The transcription factor ZNF469 regulates collagen production in liver fibrosis.

Non-alcoholic fatty liver disease (NAFLD) - characterized by excess accumulation of fat in the liver - now affects one third of the world's population. As NAFLD progresses, extracellular matrix components including collagen accumulate in the liver causing tissue fibrosis, a major determinant of disease severity and mortality. To identify transcriptional regulators of fibrosis, we computationally inferred the activity of transcription factors (TFs) relevant to fibrosis by profiling the matched transcriptomes and epigenomes of 108 human liver biopsies from a deeply-characterized cohort of patients spanning the full histopathologic spectrum of NAFLD. CRISPR-based genetic knockout of the top 100 TFs identified ZNF469 as a regulator of collagen expression in primary human hepatic stellate cells (HSCs). Gain- and loss-of-function studies established that ZNF469 regulates collagen genes and genes involved in matrix homeostasis through direct binding to gene bodies and regulatory elements. By integrating multiomic large-scale profiling of human biopsies with extensive experimental validation we demonstrate that ZNF469 is a transcriptional regulator of collagen in HSCs. Overall, these data nominate ZNF469 as a previously unrecognized determinant of NAFLD-associated liver fibrosis.

Construction of carbon nitride/zeolitic imidazolate framework-67 heterojunctions on carbon fiber cloth as the photocatalyst for various pollutants removal and hydrogen production.

With the macroscale and conductive carbon fiber cloth (CFC) as the substrate, the obtained self-supported photocatalysts hold great promise for enhancing the separation of generated carriers and the recyclability of catalysts, thereby improving the photocatalytic performance and practicality in various applications. Additionally, decorating metal-organic frameworks (MOFs) with ultrahigh surface area on the surface of effective semiconductors is a promising method to enhance the adsorption capacity and photocatalytic performance. Herein, zeolitic imidazolate framework-67 (ZIF-67) as a typical MOFs was applied to modify carbon nitride (C3N4) on the surface of macroscale and conductive CFC. CFC/C3N4/ZIF-67 (4 × 4 cm2) was obtained by a thermal condensation-chemical bath deposition two-step route, and it shows superior adsorption and photocatalytic activity toward bisphenol A (BPA), levofloxacin (LVFX), ciprofloxacin (CIP) and good hydrogen evolution activity. Besides, the recycling test for four cycles indicates the high stability of CFC/C3N4/ZIF-67 with an easy recycling process. In this study, CFC/C3N4/ZIF-67 was prepared through the hydrothermal and chemical bath deposition two-step method, which enhances light absorption and photocatalytic performance, as well as recyclability for solving environmental and energy issues.

Infection-responsive long-term antibacterial bone plates for open fracture therapy.

The infections in open fracture induce high morbidity worldwide. Thus, developing efficient anti-infective orthopedic devices is of great significance. In this work, we designed a kind of infection-responsive long-term antibacterial bone plates. Through a facile and flexible volatilization method, a multi-aldehyde polysaccharide derivative, oxidized sodium alginate, was crosslinked with multi-amino compounds, gentamycin and gelatin, to fabricate a uniform coating on Ti bone plates via Schiff base reaction, which was followed by a secondary crosslinking process by glutaraldehyde. The double-crosslinked coating was stable under normal condition, and could responsively release gentamycin by the triggering of the acidic microenvironment caused by bacterial metabolism, owning to the pH-responsiveness of imine structure. The thickness of the coating was ranging from 22.0 µm to 63.6 µm. The coated bone plates (Ti-GOGs) showed infection-triggered antibacterial properties (>99%) and high biocompatibility. After being soaked for five months, it still possessed efficient antibacterial ability, showing its sustainable antibacterial performance. The in vivo anti-infection ability was demonstrated by an animal model of infection after fracture fixation (IAFF). At the early stage of IAFF, Ti-GOGs could inhibit the bacterial infection (>99%). Subsequently, Ti-GOGs could promote recovery of fracture of IAFF. This work provides a convenient and universal strategy for fabrication of various antibacterial orthopedic devices, which is promising to prevent and treat IAFF.

Derma-like antibacterial polysaccharide gel dressings for wound care.

Large skin wound infections have high morbidity, which threaten the health of human beings severely. It is essential to develop new wound dressings that can block microbial invasion, eliminate bacteria effectively, adhere to wounds firmly, and have good biocompatibility. In this work, we designed a kind of polysaccharide gel (DLG) dressings with derma-like structure that had good wound care performances. With a facile penetration cross-linking method by the Schiff base reaction between oxidized hyaluronic acid solution and carboxymethyl chitosan solution with higher viscosity, a gradient porous structure was formed inside DLG to mimic the structure of derma, which was due to the simultaneous penetration and reaction processes between two viscous solutions. This derma-like structure endowed the gel dressings with the abilities of self-adhesion to wounds and barriers against bacteria. Through the introduction of cuttlefish juice and gentamycin, the modified gel dressings (DLG-GS) showed mild photothermal effects under the near infrared irradiation at the wavelength of 808 nm, which could reach and maintain the temperature of 45 °C. The mild heat could act together with gentamycin to produce a rapid bactericidal performance within 5 min. Meanwhile, the polysaccharide gel dressings had good biocompatibility. The in vivo anti-infection properties of DLG-GS was demonstrated by an animal model of infected full-thickness skin defect. This strategy provided a feasible solution for the prevention and treatment of infected large wounds. STATEMENT OF SIGNIFICANCE: Derma-like antibacterial gel dressings (DLG-GS) with high bacterial barrier ability, strong tissue adhesive property and good biocompatibility were constructed by a penetration cross-linking method. DLG-GS could eliminate bacterial infection within 5 min due to the rational combination of a mild photothermal effect and antibiotics. DLG-GS showed high anti-infection and wound healing properties in an animal model of infected full-thickness skin defect. This study provides a flexible and universal strategy for the development  of antibacterial wound dressings.

A Predictive Model of Metabolic Syndrome by Medical Examination: Evidence from an 8-Year Chinese Cohort.

PURPOSE: To develop a predictive model for the risk of metabolic syndrome (MetS). PATIENTS AND METHODS: Totally, 1556 residents without MetS were finally included in 2006 and they were observed for 8 years to check who developed MetS. Univariate and multivariate logistic regression analyses was adopted to explore the risk factors of MetS and develop the predictive model that used the medical examination information of MetS risk after 8 years. The receiver operating characteristic (ROC) curve was drawn to assess the predictive capacity of the model. RESULTS: The risk of MetS in overweight, prehypertension, hypertension subjects were 4.610 [95% confidence interval (CI): 2.415 to 8.800], 2.759 (95% CI: 1.519 to 5.011) and 3.589 (95% CI: 1.672 to 7.706) times higher than that in controls, respectively. The risk of MetS in people with high-density lipoprotein (HDL) <1.10 mmol/L was 3.716-fold in comparison with HDL ≥1.55 mmol/L [odds risk (OR) = 3.716, 95% CI: 1.483 to 9.313]. Individuals with fatty liver had a higher risk of MetS (OR = 2.577, 95% CI: 1.472 to 4.512). The AUC of the predictive model was 0.831 (95% CI: 0.798 to 0.865), with the sensitivity of 0.898 (95% CI: 0.831 to 0.941) and the specificity of 0.676 (95% CI: 0.651 to 0.700). CONCLUSION: The model performed well predictive power for the risk of MetS, which may provide a reference for clinicians to identify high-risk groups early.

Effects of Bunao-Fuyuan decoction serum on proliferation and migration of vascular smooth muscle cells in atherosclerotic.

Atherosclerosis (AS) is a chronic inflammatory disease, the main causes of which include abnormal lipid metabolism, endothelial injury, physical and chemical injury, hemodynamic injury, genetic factors and so on. These causes can lead to inflammatory injury of blood vessels and local dysfunction. Bunao-Fuyuan decoction (BNFY) is a traditional Chinese medicine compound that can treat cardiovascular and cerebrovascular diseases, but its effect on AS is still unknown. The aim of this study was to investigate the effect and mechanism of BNFY in proliferation and migration of vascular smooth muscle cells (VSMCs) on AS. At first, the expression of α-SMA protein in ox-LDL-induced VSMCs, which was detected by immunofluorescence staining and western blot. CCK-8 technique and cloning technique were used to detect the cell proliferation of ox-LDL-induced VSMCs after adding BNFY. Meanwhile, the expression of proliferating protein Ki67 was detected by immunofluorescence staining. Western blot was also used to detect the expression of proliferation-related proteins CDK2, CyclinE1 and P27. Flow cytometry was used to detect the effect of BNFY on cell cycle. The effects of BNFY on proliferation and migration of cells were detected by cell scratch test and Transwell. Western blot was used to detect the expression of adhesion factors ICAM1, VCAM1, muc1, VE-cadherin and RHOA/ROCK-related proteins in cells. We found that the expression of AS marker α-SMA protein increased significantly and cells shriveled and a few floated on the medium after induction of ox-LDL on VSCMs. The proliferation rate of ox-LDL VSMCs decreased significantly after adding different doses of BNFY, and BNFY can inhibit cell cycle. Meanwhile, we also found that cell invasion and migration rate were significantly inhibited and related cell adhesion factors ICAM1, VCAM1, muc1 and VE-cadherin were inhibited too by BNFY. Finally, we found that BNFY inhibited the expression of RHOA, ROCK1, ROCK2, p-MLC proteins in the RHOA/ROCK signaling pathway. Therefore, we can summarize that BNFY may inhibit the proliferation and migration of atherosclerotic vascular smooth muscle cells by inhibiting the activity of RHOA/ROCK signaling pathway.

Phthalocyanine functionalized poly(glycidyl methacrylate) nano-assemblies for photodynamic inactivation of bacteria.

The design of novel antibacterial materials has attracted increasing attention for combating bacterial infections. Herein, we conjugated zinc(ii) monoamino phthalocyanine (ZnMAPc) to poly(glycidyl methacrylate) (PGMA) via a ring-opening reaction, and the excess epoxy groups were scavenged by ethylenediamine (ED). The resultant macro-photosensitizer (PGED-Pc) can be easily dispersed in aqueous solution to form self-assembled nanoparticles and generate reactive oxygen species for inactivation of bacteria when exposed to light illumination. We found that the photodynamic pathway for the generation of singlet oxygen (1O2) was strongly inhibited in aqueous solution, and the major components for the inactivation of bacteria were superoxide anion radicals (˙O2-) and hydrogen peroxide (H2O2), which could result in the disruption of bacterial envelopes, the inactivation of vital enzymes, and the degradation of genomic DNA. PGED-Pc exhibited potent photodynamic antibacterial activity, with minimum bactericidal concentration (MBC, defined as 99.9% inactivation of bacteria) values of 128 µg mL-1 for Escherichia coli (E. coli) and 4 µg mL-1 for Staphylococcus aureus (S. aureus). As a proof-of-concept, the PGED-Pc nano-assemblies in aqueous solution can be readily immobilized on glass slides via a Schiff-base reaction, and impose potent photodynamic antibacterial activity upon light illumination. This work unveils a promising strategy for the engineering of self-sterilizing surfaces to combat bacterial infections.

Role of Bile Acids and GLP-1 in Mediating the Metabolic Improvements of Bariatric Surgery.

BACKGROUND & AIMS: Bile diversion to the ileum (GB-IL) has strikingly similar metabolic and satiating effects to Roux-en-Y gastric bypass (RYGB) in rodent obesity models. The metabolic benefits of these procedures are thought to be mediated by increased bile acids, although parallel changes in body weight and other confounding variables limit this interpretation. METHODS: Global G protein-coupled bile acid receptor-1 null (Tgr5-/-) and intestinal-specific farnesoid X receptor null (FxrΔ/E) mice on high-fat diet as well as wild-type C57BL/6 and glucagon-like polypeptide 1 receptor deficient (Glp-1r-/-) mice on chow diet were characterized following GB-IL. RESULTS: GB-IL induced weight loss and improved oral glucose tolerance in Tgr5-/-, but not FxrΔ/E mice fed a high-fat diet, suggesting a role for intestinal Fxr. GB-IL in wild-type, chow-fed mice prompted weight-independent improvements in glycemia and glucose tolerance secondary to augmented insulin responsiveness. Improvements were concomitant with increased levels of lymphatic GLP-1 in the fasted state and increased levels of intestinal Akkermansia muciniphila. Improvements in fasting glycemia after GB-IL were mitigated with exendin-9, a GLP-1 receptor antagonist, or cholestyramine, a bile acid sequestrant. The glucoregulatory effects of GB-IL were lost in whole-body Glp-1r-/- mice. CONCLUSIONS: Bile diversion to the ileum improves glucose homeostasis via an intestinal Fxr-Glp-1 axis. Altered intestinal bile acid availability, independent of weight loss, and intestinal Akkermansia muciniphila appear to mediate the metabolic changes observed after bariatric surgery and might be manipulated for treatment of obesity and diabetes.

Relationship between the NF-κB1-94ins/del ATTG locus polymorphism and risk of hypertension in the Chinese Han population.

Objective: The onset of essential hypertension is the result of a combination of genetic factors and the environment. The nuclear factor (NF)-κB1-94ins/del ATTG locus polymorphism is associated with the occurrence of various diseases. The purpose of this study was to find out the relationship between the NF-κB1-94ins/del ATTG locus polymorphism and the risk of hypertension in the Chinese Han population. Methods: A total of 585 Chinese Han patients with essential hypertension and 585 Chinese Han healthy volunteers were recruited. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to analyze the genotype of the NF-κB1-94ins/del ATTG locus in all the subjects. Results: For the NF-κB1-94ins/del ATTG locus, the dominant (adjusted odds ratio [OR] = 1.31, 95% confidence interval [CI] = 1.13-1.54, P < 0.001), recessive (adjusted OR = 1.17, 95% CI = 1.02-1.32, P = 0.03) and additive (adjusted OR = 1.19, 95% CI = 1.03-1.36, P = 0.01) models showed significant increase in the risk of hypertension. The NF-κB1-94ins/del ATTG locus II genotype was an independent risk factor for hypertension (OR = 1.15, 95% CI = 0.78-1.69, P = 0.02). The interaction between the NF-κB1-94ins/del ATTG locus polymorphism and BMI, alcohol consumption, and diabetes significantly increased the risk of hypertension (OR = 1.71, 95% CI = 1.26-1.86, P < 0.01). Conclusion: The NF-κB1-94ins/del ATTG polymorphism is an independent risk factor for essential hypertension. The NF-κB1-94ins/del ATTG locus, obesity, drinking, and diabetes also interact to yield a higher risk of hypertension.

CD36 Modulates Fasting and Preabsorptive Hormone and Bile Acid Levels.

Context: Abnormal fatty acid (FA) metabolism contributes to diabetes and cardiovascular disease. The FA receptor CD36 has been linked to risk of metabolic syndrome. In rodents CD36 regulates various aspects of fat metabolism, but whether it has similar actions in humans is unknown. We examined the impact of a coding single-nucleotide polymorphism in CD36 on postprandial hormone and bile acid (BA) responses. Objective: To examine whether the minor allele (G) of coding CD36 variant rs3211938 (G/T), which reduces CD36 level by ∼50%, influences hormonal responses to a high-fat meal (HFM). Design: Obese African American (AA) women carriers of the G allele of rs3211938 (G/T) and weight-matched noncarriers (T/T) were studied before and after a HFM. Setting: Two-center study. Participants: Obese AA women. Intervention: HFM. Main Outcome Measures: Early preabsorptive responses (10 minutes) and extended excursions in plasma hormones [C-peptide, insulin, incretins, ghrelin fibroblast growth factor (FGF)19, FGF21], BAs, and serum lipoproteins (chylomicrons, very-low-density lipoprotein) were determined. Results: At fasting, G-allele carriers had significantly reduced cholesterol and glycodeoxycholic acid and consistent but nonsignificant reductions of serum lipoproteins. Levels of GLP-1 and pancreatic polypeptide (PP) were reduced 60% to 70% and those of total BAs were 1.8-fold higher. After the meal, G-allele carriers displayed attenuated early (-10 to 10 minute) responses in insulin, C-peptide, GLP-1, gastric inhibitory peptide, and PP. BAs exhibited divergent trends in G allele carriers vs noncarriers concomitant with differential FGF19 responses. Conclusions: CD36 plays an important role in the preabsorptive hormone and BA responses that coordinate brain and gut regulation of energy metabolism.

Limiting Injury During Saphenous Vein Graft Preparation For Coronary Arterial Bypass Prevents Metabolic Decompensation.

Standard harvest and preparation of human saphenous vein (HSV) for autologous coronary and peripheral arterial bypass procedures is associated with injury and increased oxidative stress that negatively affect graft performance. In this study we investigated the global metabolomic profiles of HSV before (unprepared; UP) and after standard vein graft preparation (AP). AP-HSV showed impaired vasomotor function that was associated with increased oxidative stress, phospholipid hydrolysis and energy depletion that are characteristic of mechanical and chemical injury. A porcine model (PSV) was utilized to validate these metabolomic changes in HSV and to determine the efficacy of an improved preparation technique (OP) using pressure-regulated distension, a non-toxic vein marker, and graft storage in buffered PlasmaLyte solution in limiting metabolic decompensation due to graft preparation. Deficits in vasomotor function and metabolic signature observed in AP-PSV could be largely mitigated with the OP procedure. These findings suggest that simple strategies aimed at reducing injury during graft harvest and preparation represents a straightforward and viable strategy to preserve conduit function and possibly improve graft patency.

Comprehensive evaluation of extracellular small RNA isolation methods from serum in high throughput sequencing.

BACKGROUND: DNA and RNA fractions from whole blood, serum and plasma are increasingly popular analytes that are currently under investigation for their utility in the diagnosis and staging of disease. Small non-coding ribonucleic acids (sRNAs), specifically microRNAs (miRNAs) and their variant isoforms (isomiRs), and transfer RNA (tRNA)-derived small RNAs (tDRs) comprise a repertoire of molecules particularly promising in this regard. RESULTS: In this designed study, we compared the performance of various methods and kits for isolating circulating extracellular sRNAs (ex-sRNAs). ex-sRNAs from one healthy individual were isolated using five different isolation kits: Qiagen Circulating Nucleic Acid Kit, ThermoFisher Scientific Ambion TRIzol LS Reagent, Qiagen miRNEasy, QiaSymphony RNA extraction kit and the Exiqon MiRCURY RNA Isolation Kit. Each isolation method was repeated four times. A total of 20 small RNA sequencing (sRNAseq) libraries were constructed, sequenced and compared using a rigorous bioinformatics approach. The Circulating Nucleic Acid Kit had the greatest miRNA isolation variability, but had the lowest isolation variability for other RNA classes (isomiRs, tDRs, and other miscellaneous sRNAs (osRNA). However, the Circulating Nucleic Acid Kit consistently generated the fewest number of reads mapped to the genome, as compared to the best-performing method, Ambion TRIzol, which mapped 10% of the miRNAs, 7.2% of the tDRs and 23.1% of the osRNAs. The other methods performed intermediary, with QiaSymphony mapping 14% of the osRNAs, and miRNEasy mapping 4.6% of the tDRs and 2.9% of the miRNAs, achieving the second best kit performance rating overall. CONCLUSIONS: In summary, each isolation kit displayed different performance characteristics that could be construed as biased or advantageous, depending upon the downstream application and number of samples that require processing.

Pancreatic stellate cell secreted IL-6 stimulates STAT3 dependent invasiveness of pancreatic intraepithelial neoplasia and cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a dynamic tumor supported by several stromal elements such as pancreatic stellate cells (PSC). Significant crosstalk exists between PSCs and tumor cells to stimulate oncogenic signaling and malignant progression of PDAC. However, how PSCs activate intercellular signaling in PDAC cells remains to be elucidated. We have previously shown that activated signal transducer and activator of transcription 3 (STAT3) signaling is a key component in the progression of pancreatic neoplasia. We hypothesize that PSC secreted IL-6 activates STAT3 signaling to promote PanIN progression to PDAC. Human PDAC and mouse PanIN cells were treated with PSC-conditioned media (PSC-CM), and phospho- and total-STAT3 levels by immunoblot analysis were determined. IL-6 was quantified in PSC-CM and cell invasion and colony formation assays were performed in the presence or absence of a neutralizing IL-6 antibody and the JAK/STAT3 inhibitor AZD1480. Serum from Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox (PKT) and LSL-KrasG12D/+; Trp53R172H/+; Pdx1Cre/+ (KPC) mice demonstrated increased levels of IL-6 compared to serum from non-PDAC bearing KC and PK mice. PSC secreted IL-6 activated STAT3 signaling in noninvasive, precursor PanIN cells as well as PDAC cells, resulting in enhanced cell invasion and colony formation in both cell types. There was a significant positive linear correlation between IL-6 concentration and the ratio of phosphorylated STAT3/total STAT3. IL-6 neutralization or STAT3 inhibition attenuated PSC-CM induced activation of STAT3 signaling and tumorigenicity. These data provide evidence that PSCs are directly involved in promoting the progression of PanINs towards invasive carcinoma. This study demonstrates a novel role of PSC secreted IL-6 in transitioning noninvasive pancreatic precursor cells into invasive PDAC through the activation of STAT3 signaling.

A micro-RNA expression signature for human NAFLD progression.

BACKGROUND: The spectrum of nonalcoholic fatty liver disease (NAFLD) describes disease conditions deteriorating from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) to cirrhosis (CIR) to hepatocellular carcinoma (HCC). From a molecular and biochemical perspective, our understanding of the etiology of this disease is limited by the broad spectrum of disease presentations, the lack of a thorough understanding of the factors contributing to disease susceptibility, and ethical concerns related to repeat sampling of the liver. To better understand the factors associated with disease progression, we investigated by next-generation RNA sequencing the altered expression of microRNAs (miRNAs) in liver biopsies of class III obese subjects (body mass index ≥40 kg/m(2)) biopsied at the time of elective bariatric surgery. METHODS: Clinical characteristics and unbiased RNA expression profiles for 233 miRs, 313 transfer RNAs (tRNAs), and 392 miscellaneous small RNAs (snoRNAs, snRNAs, rRNAs) were compared among 36 liver biopsy specimens stratified by disease severity. RESULTS: The abundances of 3 miRNAs that were found to be differentially regulated (miR-301a-3p and miR-34a-5p increased and miR-375 decreased) with disease progression were validated by RT-PCR. No tRNAs or miscellaneous RNAs were found to be associated with disease severity. Similar patterns of increased miR-301a and decreased miR-375 expression were observed in 134 hepatocellular carcinoma (HCC) samples deposited in The Cancer Genome Atlas (TCGA). CONCLUSIONS: Our analytical results suggest that NAFLD severity is associated with a specific pattern of altered hepatic microRNA expression that may drive the hallmark of this disorder: altered lipid and carbohydrate metabolism. The three identified miRNAs can potentially be used as biomarkers to access the severity of NAFLD. The persistence of this miRNA expression pattern in an external validation cohort of HCC samples suggests that specific microRNA expression patterns may permit and/or sustain NAFLD development to HCC.

Loss of the histidine kinase DhkD results in mobile mounds during development of Dictyostelium discoideum.

BACKGROUND: Histidine kinases are receptors for sensing cellular and environmental signals, and in response to the appropriate cue they initiate phosphorelays that regulate the activity of response regulators. The Dictyostelium discoideum genome encodes 15 histidine kinases that function to regulate several processes during the multicellular developmental program, including the slug to culmination transition, osmoregulation, and spore differentiation. While there are many histidine kinases, there is only a single response regulator, RegA. Not surprisingly given the ubiquitous involvement of cAMP in numerous processes of development in Dictyostelium, RegA is a cAMP phosphodiesterase that is activated upon receiving phosphates through a phosphorelay. Hence, all of the histidine kinases characterized to date regulate developmental processes through modulating cAMP production. Here we investigate the function of the histidine kinase DhkD. PRINCIPAL FINDINGS: The dhkD gene was disrupted, and the resulting cells when developed gave a novel phenotype. Upon aggregation, which occurred without streaming, the mounds were motile, a phenotype termed the pollywog stage. The pollywog phenotype was dependent on a functional RegA. After a period of random migration, the pollywogs attempted to form fingers but mostly generated aberrant structures with no tips. While prestalk and prespore cell differentiation occurred with normal timing, proper patterning did not occur. In contrast, wild type mounds are not motile, and the cAMP chemotactic movement of cells within the mound facilitates proper prestalk and prespore patterning, tip formation, and the vertical elongation of the mound into a finger. CONCLUSIONS: We postulate that DhkD functions to ensure the proper cAMP distribution within mounds that in turn results in patterning, tip formation and the transition of mounds to fingers. In the absence of DhkD, aberrant cell movements in response to an altered cAMP distribution result in mound migration, a lack of proper patterning, and an inability to generate normal finger morphology.

eIF2α kinases regulate development through the BzpR transcription factor in Dictyostelium discoideum.

BACKGROUND: A major mechanism of translational regulation in response to a variety of stresses is mediated by phosphorylation of eIF2α to reduce delivery of initiator tRNAs to scanning ribosomes. For some mRNAs, often encoding a bZIP transcription factor, eIF2α phosphorylation leads to enhanced translation due to delayed reinitiation at upstream open reading frames. Dictyostelium cells possess at least three eIF2α kinases that regulate various portions of the starvation-induced developmental program. Cells possessing an eIF2α that cannot be phosphorylated (BS167) show abnormalities in growth and development. We sought to identify a bZIP protein in Dictyostelium whose production is controlled by the eIF2α regulatory system. PRINCIPAL FINDINGS: Cells disrupted in the bzpR gene had similar developmental defects as BS167 cells, including small entities, stalk defects, and reduced spore viability. ß-galactosidase production was used to examine translation from mRNA containing the bzpR 5' UTR. While protein production was readily apparent and regulated temporally and spatially in wild type cells, essentially no ß-galactosidase was produced in developing BS167 cells even though the lacZ mRNA levels were the same as those in wild type cells. Also, no protein production was observed in strains lacking IfkA or IfkB eIF2α kinases. GFP fusions, with appropriate internal controls, were used to directly demonstrate that the bzpR 5' UTR, possessing 7 uORFs, suppressed translation by 12 fold. Suppression occurred even when all but one uORF was deleted, and translational suppression was removed when the ATG of the single uORF was mutated. CONCLUSIONS: The findings indicate that BzpR regulates aspects of the development program in Dictyostelium, serving as a downstream effector of eIF2α phosphorylation. Its production is temporally and spatially regulated by eIF2α phosphorylation by IfkA and IfkB and through the use of uORFs within the bzpR 5' UTR.

eIF2α kinases control chalone production in Dictyostelium discoideum.

Growing Dictyostelium cells secrete CfaD and AprA, two proteins that have been characterized as chalones. They exist within a high-molecular-weight complex that reversibly inhibits cell proliferation, but not growth, via cell surface receptors and a signaling pathway that includes G proteins. How the production of these two proteins is regulated is unknown. Dictyostelium cells possess three GCN2-type eukaryotic initiation factor 2 α subunit (eIF2α) kinases, proteins that phosphorylate the translational initiation factor eIF2α and possess a tRNA binding domain involved in their regulation. The Dictyostelium kinases have been shown to function during development in regulating several processes. We show here that expression of an unregulated, activated kinase domain greatly inhibits cell proliferation. The inhibitory effect on proliferation is not due to a general inhibition of translation. Instead, it is due to enhanced production of a secreted factor(s). Indeed, extracellular CfaD and AprA proteins, but not their mRNAs, are overproduced in cells expressing the activated kinase domain. The inhibition of proliferation is not seen when the activated kinase domain is expressed in cells lacking CfaD or AprA or in cells that contain a nonphosphorylatable eIF2α. We conclude that production of the chalones CfaD and AprA is translationally regulated by eIF2α phosphorylation. Both proteins are upregulated at the culmination of development, and this enhanced production is lacking in a strain that possesses a nonphosphorylatable eIF2α.

Subcellular localization of ammonium transporters in Dictyostelium discoideum.

BACKGROUND: With the exception of vertebrates, most organisms have plasma membrane associated ammonium transporters which primarily serve to import a source of nitrogen for nutritional purposes. Dictyostelium discoideum has three ammonium transporters, Amts A, B and C. Our present work used fluorescent fusion proteins to determine the cellular localization of the Amts and tested the hypothesis that the transporters mediate removal of ammonia generated endogenously from the elevated protein catabolism common to many protists. RESULTS: Using RFP and YFP fusion constructs driven by the actin 15 promoter, we found that the three ammonium transporters were localized on the plasma membrane and on the membranes of subcellular organelles. AmtA and AmtB were localized on the membranes of endolysosomes and phagosomes, with AmtB further localized on the membranes of contractile vacuoles. AmtC also was localized on subcellular organelles when it was stabilized by coexpression with either the AmtA or AmtB fusion transporter. The three ammonium transporters exported ammonia linearly with regard to time during the first 18 hours of the developmental program as revealed by reduced export in the null strains. The fluorescently tagged transporters rescued export when expressed in the null strains, and thus they were functional transporters. CONCLUSION: Unlike ammonium transporters in most organisms, which import NH3/NH4+ as a nitrogen source, those of Dictyostelium export ammonia/ammonium as a waste product from extensive catabolism of exogenously derived and endogenous proteins. Localization on proteolytic organelles and on the neutral contractile vacuole suggests that Dictyostelium ammonium transporters may have unique subcellular functions and play a role in the maintenance of intracellular ammonium distribution. A lack of correlation between the null strain phenotypes and ammonia excretion properties of the ammonium transporters suggests that it is not the excretion function that is important for coupling ammonia levels to the slug versus culmination choice, but rather a sensor and/or signaling function of these proteins that is important.

Disruption of the ifkA and ifkB genes results in altered cell adhesion, morphological defects and a propensity to form pre-stalk O cells during development of Dictyostelium.

IfkA and ifkB are two GCN2-like genes present in Dictyostelium. Disruption of either gene alone results in subtle developmental defects. However, disruption of ifkA and ifkB within the same strain results in severe morphological and patterning defects in the developing double null cells. The mutant cells aggregate in streams that give tightly clumped mounds. Fingers form from the mounds but remain attached to one another, especially at their bases. The fingers culminate to give fused and entangled structures lacking proper stalk but containing some spores. The morphological defects are consistent with an enhanced cell-cell and cell-substrate adhesiveness of the developing double null cells, which may result in inappropriate cell contacts and altered cell motility and sorting properties. In ifkA/ifkB nulls, cell type proportioning and patterning is altered in favor of ALC/pstO cell types. The bias toward the ALC/pstO cell types may be due, in part, to the nuclear localization of the transcription factor STATc in growing ifkA/ifkB null cells. STATc normally becomes localized to the nucleus during finger formation and only within the pre-stalk O zone. The precocious nuclear localization seen in the mutant cells may predispose the cells to a ALC/pstO cell fate. The findings indicate that IfkA and IfkB have redundant functions in Dictyostelium morphogenesis that involve maintaining proper cell-cell and cell-substrate adhesion and the equilibrium between different cell types for proper spatial patterning.

Ammonium transporter C of Dictyostelium discoideum is required for correct prestalk gene expression and for regulating the choice between slug migration and culmination.

Ammonium transporter C (AmtC) is one of three transporters in Dictyostelium that have been proposed to regulate entry and exit of ammonia in a cell type dependent manner and to mediate ammonia signaling. Previous work demonstrated that disruption of the amtC gene results in a slugger phenotype in which the cells remain as migrating slugs when they should form fruiting bodies. More detailed studies on the null strain revealed that differentiation of prestalk cell types was delayed and maintenance of prestalk cell gene expression was defective. There was little or no expression of ecmB, a marker for the initiation of culmination. Normal expression of CudA, a nuclear protein required for culmination, was absent in the anterior prestalk zone. The absence of CudA within the tip region was attributable to the lack of nuclear localization of the transcription factor STATa, despite expression of adenylyl cyclase A mRNA in the slug tips. Disruption of the histidine kinase gene dhkC in the amtC null strain restored STATa and CudA expression and the ability to culminate. The results suggest that the lack of nuclear translocation of STATa results from low cAMP due to a misregulated and overactive DhkC phosphorelay in the amtC null strain.