.The differential expression of homing molecules such as CD62L and CCR7 on subsets of memory T cells indicates their preferential migration to lymphoid or non-lymphoid tissues during homeostasis. However, studies using parabiotic mice have shown that memory T cells that reside in peripheral tissues such as the brain and gut do not participate in homeostatic circulation; rather, they appear to be maintained independently of circulating memory T cells⁷. By surgically joining VACV-immunized mice with unimmunized partners, the authors found that T_CM populations in tissues such as the spleen and lymph node equilibrated between the parabionts, while T_RM populations in the skin did not. This indicated that like the brain and gut, skin T_RM populations were excluded from the pool of circulating memory T cells. Additionally, this parabiosis study indicates that like the gut, the skin must be seeded relatively early in the T cell response in order to establish a T_RM population¹¹. Jiang et al. also note that T_RM populations, while fully capable of effector function like their T_EM and T_CM counterparts, can be further distinguished by their differential expression of surface molecules, such as the lack of markers CD127 and CD122, and high expression of CD69 and CD103.Other viral infection models such as HSV-1 have suggested that local memory CD8 T cells can be directly activated in peripheral tissues¹² and participate in controlling viral replication³. However, it has been difficult to separate the relative contributions of recruited vs. resident memory T cells in mediating protection. To address this issue, Jiang et al. separated immunized and unimmunized parabiotic pairs once the T_CM compartment had equilibrated and gave the mice a skin challenge with VACV. The challenged mice were then treated with FTY720, an S1P₁ modulator that sequesters lymphocytes in secondary lymphoid organs, thus preventing the recruitment of circulating T cells to the site of infection¹³. They discovered that T_RM were superior to T_CM in controlling a secondary infection, as the immunized parabiont controlled viral replication significantly better than the unimmunized partner. Furthermore, they found no difference in the viral titers of the untreated and FTY720-treated immunized parabionts. This suggests that in the VACV infection model, T_RM are sufficient to mediate protection against reinfection, even in the absence of any input from circulating T cells.When a host is immunized through the skin by methods such as scarification, previous studies have implied that the T_RM will remain local and patrol an area of tissue that is directly adjacent to the site of infection. For example, in the HSV-1 infection model, it has been shown that when mice are infected on one flank, that flank is better protected against subsequent challenge than the contralateral, unimmunized flank³. Migration of T cells into tissues such as the skin and gut requires the expression of particular homing markers that are most strongly upregulated when T cells are activated via immunization routes that involve the target tissue¹⁴. Ligands to some of the skin homing markers upregulated by activated T cells are constitutively expressed in blood vessel endothelium, albeit at a lower level than inflamed tissue. This raises questions as to whether T cells activated through local skin infection can accumulate in distal unimmunized skin, and whether these T cells can provide preferential protection compared to T cells activated through other immunization routes. To address these questions, Jiang et al. immunized mice on one ear using VACV and then examined the accumulation of T cells in the other ear. They found that activated OT-I cells were capable of migrating to the uninfected ear, and that the number of cells found in the uninfected ear could be boosted by immunizing mice multiple times through various skin routes. Furthermore, when mice were challenged in the previously uninfected ear, viral titers were significantly lower in mice immunized via skin scarification compared to mice immunized intraperitoneally (Figure 1). Together with the T cell number data, the authors suggest that T_RM in the skin can be established at sites distal to the initial infection, and that these T_RM may play a role in providing widespread protection against reinfection.Open in a separate windowFigure 1Skin epidermal infection by virus leads to T_RM residency and local or global antiviral protection. When the host is infected via skin scarification, the resulting inflammation increases expression of chemoattractants and adhesion molecules such as P- and E-selectin on the endothelium of the blood vessels. T cells activated through this route upregulate skin homing markers such as P- and E-selectin ligands. Collectively, these events lead to the recruitment of circulating, antigen-specific effector/effector memory (T_EM) CD8 T cells into the infected tissue. The recruited CD8 T cells establish a pool of resident memory T cells (T_RM) that can provide protection against future skin infections. Jiang et al. show that upon multiple skin immunizations, circulating effector/T_EM CD8 T cells also accumulate at uninfected sites. These CD8 T cells can then form a T_RM population that is immunologically protective. However, the establishment of T_RM in uninfected skin is dependent on the route of immunization, as skin-independent methods such as intraperitoneal injection did not lead to protection against a secondary skin challenge.In summary, the study by Jiang and colleagues show that T_RM established in the skin are maintained independently of circulating T_CM. Furthermore, they demonstrate that T_RM alone are sufficient to protect against pathogens such as vaccinia virus, although the role that different subsets of memory T cells play in mediating immunity will likely vary depending on the infectious agent^10,15. Importantly, Jiang et al. show that through prime-boosting, a substantial T_RM population can be established at distal skin sites absent of infection or inflammation. This suggests that the frontline defense that T_RM can provide in barrier tissues does not have to be limited by the location of immunization (Figure 1). In future studies, it will be important to continue to gain an understanding of T_RM, including the factors that lead to their establishment and maintenance. Despite the variety of tissues in which T_RM have been described, T_RM appear to share certain phenotypic and functional characteristics, such as the expression of surface markers CD103 and CD69, as well as the ability to produce effector molecules such as IFNγ and granzyme B^5,6,8,16. Thus, it will be interesting to determine whether there is a common pathway by which T_RM differentiate, or whether each tissue has its own individual requirements. Beyond establishment, the mechanism that mediates the long-term survival of T_RM is unknown. Previous studies have shown that T_RM from tissues such as the brain do not survive well when taken out of their tissue of residence⁶. This suggests that the signals that sustain T_RM may be locally provided and may even be tissue specific, different from the cytokines that maintain systemic T_CM and T_EM. Understanding how T_RM are established and the roles that they play in protecting against invading pathogens will be critical in designing vaccines and immunotherapies that target specific tissues.     

Adipocyte hypertrophy is associated with lysosomal permeability both in vivo and in vitro: role in adipose tissue inflammation

Obesity in both humans and rodents is characterized by adipocyte hypertrophy and the presence of death adipocytes surrounded by macrophages forming "crown-like structures." However, the biochemical pathways involved in triggering adipocyte death as well as the role of death adipocytes in adipose tissue remodeling and macrophage infiltration remain poorly understood. We now show that induction of adipocyte hypertrophy by incubation of mature adipocytes with saturated fatty acids results in lysosomal destabilization and cathepsin B (ctsb), a key lysosomal cysteine protease, activation and redistribution into the cytosol. ctsb activation was required for the lysosomal permeabilization, and its inhibition protected cells against mitochondrial dysfunction. With the use of a dietary murine model of obesity, ctsb activation was detected in adipose tissue of these mice. This is an early event during weight gain that correlates with the presence of death adipocytes, and precedes macrophage infiltration of adipose tissue. Moreover, ctsb-deficient mice showed decreased lysosomal permeabilization in adipocytes and were protected against adipocyte cell death and macrophage infiltration to adipose tissue independent of body weight. These data strongly suggest that ctsb activation and lysosomal permeabilization in adipocytes are key initial events that contribute to the adipocyte cell death and macrophage infiltration into adipose tissue associated with obesity. Inhibition of ctsb activation may be a new therapeutic strategy for the treatment of obesity-associated metabolic complications.     

Mediator subunits MED1 and MED24 cooperatively contribute to pubertal mammary gland development and growth of breast carcinoma cells

The Mediator subunit MED1 is essential for mammary gland development and lactation, whose contribution through direct interaction with estrogen receptors (ERs) is restricted to involvement in pubertal mammary gland development and luminal cell differentiation. Here, we provide evidence that the MED24-containing submodule of Mediator functionally communicates specifically with MED1 in pubertal mammary gland development. Mammary glands from MED1/MED24 double heterozygous knockout mice showed profound retardation in ductal branching during puberty, while single haploinsufficient glands developed normally. DNA synthesis of both luminal and basal cells were impaired in double mutant mice, and the expression of ER-targeted genes encoding E2F1 and cyclin D1, which promote progression through the G(1)/S phase of the cell cycle, was attenuated. Luciferase reporter assays employing double mutant mouse embryonic fibroblasts showed selective impairment in ER functions. Various breast carcinoma cell lines expressed abundant amounts of MED1, MED24, and MED30, and attenuated expression of MED1 and MED24 in breast carcinoma cells led to attenuated DNA synthesis and growth. These results indicate functional communications between the MED1 subunit and the MED24-containing submodule that mediate estrogen receptor functions and growth of both normal mammary epithelial cells and breast carcinoma cells.     

Microflorae of aquatic moss pillars in a freshwater lake, East Antarctica, based on fatty acid and 16S rRNA gene analyses

Aquatic mosses in the genera Bryum and Leptobryum form unique tower-like ??moss pillars?? underwater in some Antarctic lakes, in association with algae and cyanobacteria. These are communities with a two-layer structure comprising an oxidative exterior and reductive interior. Although habitats and photosynthetic properties of moss pillars have been reported, microfloral composition of the two-layer structure has not been described. Here we report fatty acid analysis of one moss pillar and molecular phylogenetic analysis, based on the 16S rRNA gene, of this and one other moss pillar. Cluster analysis of the phospholipid fatty acid composition showed three groups corresponding to the exterior, upper interior, and lower interior of the pillar. This suggested that species composition differed by section, with the exterior dominated by photosynthetic organisms such as mosses, algae, and cyanobacteria, the upper interior primarily containing gram-positive bacteria and anaerobic sulfate-reducing bacteria, and the lower interior dominated by gram-negative bacteria. Molecular phylogenetic analysis revealed that Proteobacteria dominate the moss pillar as a whole; cyanobacteria were found on the exterior and the gram-positive obligate anaerobe Clostridium in the interior, while gram-positive sulfate-reducing bacteria were present in the lowest part of the interior. Nitrogen-fixing bacteria and denitrifying bacteria were found in all sections. Thus, fatty acid analysis and genetic analysis showed similar patterns. These findings suggest that microorganisms of different phylogenetic groups inhabit different sections of a single moss pillar and form a microbial community that performs biogeochemical cycling to establish and maintain a structure in an oxidation?Creduction gradient between exterior and interior.