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Engineered bacteria can be leveraged for in situ tumor vaccinations with their tumor-targeting ability and adjuvanticity. Engineering strategies like chemical modification, nanotechnology, and genetic engineering improve their safety and efficacy.

Injectable poly- l -lactic acid (PLLA) is a new type of biodegradable dermal filler that has been utilized for soft tissue filling. However, there is no convenient and reliable method to assess the long-term safety of PLLA filler.To assess the long-term safety of PLLA injection into nasolabial folds by high-frequency ultrasound and to select the ultrasonic probes with the most appropriate frequency.After a 30-month PLLA injection into the deep dermis of the nasolabial fold, subjects were examined by high-frequency ultrasound with the 20 MHz and 50 MHz probes.Twenty subjects with nasolabial fold contour deficiency were enrolled in this study. After a 30-month PLLA injection in nasolabial folds, PLLA degraded entirely in 16 subjects (16/20, 80%), and abnormal echo in the skin was observed in 4 subjects (4/20, 20%) caused by undegraded PLLA microparticles, PLLA microparticles deposition, fibrous nodules, and granuloma. The 20-MHz probe is more appropriate than the 50-MHz probe for evaluating the adverse effects of PLLA injection.High-frequency ultrasound is a rapid, reliable, and noninvasive method to monitor the degradation condition of PLLA and the formation of papules and nodules associated with PLLA injection.

Muscle-specific tyrosine kinase myasthenia gravis (MuSK MG) is an autoimmune disease that causes life-threatening muscle weakness due to anti-MuSK autoantibodies that disrupt neuromuscular junction signaling. To avoid chronic immunosuppression from current therapies, we engineered T cells to express a MuSK chimeric autoantibody receptor with CD137-CD3ζ signaling domains (MuSK-CAART) for precision targeting of B cells expressing anti-MuSK autoantibodies. MuSK-CAART demonstrated similar efficacy as anti-CD19 chimeric antigen receptor T cells for depletion of anti-MuSK B cells and retained cytolytic activity in the presence of soluble anti-MuSK antibodies. In an experimental autoimmune MG mouse model, MuSK-CAART reduced anti-MuSK IgG without decreasing B cells or total IgG levels, reflecting MuSK-specific B cell depletion. Specific off-target interactions of MuSK-CAART were not identified in vivo, in primary human cell screens or by high-throughput human membrane proteome array. These data contributed to an investigational new drug application and phase 1 clinical study design for MuSK-CAART for the treatment of MuSK autoantibody-positive MG.

Chinese biochemical engineering is committed to supporting the chemical and food industries, to advance science and technology frontiers, and to meet major demands of Chinese society and national economic development. This paper reviews the development of biochemical engineering, strategic deployment of these technologies by the government, industrial demand, research progress, and breakthroughs in key technologies in China. Furthermore, the outlook for future developments in biochemical engineering in China is also discussed.

To systematically review the efficacy of photodynamic therapy (PDT) in the treatment of rosacea.PubMed, Embase, and Cochrane Library databases were searched for articles published by February 5, 2022, using "photodynamic therapy" and "rosacea" as the keywords.Nine studies were included in the review. The number of patients varied from 1 to 30 in each study, with ages ranging from 18 to 76 years. Methyl aminolevulinate (MAL) and aminolevulinic acid (ALA) were used as the photosensitizer, and red light, blue light, intense pulsed light (IPL), long-pulsed dye laser (LPDL), pulsed dye laser (PDL), and tungsten lamp were used as the light or laser source. The follow-up time ranged from one month to 25 months. Most of the studies showed a satisfactory clinical response, and the side effects were tolerant and temporary.Current studies have provided preliminary evidence that PDT is an efficient and safe therapy in treating rosacea. However, rigorous randomized control trials (RCTs) with a larger sample size and longer follow-up time are warranted to verify the curative effects of PDT in treating rosacea and explore the most appropriate treatment schedule.

BACKGROUND: Preliminary observation in clinical practice showed that subjective neurocognitive complaints are relatively common in bullous pemphigoid (BP) patients. Yet, little has been done to investigate the neurocognitive status in BP. METHODS: This is a multicenter observational case-control study comprised of 61 BP patients and 65 matched control subjects from 3 medical centers in China from 2014 to 2019. To evaluate the cognitive function between BP patients and matched controls, all the subjects finished the mini-mental state examination (MMSE) and montreal cognitive assessment (MoCA). RESULTS: The overall scores were significantly lower in BP than in controls (P(MoCA)

Desmoglein 3 chimeric autoantibody receptor T cells (DSG3-CAART) expressing the pemphigus vulgaris (PV) autoantigen DSG3 fused to CD137-CD3ζ signaling domains, represent a precision cellular immunotherapy approach for antigen-specific B cell depletion. Here, we present definitive preclinical studies enabling a first-in-human trial of DSG3-CAART for mucosal PV. DSG3-CAART specifically lysed human anti-DSG3 B cells from PV patients and demonstrated activity consistent with a threshold dose in vivo, resulting in decreased target cell burden, decreased serum and tissue-bound autoantibodies, and increased DSG3-CAART engraftment. In a PV active immune model with physiologic anti-DSG3 IgG levels, DSG3-CAART inhibited antibody responses against pathogenic DSG3 epitopes and autoantibody binding to epithelial tissues, leading to clinical and histologic resolution of blisters. DSG3 autoantibodies stimulated DSG3-CAART IFN-γ secretion and homotypic clustering, consistent with an activated phenotype. Toxicology screens using primary human cells and high-throughput membrane proteome arrays did not identify off-target cytotoxic interactions. These preclinical data guided the trial design for DSG3-CAART and may help inform CAART preclinical development for other antibody-mediated diseases.

Objectives: The clinical outcome of bullous pemphigoid appears worse in patients with infectious complications, and assessment of the prevalence and risk factors of infectious complications could be necessary to plan preventative strategies and to instruct the treatment plans. We sought to determine the risk factors of infection and compare associated factors in inpatients and outpatients with different system infections.Design: This is a single-centered retrospective study on the medical records of 252 patients from 2010 to 2018 at the dermatology department, Peking Union Medical College. Medical profiles of medical history, diagnosis, infectious complications, and treatment plans were analyzed. The associated factors were compared between the subgroups, including inpatients and outpatients, different body sites of infection.Results: Of the total 252 patients with bullous pemphigoid (BP), 81 patients (81/252, 32.1%) had infectious complications. Forty-eight patients died from pulmonary infections (11/48, 22.9%), cardiovascular diseases (6/48, 12.5%), and other diseases. Infections were most frequently found in skin/mucosa (44/252, 17.5%), respiratory system (32/252, 12.7%), and blood (10/252, 4.0%). On multivariate analysis, risk factors of infections in BP were maximal control dose of corticosteroids (OR 2.539, 95% CI 1.456–4.430, p = 0.001), low serum albumin level (OR 2.557, 95% CI 1.283, 5.092, p = 0.007), hospitalization (OR 4.025, 95% CI 2.289, 7.079, p < 0.001), comorbidities including respiratory disease (OR 4.060, 95% CI, 1.861, 8.858, p < 0.001), eye disease (OR 4.431, 95% CI 1.864, 10.532, p < 0.001), and diabetes (OR 2.667, 95% CI 1.437, 4.949, p = 0.002). The rate of infection was significantly higher in inpatients compared to that in outpatients (54.0 vs. 20.6%, p < 0.001), with diverse risk factors. Mucocutaneous infections were associated with a maximal control dose of corticosteroid and other dermatoses. Respiratory infections were related to respiratory disease and old age, and hematologic infection was associated with low serum hemoglobin levels and mucosal involvement of BP. Both of them were associated with mucosal involvement of BP and high titer anti-BP180 antibody.Conclusions: Infectious complications of bullous pemphigoid are common and are associated with mucosal involvement of BP, more comorbidities, the higher dose of corticosteroids, and the lower level of serum albumin.

Bullous pemphigoid (BP) is a senile autoimmune blistering disease with autoantibodies against the basement membrane. Less than 20 cases of localized BP in young adults have been reported and the understanding of localized BP is very limited. An unusual location of localized BP is here described. A 30-year-old woman presented with a 4-month history of itchy erythema on her trunk. The lesion, well-demarcated erythema and maculopapules in bra's shape, had been misdiagnosed as contact dermatitis. Laboratory finding was notable for serum autoantibodies to BP antigen 180 (BP180). Histopathological examination revealed a subepidermal blister with eosinophils and neutrophils infiltration. Salt-split indirect immunofluorescence revealed linear deposition of IgG at the dermoepidermal junction. After treating her with minocycline 200 mg and nicotinamide 1,500 mg per day, all lesions resolved within 1 month. Localized BP is usually misdiagnosed. It starts from various triggers and has a more benign disease course. It should be emphasized that a long-term follow-up of patients with localized BP may be important for management of the chronic disease, given a relatively high risk of developing generalized BP.

Paramyxovirus establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Of the various pattern recognition receptors in the host, the cytosolic RNA helicases interact with viral RNA to activate the mitochondrial antiviral signaling protein (MAVS) and subsequent cellular interferon (IFN) response. On the other hand, viruses explore multiple strategies to resist host immunity. In this study, we found that Newcastle disease virus (NDV) infection induced MAVS degradation. Further analysis showed that NDV V protein degraded MAVS through the ubiquitin-proteasome pathway to inhibit IFN-β production. Moreover, NDV V protein led to proteasomal degradation of MAVS through Lys362 and Lys461 ubiquitin to prevent IFN production. Further studies showed that NDV V protein recruited E3 ubiquitin ligase RNF5 to polyubiquitinate and degrade MAVS. Compared with levels for wild-type NDV infection, V-deficient NDV induced attenuated MAVS degradation and enhanced IFN-β production at the late stage of infection. Several other paramyxovirus V proteins showed activities of degrading MAVS and blocking IFN production similar to those of NDV V protein. The present study revealed a novel role of NDV V protein in targeting MAVS to inhibit cellular IFN production, which reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit. IMPORTANCE Host anti-RNA virus innate immunity relies mainly on the recognition by retinoic acid-inducible gene I and melanoma differentiation-associated protein 5 and subsequently initiates downstream signaling through interaction with MAVS. On the other hand, viruses have developed various strategies to counteract MAVS-mediated signaling. The mechanism for paramyxoviruses regulating MAVS to benefit their infection remains unknown. In this article, we demonstrate that the V proteins of NDV and several other paramyxoviruses target MAVS for ubiquitin-mediated degradation through E3 ubiquitin ligase RING-finger protein 5 (RNF5). MAVS degradation leads to the inhibition of the downstream IFN-β pathway and therefore benefits virus proliferation. Our study reveals a novel mechanism of NDV evading host innate immunity and provides insight into the therapeutic strategies for the control of paramyxovirus infection.

BackgroundCurrent evidence has revealed a significant association between bullous pemphigoid (BP) and neurological diseases (ND), including stroke, but the incidence of BP autoantibodies in patients with stroke has not previously been investigated.ObjectiveOur study aims to assess BP antigen-specific antibodies in stroke patients.Methods100 patients with stroke and 100 healthy controls were randomly selected to measure anti-BP180/230 IgG autoantibodies by enzyme-linked immunosorbent assay (ELISA), salt split indirect immunofluorescence (IIF) and immunoblotting against human cutaneous BP180 and BP180-NC16A.ResultsAnti-BP180 autoantibodies were found in 14(14.0%) patients with stroke and 5(5.0 %) of controls by ELISA (pLimitationsLongitudinal changes in antibody titers and long-term clinical outcome for a long duration were not fully investigated.ConclusionDevelopment of anti-BP180 autoantibodies occur at a higher frequency after stroke, suggesting BP180 as a shared autoantigen in stroke with BP and providing novel insights into BP pathogenesis in aging.

Cilia-related proteins are believed to be involved in a broad range of cellular processes. Retinitis pigmentosa GTPase regulator interacting protein 1-like (RPGRIP1L) is a ciliary protein required for ciliogenesis in many cell types, including epidermal keratinocytes. Here we report that RPGRIP1L is also involved in the maintenance of desmosomal junctions between keratinocytes. Genetically disrupting the Rpgrip1l gene in mice caused intraepidermal blistering, primarily between basal and suprabasal keratinocytes. This blistering phenotype was associated with aberrant expression patterns of desmosomal proteins, impaired desmosome ultrastructure, and compromised cell-cell adhesion in vivo and in vitro. We found that disrupting the RPGRIP1L gene in HaCaT cells, which do not form primary cilia, resulted in mislocalization of desmosomal proteins to the cytoplasm, suggesting a cilia-independent function of RPGRIP1L. Mechanistically, we found that RPGRIP1L regulates the endocytosis of desmogleins such that RPGRIP1L-knockdown not only induced spontaneous desmoglein endocytosis, as determined by AK23 labeling and biotinylation assays, but also exacerbated EGTA- or pemphigus vulgaris IgG-induced desmoglein endocytosis. Accordingly, inhibiting endocytosis with dynasore or sucrose rescued these desmosomal phenotypes. Biotinylation assays on cell surface proteins not only reinforced the role of RPGRIP1L in desmoglein endocytosis, but also suggested that RPGRIP1L may be more broadly involved in endocytosis. Thus, data obtained from this study advanced our understanding of the biological functions of RPGRIP1L by identifying its role in the cellular endocytic pathway., Author summary The desmosome is a type of intercellular junction, essential for cells to adhere to one another. Abnormalities in desmosomes can cause disorders in the hair, skin, and heart, some of which are severe or even fatal. Here, we discovered that RPGRIP1L, a protein known to regulate cilia formation and function, is essential for stabilizing desmosomes of skin keratinocytes. Specifically, suppressing the Rpgrip1l gene in mice or in keratinocytes disrupted the ultrastructure of desmosomes, and compromised cell-cell adhesion in vivo and in vitro. We found that knocking down RPGRIP1L in keratinocytes aberrantly accelerated the internalization of cell membrane desmogleins, key desmosomal cadherins. Inhibiting endocytosis effectively rescued these phenotypes. Biotinylation assays confirmed that desmogleins are likely the primary targets of RPGRIP1L. Interestingly, membrane proteins that are not directly associated with the desmosomes were also found to be internalized in RPGRIP1L-knockdown cells, raising the possibility that RPGRIP1L might regulate endocytosis more broadly. Findings from this study not only identified RPGRIP1L as a regulator of the desmosomes, but also expanded our understanding of cilia-related proteins in the formation of the desmosomal junctions.

Chimeric autoantibody receptors (CAARs) direct T cells to kill autoantigen-specific B cells. We previously established proof-of-concept for the CAAR approach using gene-engineered T cells expressing CAARs comprising DSG3, the autoantigen in the autoimmune blistering disease mucosal pemphigus vulgaris, fused to CD137-CD3ζ signaling domains. Here, we present results of preclinical studies that supported the DSG3-CAART investigational new drug application. DSG3-CAART demonstrated activity related to dose in a passive transfer polyclonal anti-DSG3 hybridoma mouse model of mucosal pemphigus vulgaris, resulting in reversal of the rising serum anti-DSG3 antibody titers, target cell burden, and IgG deposition in epithelial tissues, as well as increased DSG3-CAART engraftment relative to dose. We further evaluated DSG3-CAART efficacy in a novel exploratory active immune mouse model with physiologic levels of circulating anti-DSG3 IgG, which demonstrated decreased antibody responses against pathogenic DSG3 epitopes and autoantibody binding to epithelial tissues, leading to clinical and histologic resolution of blisters. High-throughput screening of a membrane protein array with soluble DSG3 CAAR did not identify verifiable off-target toxicities. These preclinical studies have enabled a first-in-human clinical trial of DSG3-CAART in mucosal pemphigus vulgaris and provide a foundation that may inform the preclinical development of future CAAR T cell therapies for antigen-specific B cell depletion in other autoantibody-mediated diseases.

Background: Pemphigus vulgaris (PV) is a potentially life-threatening mucocutaneous autoimmune blistering disease characterized by suprabasal acantholysis, causing painful mucocutaneous blisters and erosions. Current mainstay therapy for pemphigus is systemic corticosteroids in combination with or without immunosuppressive adjuvants, which may cause severe adverse effects and seriously impact on the quality of life in pemphigus patients. The objective of this study was to evaluate the efficacy and safety of thalidomide therapy in patients with PV. Methods: This study examined six PV patients from June 5, 2017, to November 11, 2018, in the dermatology department of Peking Union Medical College Hospital. Treatment with thalidomide was applied at a dose of 50–100 mg/day for disease control. Results: The mean age of the six patients (two male and four female patients) at the time of thalidomide therapy initiation was 50.2 years (range: 38–67 years), and the total duration of follow-up after thalidomide therapy was 13.2 months (range: 5–25 months). All patients responded favorably to thalidomide treatment, and three patients showed a dramatic reduction in anti-Dsg3 autoantibodies in the serologic examinations within 1 year. Five patients were found to have mucosal involvement. Mild adverse effects were noted in three patients, which could be managed after the application of symptomatic treatment and did not interfere with the pemphigus therapy. Conclusion: These results demonstrate that thalidomide could be an effective and safe option for PV patients, especially those who are concerned about steroid-induced severe complications, and have mucosal diseases.

Mitochondrial antiviral-signaling protein (MAVS) is an essential adaptor protein in retinoic acid-inducible gene I (RIG-I)-mediated antiviral innate immunity in mammals. In this study, the goose MAVS gene (goMAVS) was identified. The 2019 bp-long goMAVS exhibits 96.2% amino acid similarity compared to the predicted goMAVS. Quantitative real-time polymerase chain reactions showed that goMAVS mRNA was widely expressed in different tissues. The overexpression of goMAVS in goose embryo fibroblast cells up-regulated the mRNA levels of goose interferon-stimulated genes. We concluded that MAVS mediates the activation of type I interferon (IFN) pathway in a species-specific manner. We further demonstrated that a CARD-like domain, transmembrane domain and two previously unidentified domains of goMAVS were essential for the activation of type I IFN pathway. GoMAVS inhibited Newcastle disease virus replication by activating type I IFN pathways, especially at the early stages of infection. Finally, the interaction between goMAVS and goose RIG-I was confirmed. The CARD domain of goMAVS plays a vital role in the interaction. Together, we identified goMAVS as a goRIG-I interactive protein and concluded that goMAVS is involved in the activation of type I IFN pathways in goose cells.

Lineage analysis of autoreactive B cells can reveal the origins of autoimmunity. In the autoimmune disease pemphigus vulgaris (PV), desmoglein 3 (DSG3) and DSG1 autoantibodies are predominantly of the IgG4 subclass and less frequently of IgG1 and IgA subclasses, prompting us to investigate whether anti-DSG IgG4 B cells share lineages with IgG1, IgA1, and IgA2. Combining subclass-specific B cell deep sequencing with high-throughput antibody screening, we identified 80 DSG-reactive lineages from 4 PV patients. Most anti-DSG IgG4 B cells lacked clonal relationships to other subclasses and preferentially targeted DSG adhesion domains, whereas anti-DSG IgA frequently evolved from or to other subclasses and recognized a broader range of epitopes. Our findings suggest that anti-DSG IgG4 B cells predominantly evolve independently or diverge early from other subclasses and that IgA is most often not the origin of IgG autoreactivity in PV. These data provide insight into how autoreactivity diversifies across B cell subclasses.

Background Methazolamide is used to lower intraocular pressure in patients with glaucoma. Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) associated with methazolamide treatment have been diagnosed in Korean, Japanese, and Japanese-American patients. According to recent research, the human leukocyte antigen (HLA) allele HLA-B*59:01 is strongly linked to SJS/TEN associated with methazolamide treatment. Objective A patient of Chinese–Korean ethnicity was diagnosed with TEN associated with methazolamide treatment. The purpose of this study was to examine the potential genetic basis of this disease. Methods A polymerase chain reaction sequence-specific primer (PCR-SSP) typing system was used to genotype this patient's peripheral blood DNA for HLA-B*59. Results The genotype HLA-B*59:01 was detected in the patient. Conclusions This study demonstrates the genotype of HLA-B*59:01 in a patient with TEN associated with methazolamide treatment and thus supports the possible correlation between genetic background and methazolamide-associated SJS/TEN.

Pemphigus vulgaris (PV) is an autoimmune blistering disease characterized by autoantibodies to the keratinocyte adhesion protein desmoglein 3 (Dsg3). Previous studies suggest that PV pathogenesis involves p38 mitogen-activated protein kinase-dependent and -independent pathways. However, p38 is a difficult protein to study and therapeutically target because it has four isoforms and multiple downstream effectors. In this study, we identify MAPKAP (mitogen-activated protein kinase-activated protein) kinase 2 (MK2) as a downstream effector of p38 signaling in PV and describe MK2-dependent and -independent mechanisms of blister formation using passive transfer of human anti-Dsg IgG4 mAbs to neonatal mice. In human keratinocytes, PV mAbs activate MK2 in a dose-dependent manner. MK2 is also activated in human pemphigus skin blisters, causing translocation of MK2 from the nucleus to the cytosol. Small-molecule inhibition of MK2 and silencing of MK2 expression block PV mAb-induced Dsg3 endocytosis in human keratinocytes. In addition, small-molecule inhibition and genetic deletion of p38α and MK2 inhibit spontaneous but not induced suprabasal blisters by PV mAbs in mouse passive transfer models. Collectively, these data suggest that MK2 is a key downstream effector of p38 that can modulate PV autoantibody pathogenicity. MK2 inhibition may be a valuable adjunctive therapy for control of pemphigus blistering.

We recently found that the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL)1 is essential for mediating adiponectin signal to induce liver kinase B (LKB)1 cytosloic translocation, an essential step for activation of AMP-activated protein kinase (AMPK) in cells. However, the underlying molecular mechanisms remain unknown. Here, we demonstrate that treating C2C12 myotubes with adiponectin promoted APPL1 interaction with protein phosphatase 2A (PP2A) and protein kinase Cζ (PKCζ), leading to the activation of PP2A and subsequent dephosphorylation and inactivation of PKCζ. The adiponectin-induced inactivation of PKCζ results in dephosphorylation of LKB1 at Ser307 and its subsequent translocation to the cytosol, where it stimulates AMPK activity. Interestingly, we found that metformin also induces LKB1 cytosolic translocation, but the stimulation is independent of APPL1 and the PP2A-PKCζ pathway. Together, our study uncovers a new mechanism underlying adiponectin-stimulated AMPK activation in muscle cells and shed light on potential targets for prevention and treatment of insulin resistance and its associated diseases.

Pemphigus vulgaris (PV) is a potentially fatal blistering disease characterized by autoantibodies against the desmosomal adhesion protein desmoglein (Dsg) 3. Whether autoantibody steric hindrance or signaling through pathways such as p38 MAPK is primary in disease pathogenesis is controversial. PV mAbs that cause endocytosis of Dsg3 but do not dissociate keratinocytes because of compensatory adhesion by Dsg1 do not activate p38. The same mAbs plus exfoliative toxin to inactivate Dsg1 but not exfoliative toxin alone activate p38, suggesting that p38 activation is secondary to loss of adhesion. Mice with epidermal p38α deficiency blister after passive transfer of PV mAbs; however, acantholytic cells retain cell surface Dsg3 compared with wild-type mice. In cultured keratinocytes, p38 knockdown prevents loss of desmosomal Dsg3 by PV mAbs, and exogenous p38 activation causes internalization of Dsg3, desmocollin 3, and desmoplakin. p38α MAPK is therefore not required for the loss of intercellular adhesion in PV, but may function downstream to augment blistering via Dsg3 endocytosis. Treatments aimed at increasing keratinocyte adhesion could be used in conjunction with immunosuppressive agents, potentially leading to safer and more effective combination therapy regimens.

To address the functions of SigN (SCO4034) in Streptomyces coelicolor, we constructed a sigN null mutant M145Z, which showed a defect in sporulation. The differential proteomic profiles of wild-type S. coelicolors M145 and M145Z were demonstrated by two-dimensional polyacrylamide gel electrophoresis (2D PAGE), which identified 24 different spots that were up- or down-regulated due to sigN disruption. Among them, 22 proteins were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). The up-regulated proteins were involved in energy metabolism, stress responses, ATP binding, ppGpp stringent stress response and transcriptional anti-termination, etc. The down-regulated proteins were related to antibiotic synthesis and differentiation. The results gave a new insight into the regulatory mechanism of SigN in S. coelicolor. Furthermore, deletion of sigN caused growth retardation under all stress conditions examined including heat, cold, acid, oxidation, salt and ethanol. These results indicated that SigN was involved in morphological development, secondary metabolism and stress responses in S. coelicolor.

APPL1 is a newly identified adiponectin receptor-binding protein that positively mediates adiponectin signaling in cells. Here we report that APPL2, an isoform of APPL1 that forms a dimer with APPL1, can interacts with both AdipoR1 and AdipoR2 and acts as a negative regulator of adiponectin signaling in muscle cells. Overexpression of APPL2 inhibits the interaction between APPL1 and AdipoR1, leading to down-regulation of adiponectin signaling in C2C12 myotubes. In contrast, suppressing APPL2 expression by RNAi significantly enhances adiponectin-stimulated glucose uptake and fatty acid oxidation. In addition to targeting directly to and competing with APPL1 in binding with the adiponectin receptors, APPL2 also suppresses adiponectin and insulin signaling by sequestrating APPL1 from these two pathways. In addition to adiponectin, metformin also induces APPL1-APPL2 dissociation. Taken together, our results reveal that APPL isoforms function as an integrated Yin-Yang regulator of adiponectin signaling and mediate the cross-talk between adiponectin and insulin signaling pathways in muscle cells.

The binding of the adaptor protein APPL1 to adiponectin receptors is necessary for adiponectin-induced AMP-activated protein kinase (AMPK) activation in muscle, yet the underlying molecular mechanism remains unknown. Here we show that in muscle cells adiponectin and metformin induce AMPK activation by promoting APPL1-dependent LKB1 cytosolic translocation. APPL1 mediates adiponectin signaling by directly interacting with adiponectin receptors and enhances LKB1 cytosolic localization by anchoring this kinase in the cytosol. Adiponectin also activates another AMPK upstream kinase Ca2+/calmodulin-dependent protein kinase kinase by activating phospholipase C and subsequently inducing Ca2+ release from the endoplasmic reticulum, which plays a minor role in AMPK activation. Our results show that in muscle cells adiponectin is able to activate AMPK via two distinct mechanisms as follows: a major pathway (the APPL1/LKB1-dependent pathway) that promotes the cytosolic localization of LKB1 and a minor pathway (the phospholipase C/Ca2+/Ca2+/calmodulin-dependent protein kinase kinase-dependent pathway) that stimulates Ca2+ release from intracellular stores.

Here we reported that, in Saccharomyces cerevisiae, deleting Swi1 (ScSwi1), a core component in Swi/Snf complex, caused defects of invasive growth, pseudohyphal growth, FLO11 expression, and proper cell separation. Re-introduction of SWI1 into the swi1 mutants could suppress all defects observed. We also showed that overproducing Swi1 could suppress the defect of flo8 cells in pseudohyphal growth in diploids, but not invasive growth in haploids. Overexpression of SWI1 could not bypass the requirement of Ste12 or Tec1 in invasive growth or pseudohyphal growth. We concluded that the Swi/Snf complex was required for FLO11 expression and proper cell separation, and both the FLO8 and STE12 genes should be present for the complex to function for the invasive growth but only the STE12 gene was required for the pseudohyphal growth. Ectopic expression of Candida albicans SWI1 (CaSWI1) could partially complement the defects examined of haploid Scswi1 mutants, but failed to complement the defects examined of diploid Scswi1/Scswi1 mutants. Overexpressing CaSwi1 mitigated invasive and pseudohyphal growth defects resulting from deletions in the MAP kinase and cAMP pathways. The integrity of S. cerevisiae Swi/Snf complex is required for invasive and filamentous growth promoted by overexpressing CaSwi1.

The intercellular interactions of the desmosomal cadherins, desmoglein and desmocollin, are required for epidermal cell adhesion. Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by autoantibodies against desmoglein (Dsg) 3. During calcium-induced desmosome assembly, treatment of primary human keratinocytes with pathogenic monovalent anti-Dsg3 mAbs produced from a PV patient causes a decrease of Dsg3 and desmoplakin but not desmocollin (Dsc) 3 in the Triton-insoluble fraction of cell lysates within 2 hours. Immunofluorescence and antibody ELISA studies suggest that pathogenic mAbs cause internalization of cell-surface Dsg3 but not Dsc3 through early endosomes. Electron microscopy demonstrated a lack of well-formed desmosomes in keratinocytes treated with pathogenic compared to nonpathogenic mAbs. In contrast, pathogenic mAbs caused late depletion of Dsg3 from preformed desmosomes at 24 hours, with effects on multiple desmosomal proteins including Dsc3 and plakoglobin. Together, these studies indicate that pathogenic PV mAbs specifically cause internalization of newly synthesized Dsg3 during desmosome assembly, correlating with their pathogenic activity. Monovalent human PV anti-Dsg mAbs reproduce the effects of polyclonal PV IgG on Dsg3 and will facilitate future studies to further dissect the cellular mechanisms for the loss of cell adhesion in pemphigus.

Epidermal cell adhesion depends on the intercellular interactions of transmembrane cadherin glycoproteins, which form the basis of adherens junctions and desmosomes. Pemphigus is a blistering disease of the skin and mucous membranes characterized by autoantibodies against the cell surface desmosomal cadherins, desmoglein (Dsg) 3 and Dsg1. An unanswered question in pemphigus pathophysiology is the mechanism of acantholysis, or loss of keratinocyte cell adhesion. One longstanding theory for pemphigus pathogenesis is the concept of steric hindrance, in which pathogenic pemphigus autoantibodies cause loss of intercellular adhesion by directly interfering with desmosomal cadherin trans-interactions. However, several recent studies have demonstrated that modulation of p38MAPK, Rho family GTPase, c-myc, protein kinase C, and phospholipase C signaling pathways prevents keratinocyte dissociation induced by pemphigus autoantibodies. As it is unlikely that desmosomal signaling would occur only in response to pemphigus autoantibodies, these studies suggest that numerous different signaling molecules may play a role in desmosomal homeostasis. Many of these same signaling pathways regulate classical cadherins in adherens junctions. Given the recent discovery of bidirectional crosstalk between adherens junctions and desmosomes, it would be valuable to understand how signaling pathways implicated in pemphigus pathogenesis may be involved in more general mechanisms of desmosome and adherens junction regulation. In this review, we will summarize the evidence supporting a role for steric hindrance and signaling mechanisms in the pathogenesis of pemphigus acantholysis and discuss potential analogues in the classical cadherin literature.

Adiponectin functions as an insulin sensitizer, and yet the underlying molecular mechanism(s) remains largely unknown. We found that treating C2C12 myotubes with adiponectin or rapamycin enhanced the ability of insulin to stimulate IRS-1 tyrosine phosphorylation and Akt phosphorylation, concurrently with reduced p70 S6 kinase phosphorylation at Thr389 as well as IRS-1 phosphorylation at Ser302 and Ser636/639. Overexpression of dominant-negative AMP kinase (AMPK), but not dominant-negative p38 MAPK, reduced the insulin-sensitizing effect of adiponectin. Rapamycin, but not adiponectin, enhanced insulin-stimulated Akt phosphorylation in HeLa cells, which lack LKB1, and exogenous expression of LKB1 in HeLa cells rescued the insulin-sensitizing effect of adiponectin. Finally, overexpression of wild-type Rheb (Ras homology-enriched in brain) or the TSC2 mutant lacking the AMPK phosphorylation site (TSC2S1345A) inhibited the insulin-sensitizing effect of adiponectin in C2C12 cells. These results indicate that activation of the LKB1/AMPK/TSC1/2 pathway alleviates the p70 S6 kinase-mediated negative regulation of insulin signaling, providing a mechanism by which adiponectin increases insulin sensitivity in cells.

There appear to be compelling evidences presenting adiponectin as a key regulator of energy homeostasis. Over the past 10 years, much work has been done to identify the molecular mechanisms by which adiponectin functions in the body. We and other groups have demonstrated that adiponectin activates multiple signaling pathways, which mediate its anti-diabetic, anti-atherogenic and anti-inflammatory functions. Comprehensive analysis of the mechanism of adiponectin action may allow us to elucidate the etiology of metabolic syndrome associated diseases including diabetes and cardiovascular diseases, where dysfunction of adiponectin may contribute to pathogenesis of diseases. While regulation of adiponectin gene expression or secretion remains an interesting topic in studies of cell metabolism, further intensive studies are necessary to illustrate the molecular mechanisms. Importantly, identification of molecules in the adiponectin signaling pathways and in the regulation of adiponectin gene expression may provide novel targets for therapeutic drugs.