Genomic and functional features of yeast species in Korean traditional fermented alcoholic beverage and soybean products.

In this review, we describe the genomic and physiological features of the yeast species predominantly isolated from Nuruk, a starter for traditional Korean rice wines, and Jang, a traditional Korean fermented soy product. Nuruk and Jang have several prevalent yeast species, including Saccharomycopsis fibuligera, Hyphopichia burtonii, and Debaryomyces hansenii complex, which belong to the CUG clade showing high osmotic tolerance. Comparative genomics revealed that the interspecies hybridization within yeast species for generating heterozygous diploid genomes occurs frequently as an evolutional strategy in the fermentation environment of Nuruk and Jang. Through gene inventory analysis based on the high-quality reference genome of S. fibuligera, new genes involved in cellulose degradation and volatile aroma biosynthesis and applicable to the production of novel valuable enzymes and chemicals can be discovered. The integrated genomic and transcriptomic analysis of Hyphopichia yeasts, which exhibit strong halotolerance, provides insights into the novel mechanisms of salt and osmo-stress tolerance for survival in fermentation environments with a low-water activity and high-concentration salts. In addition, Jang yeast isolates, such as D. hansenii, show probiotic potential for the industrial application of yeast species beyond fermentation starters to diverse human health sectors.

Genomic features, aroma profiles, and probiotic potential of the Debaryomyces hansenii species complex strains isolated from Korean soybean fermented food.

Fermented soybean products are gaining attention in the food industry owing to their nutritive value and health benefits. In this study, we performed genomic analysis and physiological characterization of two Debaryomyces spp. yeast isolates obtained from a Korean traditional fermented soy sauce "ganjang". Both Debaryomyces hansenii ganjang isolates KD2 and C11 showed halotolerance to concentrations of up to 15% NaCl and improved growth in the presence of salt. Ploidy and whole-genome sequencing analyses indicated that the KD2 genome is haploid, whereas the C11 genome is heterozygous diploid with two distinctive subgenomes. Interestingly, phylogenetic analysis using intron sequences indicated that the C11 strain was generated via hybridization between D. hansenii and D. tyrocola ancestor strains. The D. hansenii KD2 and D. hansenii-hybrid C11 produced various volatile flavor compounds associated with butter, caramel, cheese, and fruits, and showed high bioconversion activity from ferulic acid to 4-vinylguaiacol, a characteristic flavor compound of soybean products. Both KD2 and C11 exhibited viability in the presence of bile salts and at low pH and showed immunomodulatory activity to induce high levels of the anti-inflammatory cytokine IL-10. The safety of the yeast isolates was confirmed by analyzing virulence and acute oral toxicity. Together, the D. hansenii ganjang isolates possess physiological properties beneficial for improving the flavor and nutritional value of fermented products.

Hypoxia-Inducible Factor-2 Alpha Regulates the Migration of Fibroblast-like Synoviocytes via Oxidative Stress-Induced CD70 Expression in Patients with Rheumatoid Arthritis.

This study aimed to examine the role of CD70, which is highly expressed on fibroblast-like synoviocytes (FLS), in rheumatoid arthritis (RA) patients. FLS isolated from RA (n = 14) and osteoarthritis (OA, n = 4) patients were stimulated with recombinant interleukin-17 (IL-17; 5 ng/mL) and tumor necrosis factor alpha (TNF-α; 5 ng/mL) for 24 h. Expression of CD70, CD27/soluble CD27 (sCD27), and hypoxia-inducible factor-2 alpha (HIF-2α) was analyzed by RT-qPCR, flow cytometry, and ELISA assays, respectively. Reactive oxygen species (ROS) expression and cell migration were also examined. The HIF-2α inhibitor PT-2385 and CD70 inhibitor BU69 were used to specifically suppress these pathways. Stimulation with IL-17 and TNF-α significantly induced CD70 expression in RA FLS. Although the synovial fluids from patients with RA contained high levels of sCD27, surface expression of CD27, a ligand of CD70, was rarely detected in RA FLS. Cytokine-induced CD70 expression was significantly decreased following antioxidant treatment. Following HIF-2α inhibition, RA FLS had decreased expression of CD70 and ROS levels. Migration of RA FLS was also inhibited by inhibition of CD70 or HIF-2α. The surface expression of CD70 is regulated by HIF-2α and ROS levels and is a key contributor to cytokine-enhanced migration in RA FLS.

Reduction of Oxidative Stress in Peripheral Blood Mononuclear Cells Attenuates the Inflammatory Response of Fibroblast-like Synoviocytes in Rheumatoid Arthritis.

The production and oxidation mechanism of reactive oxygen species (ROS) are out of balance in rheumatoid arthritis (RA). However, the correlation between ROS and T cell subsets in RA remains unclear. Peripheral blood mononuclear cells (PBMCs) from patients with RA (n = 40) and healthy controls (n = 10) were isolated from whole blood samples. Synovial tissues (n = 3) and synovial fluid (n = 10) were obtained from patients with RA. The repartition of T cell subsets and expression of ROS and cytokines were examined according to RA severity. Fibroblast-like synoviocytes (FLSs) from patients with RA were stimulated with PBMCs and the expression of inflammation-related molecules were measured by RT-PCR and cytokine array. Regulatory T cells from patients with moderate (5.1 > DAS28 ≥ 3.2) RA showed the highest expression of mitochondrial ROS among the groups based on disease severity. Although ROS levels steadily increased with RA severity, there was a slight decline in severe RA (DAS28 ≥ 5.1) compared with moderate RA. The expression of inflammatory cytokines in RA FLSs were significantly inhibited when FLSs were co-cultured with PBMCs treated with ROS inhibitor. These findings provide a novel approach to suppress inflammatory response of FLSs through ROS regulation in PBMCs.

Short bZIP homologue of sulfur regulator Met4 from Ogataea parapolymorpha does not depend on DNA-binding cofactors for activating genes in sulfur starvation.

The acquisition of sulfur from environment and its assimilation is essential for fungal growth and activities. Here, we describe novel features of the regulatory network of sulfur metabolism in Ogataea parapolymorpha, a thermotolerant methylotrophic yeast with high resistance to harsh environmental conditions. A short bZIP protein (OpMet4p) of O. parapolymorpha, displaying the combined structural characteristics of yeast and filamentous fungal Met4 homologues, plays a key role as a master regulator of cell homeostasis during sulfur limitation, but also its function is required for the tolerance of various stresses. Domain swapping analysis, combined with deletion analysis of the regulatory domains and genes encoding OpCbf1p, OpMet28p, and OpMet32p, indicated that OpMet4p does not require the interaction with these DNA-binding cofactors to induce the expression of sulfur genes, unlike the Saccharomyces cerevisiae Met4p. ChIP analysis confirmed the notion that OpMet4p, which contains a canonical bZIP domain, can bind the target DNA in the absence of cofactors, similar to homologues in other filamentous fungi. Collectively, the identified unique features of the O. parapolymorpha regulatory network, as the first report on the sulfur regulation by a short yeast Met4 homologue, provide insights into conservation and divergence of the sulfur regulatory networks among diverse ascomycetous fungi.

Yeast synthetic biology for designed cell factories producing secretory recombinant proteins.

Yeasts are prominent hosts for the production of recombinant proteins from industrial enzymes to therapeutic proteins. Particularly, the similarity of protein secretion pathways between these unicellular eukaryotic microorganisms and higher eukaryotic organisms has made them a preferential host to produce secretory recombinant proteins. However, there are several bottlenecks, in terms of quality and quantity, restricting their use as secretory recombinant protein production hosts. In this mini-review, we discuss recent developments in synthetic biology approaches to constructing yeast cell factories endowed with enhanced capacities of protein folding and secretion as well as designed targeted post-translational modification process functions. We focus on the new genetic tools for optimizing secretory protein expression, such as codon-optimized synthetic genes, combinatory synthetic signal peptides and copy number-controllable integration systems, and the advanced cellular engineering strategies, including endoplasmic reticulum and protein trafficking pathway engineering, synthetic glycosylation, and cell wall engineering, for improving the quality and yield of secretory recombinant proteins.

Intravitreal aflibercept for submacular hemorrhage secondary to neovascular age-related macular degeneration and polypoidal choroidal vasculopathy.

PURPOSE: To evaluate the efficacy of intravitreal aflibercept monotherapy for submacular hemorrhage secondary to neovascular age-related macular degeneration (AMD) and polypoidal choroidal vasculopathy (PCV). METHODS: This prospective, phase 4 clinical trial included 29 patients diagnosed with fovea-involving submacular hemorrhage secondary to neovascular AMD (7 patients) or PCV (22 patients). Patients were initially administered 3 monthly aflibercept injections, followed by 1 injection every 2 months. The primary outcome measure was changes in Early Treatment Diabetic Retinopathy Study (ETDRS) best-corrected visual acuity (BCVA) during the 56-week study period. Other key outcome measures were the proportion of patients who exhibited changes in BCVA of ≥ 15 ETDRS letters from baseline and changes in central retinal thickness (CRT). RESULTS: The mean size of hemorrhage was 6.2 ± 4.8-disc-diameter area. The mean BCVA significantly improved from 52.9 ± 17.8 ETDRS letters at week 0 (baseline) to 71.8 ± 16.1 letters at week 56 (P < 0.001). At week 56, improvement in BCVA of ≥ 15 letters was noted in 16 patients (55.2%), whereas none of the patients experienced a loss of ≥ 15 letters. The mean CRT significantly decreased from 498.9 ± 194.2 µm at week 0 to 248.3 ± 45.0 µm at week 56 (P < 0.001). During the study period, retinal break developed in one patient. CONCLUSIONS: Intravitreal aflibercept administered every 2 months after the 3 initial monthly doses was found to be an effective and safe treatment method for submacular hemorrhage secondary to neovascular AMD.

Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression.

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera ß-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.

Intrinsic changes of left ventricular function in patients with Behçet disease and comparison according to systemic disease activity.

PURPOSE: Although cardiac manifestation of Behçet disease (BD) has been described in sporadic reports, its timely diagnosis remains difficult. The objective of this study was to describe early cardiac manifestations of BD. We also performed a comprehensive classification of systemic BD activity and compared their cardiac manifestations. METHODS: A prospective screening using speckle tracking echocardiography was performed in 85 patients with BD who had no history of heart disease. After excluding subjects with left ventricular (LV) ejection fraction (LVEF) <50% (n = 1), atrial fibrillation (n = 2), or inadequate echocardiographic images (n = 1), we analyzed their clinical and echocardiographic parameters including LV global longitudinal strains (GLS) and compared them with those of an age- and gender-matched control group (n = 145). Systemic BD activity was classified as minimal (Group A), controlled (Group B), and active (Group C). RESULTS: In 81 study patients (59 females, age of 51 ± 11 years), echocardiography revealed a mean LVEF of 64 ± 5% without any significant valvular dysfunction or aortic aneurysm. Although there was no difference in LVEF between the control group and the patient group, the patient group showed significant reduction in GLS (-17.1 ± 2.9% vs -20.8 ± 2.2%, P < .001). Groups A (n = 21, 26%), B (n = 47, 58%), and C (n = 13, 58%) consistently showed reduction in GLS compared with the control group. However, there was no significant difference in cardiac manifestations among these groups according to systemic disease activity. CONCLUSION: Patients with BD present intrinsic LV dysfunction despite no apparent abnormality on routine echocardiography. However, their cardiac manifestations are not proportional to systemic BD activity.

Preferential Induction of the T Cell Auxiliary Signaling Molecule B7-H3 on Synovial Monocytes in Rheumatoid Arthritis.

B7-H3, a newly identified B7 family member, has functional duality as a co-stimulator and co-inhibitor that fine-tunes T cell-mediated immune responses. Given that B7-H3 expression on human monocytes and dendritic cells is enhanced by inflammatory cytokines, its potential inmmunoregulatory role at sites of inflammation has been suggested. Further, monocytes play crucial roles in the pathophysiology of various inflammatory disorders including autoimmune diseases; however, the immunological role of B7-H3 in rheumatoid arthritis (RA) has not been defined. Thus, we aimed to investigate the possible roles of monocyte B7-H3 in the pathogenesis of RA. Synovial monocytes, but not peripheral monocytes, in RA patients predominantly express surface B7-H3. The 4Ig isoform of B7-H3 is exclusively induced on the cell surface, whereas the 2Ig B7-H3 isoform is constitutively expressed in the intracytoplasmic region of both peripheral and synovial monocytes. B7-H3 knockdown experiments reveal that surface B7-H3 has an inhibitory effect on IFN-γ production in CD4 memory cells. Moreover, surface B7-H3 expression on synovial monocytes inversely correlates with RA clinical parameters. Our findings demonstrate that activation-induced B7-H3 expression on synovial monocytes has the potential to inhibit Th1-mediated immune responses and immunomodulatory roles affecting RA pathogenesis.

TNFα and IL-1ß in the synovial fluid facilitate mucosal-associated invariant T (MAIT) cell migration.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that primarily affect the joints and inflammatory cell migration into inflamed articular sites contribute to this disease. Among the inflammatory cells, human mucosal-associated invariant T (MAIT) cells were recently recognized as critical cellular component with a pathological role in RA. However, their migratory characteristics are poorly understood. The aim of this study was to determine whether human MAIT cells preferentially traffick to inflamed synovial sites in rheumatoid arthritis patients and to elucidate the underlying mechanism. First, we found that TNFα and IL-1ß were elevated in synovial fluid (SF) of RA patients, which resulted in increased expression of E-selectin, ICAM-1 and V-CAM-1 on blood vessel endothelial cells. To understand whether TNFα and IL-1ß in the SF facilitated MAIT cell migration, we analyzed CD161+ TCRα7.2+ MAIT and other CD3+ T cells for differences in migratory capacity. Collectively, our results demonstrate that TNFα and IL-1ß in the SF facilitated MAIT cell migration dependent on expression of selectin ligand, sialyl LewisX (sLeX) and CCR6 on MAIT cells. We also showed that MAIT cells in the SF from RA patients equipped upregulated sLeX compared to the peripheral blood of RA patients and healthy persons, which suggest that TNFα and IL-1ß mediated expression of E-selectin preferentially attract sLeX mediated MAIT cell migration into the SF of RA patients.

Association of Single Nucleotide Polymorphisms of PADI4 and HLA-DRB1 Alleles with Susceptibility to Rheumatoid Arthritis-Related Lung Diseases.

OBJECTIVE: Lung diseases (LD) are common extra-articular manifestations in rheumatoid arthritis (RA). However, little is known about factors associated with susceptibility to rheumatoid arthritis-related lung diseases (RA-LD). The aim of the present study was to investigate whether the single nucleotide polymorphisms (SNPs) of PADI4 and HLA-DRB1 alleles were associated with RA-LD. METHODS: Blood samples and clinical data were collected from 116 consecutive RA patients who satisfied the 1987 American College of Rheumatology classification criteria. RA-LD was diagnosed using high-resolution computed tomography of the chest. All patients were genotyped for SNPs of PADI4 and HLA-DRB1 alleles and analyzed for full amino acid sequence of the HLA protein corresponding to a 4-digit HLA typing. Data were analyzed by independent t test (or Mann-Whitney test) for continuous variables, Chi-square test (or Fisher's exact test) and trend test for categorical variables, and logistic regression analysis. RESULTS: Ninety-four (81.0 %) RA patients had LD, of which eight (6.9 %) had interstitial lung disease (ILD) and 92 (79.3 %) had airway abnormalities in which 64 (55.2 %) showed bronchiectasis and 47 (40.5 %) revealed bronchial wall thickening. The recessive genotype of padi4_92 was susceptible to airway abnormalities (OR = 2.22, 95 % CI = 1.05-4.49, p = 0.034). Tryptophan at position 9 of HLA-DRB1 sequence was associated with the susceptibility to RA-ILD (OR = 22.89, 95 % CI = 1.20-432.56, p = 0.037). CONCLUSION: PADI4 polymorphisms and HLA-DRB1 alleles could attribute differently to the development of airway abnormalities and ILD, respectively, in RA.

Yeast synthetic biology for the production of recombinant therapeutic proteins.

The production of recombinant therapeutic proteins is one of the fast-growing areas of molecular medicine and currently plays an important role in treatment of several diseases. Yeasts are unicellular eukaryotic microbial host cells that offer unique advantages in producing biopharmaceutical proteins. Yeasts are capable of robust growth on simple media, readily accommodate genetic modifications, and incorporate typical eukaryotic post-translational modifications. Saccharomyces cerevisiae is a traditional baker's yeast that has been used as a major host for the production of biopharmaceuticals; however, several nonconventional yeast species including Hansenula polymorpha, Pichia pastoris, and Yarrowia lipolytica have gained increasing attention as alternative hosts for the industrial production of recombinant proteins. In this review, we address the established and emerging genetic tools and host strains suitable for recombinant protein production in various yeast expression systems, particularly focusing on current efforts toward synthetic biology approaches in developing yeast cell factories for the production of therapeutic recombinant proteins.

Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha.

OBJECTIVES: The promoter of HpMET3, encoding an ATP sulfurylase, was evaluated for its potential as a repressible promoter to downregulate the expression of target genes in the thermotolerant, methylotrophic yeast Hansenula polymorpha. RESULTS: The expression of lacZ under the control of the 0.6 kb HpMET3 promoter was efficiently downregulated by cysteine, but not by methionine or sulfate. The HpMET3 promoter was used to generate a conditional mutant of the HpPMT2 gene encoding an O-mannosyltransferase, which is involved in post-translational protein modification. The addition of 0.5 mM cysteine adversely affected the growth of the conditional HpMET3(p)-Hppmt2 mutant strain by downregulating transcription of HpPMT2 to approx. 40 % of the normal levels, indicating that the HpPMT2 gene is essential for cell viability. However, the HpMET3 promoter was neither induced nor repressed in the heterologous host Saccharomyces cerevisiae. CONCLUSION: Our results reveal that the cysteine-repressible HpMET3 promoter is a useful tool that downregulates the expression of various genes in H. polymorpha.

Intravitreal anti-vascular endothelial growth factor for submacular hemorrhage from choroidal neovascularization.

PURPOSE: To evaluate the efficacy of intravitreal anti-vascular endothelial growth factor (VEGF) monotherapy for patients diagnosed with exudative age-related macular degeneration (AMD) accompanied by submacular hemorrhage. DESIGN: Retrospective, observational case series. PARTICIPANTS: Ninety-one eyes of 91 patients who initially presented with submacular hemorrhage associated with exudative AMD from October 2009 to September 2012. Patients were followed up for at least 6 months after treatment. METHODS: Best-corrected visual acuity (BCVA) was measured at diagnosis and at 1, 3, and 6 months after treatment. The duration of symptoms was estimated. The extent of hemorrhage was estimated using fundus photography, and central foveal thickness was measured using optical coherence tomography. Change in BCVA during 6 months after treatment was estimated. The correlation of BCVA at 6 months with duration of symptoms, extent of hemorrhage, and central foveal thickness was evaluated. MAIN OUTCOME MEASURES: The BCVA, duration of symptoms, extent of hemorrhage, and central foveal thickness. RESULTS: The mean duration of symptoms was 27.6±39.5 days. The mean extent of hemorrhage was 7.8±5.6 disc areas, and the mean central foveal thickness was 610.1±249.6 µm. All eyes were treated with 3.2±0.8 (range, 1-5) monthly intravitreal anti-VEGF injections during the 6-month follow-up period. The logarithm of the minimum angle of resolution BCVA at diagnosis and at 1, 3, and 6 months after the initial diagnosis was 1.38±0.53 (Snellen equivalent, 20/479), 1.27±0.57, 1.05±0.58, and 0.96±0.65 (Snellen equivalent, 20/182), respectively. The BCVA at 6 months significantly improved from baseline (P < 0.001). Poor BCVA at 6 months correlated with a longer duration of symptoms, greater extent of hemorrhage, and greater central foveal thickness (P = 0.008, P = 0.004, and P = 0.014, respectively). CONCLUSIONS: Anti-VEGF monotherapy was found to be a useful treatment option for exudative AMD accompanied by submacular hemorrhage. However, the limited efficacy in eyes with large hemorrhage may suggest the need for more aggressive treatment in these cases.

Functional and molecular characterization of novel Hansenula polymorpha genes, HpPMT5 and HpPMT6, encoding protein O-mannosyltransferases.

The genome of the thermotolerant methylotrophic yeast Hansenula polymorpha reveals the presence of five PMT homologues (HpPMT1, HpPMT2, HpPMT4, HpPMT5, and HpPMT6) encoding protein O-mannosyltransferases. Here, we report on the systematic characterization of HpPMT5 and HpPMT6, encoding novel PMT1 and PMT2 subfamily members, respectively. Although no apparent growth defects were detected in the Hppmt5Δ and Hppmt6Δ single mutants, the single mutants showed dramatic sensitivity to the Pmt1p inhibitor, and the Hppmt1pmt5Δ and Hppmt1pmt6Δ double mutants displayed increased susceptibility to cell wall-disturbing reagents. Activation of the cell wall integrity signaling pathway in the double mutant strains was further indicated by the markedly induced phosphorylation of MAP kinases, such as HpMpk1p and HpHog1p. Noticeably, O-mannosylation of the surface glycoproteins HpWsc1p and HpMid2p became severely defective only in the double mutants, supporting the involvement of HpPmt5p and HpPmt6p in O-mannosylation of these sensor proteins. On the other hand, co-immunoprecipitation experiments revealed only marginal interaction between HpPmt5p and HpPmt2p, even in the absence of HpPmt1p. Taken together, our results suggest that the functions of HpPmt5p and HpPmt6p are minor but become crucial upon the loss of HpPmt1p for protein O-mannosylation, which is essential for cell growth, cell wall integrity, and stress resistance in H. polymorpha.

LKB1 Regulates Inflammation of Fibroblast-like Synoviocytes from Patients with Rheumatoid Arthritis via AMPK-Dependent SLC7A11-NOX4-ROS Signaling.

Fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) patients have increased reactive oxygen species (ROS) levels and an impaired redox balance compared with FLS from control patients. Liver kinase B1 (LKB1) plays a key role in ROS scavenging and cellular metabolism in various cancers. Here, we aimed to determine the specific mechanism of LKB1 in RA pathogenesis. FLS were obtained from RA patients (n = 10). siRNA-induced LKB1 deficiency in RA FLS increased ROS levels via NADPH oxidase 4 (NOX4) upregulation. RA FLS migration and expression of inflammatory factors, including interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor-alpha (TNF-α), and vascular endothelial growth factor (VEGF), were enhanced by LKB1 deficiency. LKB1-deficient RA FLS showed increased sensitivity to oxidative stress damage caused by hydrogen peroxidase exposure. siRNA-induced solute carrier family 7 member 11 (SLC7A11) deficiency in RA FLS enhanced NOX4 and ROS expression and increased cell migration. When LKB1-deficient RA FLS were stimulated with an AMP-activated protein kinase (AMPK) activator, the LKB1-inhibition-induced cell migration significantly decreased through the restoration of SLC7A11/NOX4 expression. LKB1 regulates the AMPK-mediated SLC7A11-NOX4-ROS pathway to control cell migration and inflammation. Our data indicate that LKB1 is a key regulator of redox homeostasis in RA FLS.

Immediate Vitrectomy for Acute Endophthalmitis in Patients with a Visual Acuity of Hand Motion or Better.

PURPOSE: To determine the efficacy of immediate pars plana vitrectomy as the primary treatment for acute endophthalmitis in patients with a visual acuity (VA) of hand motion (HM) or better. METHODS: A total of 149 patients who were referred to a single center for acute endophthalmitis after cataract surgery over the 13-year study period were retrospectively analyzed. Only patients presenting with a VA of at least HM were included. Patients were initially treated with either primary vitrectomy or intravitreal antibiotic injection alone, and their visual outcomes and reintervention rates after initial treatment were compared. RESULTS: There was no significant difference in the proportion of good (final VA ≥20 / 40) and poor (VA ≤ counting finger) visual outcomes between the groups. However, subgroup analysis of patients with a VA of HM (92 eyes) showed that the incidence of reintervention (14 of 72 eyes [19.4%] vs. 9 of 20 eyes [45.0%]) and poor visual outcomes (10 of 72 eyes [13.9%] vs. 8 of 20 eyes [40.0%]) were lower after prompt vitrectomy than after intravitreal antibiotic injection alone (p = 0.019 and p = 0.022, respectively). For those with a VA of at least counting finger, no significant difference was observed between the groups. CONCLUSIONS: For patients with endophthalmitis presenting with a VA of HM, performing a prompt vitrectomy reduced the incidence of reintervention and poor visual outcomes than the administration of intravitreal antibiotics alone. Our results suggest that primary vitrectomy for patients with endophthalmitis presenting with a VA of HM could be more beneficial than intravitreal antibiotic injection alone.

Cytoplasmic zinc promotes IL-1ß production by monocytes and macrophages through mTORC1-induced glycolysis in rheumatoid arthritis.

The essential micronutrient zinc regulates immune responses by affecting signaling pathways. In activated monocytes and macrophages, signaling networks mediate the metabolic reprogramming that meets the demands of participation in immune responses. Here, we demonstrated that cytoplasmic, bioavailable zinc was essential for promoting IL-1ß production in activated human monocytes and macrophages downstream of glycolysis induced by the kinase-containing multiprotein complex mTORC1. The concentration of cytoplasmic zinc was determined by that of extracellular zinc, which was brought into cells through the zinc-specific importer Zip8. The abundance of Zip8 was increased in monocytes from patients with rheumatoid arthritis (RA), as well as in LPS-stimulated monocytes and macrophages from healthy individuals. The mTORC1-mediated phosphorylation of S6 kinase (S6K) was enhanced by zinc-mediated inhibition of PP2A, a phosphatase that targets S6K. As a result, IL-1ß production was increased due to the activation of mTORC1-induced glycolysis. In monocytes of patients with RA, the expression of Zip8 and the zinc-inducible metallothionein isoform MT2A and the phosphorylation of S6K were enhanced compared with those of healthy controls. Furthermore, Zip8 expression correlated with more severe RA clinical parameters, suggesting that Zip8-mediated zinc influx is related to inflammatory conditions. These results provide insight into the role of cytoplasmic, bioavailable zinc in the metabolic reprogramming of human monocytes and macrophages in inflammatory responses.

CD14+ monocytes and soluble CD14 of synovial fluid are associated with osteoarthritis progression.

Objectives: This study aims to investigate the role of cluster of differentiation 14 (CD14) expressed monocytes and soluble CD14-mediated pathway in the synovial inflammation of knee osteoarthritis (OA). Patients and methods: Between May 2012 and July 2013, a total of 35 patients with knee OA (9 males, 26 females; mean age: 66.3±8.8 years; range, 52 to 79 years) were included in this cross-sectional study. Synovial fluid was obtained from knee joints of 35 OA patients. The CD14+ monocytes from synovial fluid mononuclear cells (SFMCs) were isolated using the MACS. The fibroblast-like synoviocytes (FLSs) isolated from knee joint tissue were incubated with recombinant CD14 and lipopolysaccharide (LPS) for 24 h. Cytokine profiling was performed with the Luminex® Performance Assay or magnetic bead panel kit. The expression of CD14 and CD16 was analyzed by immunohistochemistry and flow cytometry. Results: The concentration of sCD14 in synovial fluid was correlated with the interleukin-6 (IL-6) level (n=35) (ρ=0.654, p<0.001). The culture supernatants of CD14+ monocytes isolated from SFMC (n=15) showed a correlation between sCD14 and IL-6 (ρ=0.784, p=0.001), along with complement component 3 (ρ=0.756, p=0.010), IL-1b (ρ=0.652, p=0.012), and tumor necrosis factor-alpha (ρ=0.806, p=0.001). Following recombinant CD14 and LPS treatment, OA FLS synergistically enhanced the secretion of IL-6, IL-8, and matrix metalloproteinase 3 (n=3, p<0.05). In five paired-samples from identical patients, the proportions of CD14+ monocytes were significantly elevated in recurred synovial fluid compared to those in initial synovial fluid (p=0.043). When monocyte subsets were analyzed in SFMC (n=26), CD14+CD16+monocytes were abundant (p=0.019) and had higher toll-like receptor 4 expression than CD14+CD16- (p<0.001). Conclusion: Our study results suggest that CD14+ monocytes and the sCD14-mediated pathway play an important role in OA aggravation through inflammatory cytokine secretion.