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TAX1BP1 is a selective macroautophagy/autophagy receptor that inhibits NFKB and RIGI-like receptor (RLR) signaling to prevent excessive inflammation and maintain homeostasis. Selective autophagy receptors such as SQSTM1/p62 and OPTN are phosphorylated by the kinase TBK1 to stimulate their selective autophagy function. However, it is unknown if TAX1BP1 is regulated by TBK1 or other kinases under basal conditions or during RNA virus infection. Here, we found that TBK1 and IKBKE/IKKi function redundantly to phosphorylate TAX1BP1 and regulate its autophagic turnover through canonical macroautophagy. TAX1BP1 phosphorylation promotes its localization to lysosomes, resulting in its degradation. Additionally, we found that during vesicular stomatitis virus infection, TAX1BP1 is targeted to lysosomes in an ATG8-family protein-independent manner. Furthermore, TAX1BP1 plays a critical role in the clearance of MAVS aggregates, and phosphorylation of TAX1BP1 controls its MAVS aggrephagy function. Together, our data support a model whereby TBK1 and IKBKE license TAX1BP1-selective autophagy function to inhibit MAVS and RLR signaling. Abbreviations: ATG: autophagy related; BafA1: bafilomycin A1; CALCOCO2: calcium binding and coiled-coil domain 2; GFP: green fluorescent protein; IFA: indirect immunofluorescence assay; IFN: interferon; IκB: inhibitor of nuclear factor kappa B; IKK: IκB kinase; IRF: interferon regulatory factor; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MOI: multiplicity of infection; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase regulatory subunit gamma; NFKB: nuclear factor kappa B; OPTN: optineurin; Poly(I:C): polyinosinic-polycytidylic acid; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RIGI: RNA sensor RIG-I; RLR: RIGI-like receptor; SDD-AGE: semi-denaturing detergent-agarose gel electrophoresis; SeV: Sendai virus; SLR: SQSTM1-like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF: TNF receptor associated factor; VSV: vesicular stomatitis virus; ZnF: zinc finger.

Objective: This study assessed the feasibility of smartphone-based colposcopy (SBC) for visual inspection of the cervix by community healthcare workers in low-resource areas., Methods: This was a retrospective study conducted in community villages in rural Vietnam, where 177 participants were enrolled for a cervical cancer screening. Cervical images were obtained by pre-trained community healthcare workers using a portable, upcycled SBC (Samsung Galaxy Note 20). Images were taken before and after the visual inspection after acetic acid (VIA) examination. Captured images were stored on a web server through an Android-based application and later reviewed independently by two experienced gynecologists. Image quality was assessed, and kappa statistics were calculated for the measurement of agreement in VIA findings., Results: Cervical images of 177 women obtained between July and August 2020 were analyzed. The mean age of women was 42 ± 9.1 years, and 20.3% were postmenopausal. The percentage of adequate visibility of the squamocolumnar junction (SCJ) in the captured images was 83.1%. The kappa value for interobserver reliability was 0.61 for VIA positivity agreement between the two gynecologists. Image clarity was rated as average or above in 77.3%. The reasons for suboptimal clarity were poor focusing (15.3%), inadequate SCJ visibility (18%), and obscuring of the transformation zone due to blood (11.3%), discharge (14.7%), or artifacts such as intrauterine devices or polyps (5.1%)., Conclusion: Upcycled SBC was feasible when performed by pre-trained healthcare workers in a low-resource setting. VIA findings by SBC showed adequate agreement between two independent assessments, suggesting its potential as a method to aid cervical cancer screening., (© 2024 The Author(s). International Journal of Gynecology & Obstetrics published by John Wiley & Sons Ltd on behalf of International Federation of Gynecology and Obstetrics.)

Background: There is controversy about whether intraoperative cholangiogram (IOC) should be performed routinely during laparoscopic cholecystectomy for patients with acute biliary pancreatitis, given significant false positive and negative rates and increased resource utilization. The aim of this study was to clarify the role of IOC in cases of mild biliary pancreatitis in patients undergoing index admission cholecystectomy, its impact on patient outcomes, and the impact of blood tests, imaging, and preoperative intervention on the detection of choledocholithiasis. Methods: A retrospective review of all patients presenting with acute mild biliary pancreatitis between January 2006 and December 2019 was conducted. Data collected included patient demographics, serum chemistry, IOC, and Endoscopic Retrograde Cholangiopancreatography (ERCP) findings, imaging findings, length of stay, operative length, and long-term follow-up outcomes. Results: 284 patients met the inclusion criteria for the study. The overall false positive IOC rate was 7.4%. Worsening bilirubin trend was a positive predictive value (PPV) for positive IOC and ERCP outcomes with a relative risk of 2.93 ( P < .01) and 2.32 ( P = .013), respectively. Improving preoperative bilirubin trend had a significant negative predictive value in IOC with a relative risk of .59 ( P = .02). Positive IOC was shown to significantly increase operative length with a relative risk of 2.03 ( P < .001). Discussion: A rising preoperative bilirubin is a predictor of a positive IOC and patients with normalizing bilirubin levels or a preoperative ERCP are less likely to have choledocholithiasis. These features may be used to select patients that would benefit from an IOC for index admission cholecystectomy., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5&#95;2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5&#95;2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris ) and biochemical characterization in vitro . Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5&#95;2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications., Competing Interests: The authors declare no conflict of interest.

The Swedish Perioperative Register (SPOR) - a national quality register - supports automatic data transfer from local medical records to its central database, without the need for further manual input. Covering 100 percent of surgical operations across all publicly run hospitals in Sweden, SPOR data show regional inequality regarding patients' waiting time from the decision to operate until the start of surgery. SPOR data can inform several areas of improvement for perioperative care, such as management of postoperative pain and nausea, increased use of the Checklist for Safe Surgery, and improved logistics. The growing number of online reports downloaded locally suggests increasing use of SPOR data for follow-up and care improvement efforts. Data from many privately run surgical units do not appear in the SPOR due to technical difficulties in reporting.

BACH2 (BTB Domain and CNC Homolog 2) is a transcription factor that serves as a central regulator of immune cell differentiation and function, particularly in T and B lymphocytes. A picture is emerging that BACH2 may function as a master regulator of cell fate that is exquisitely sensitive to cell activation status. In particular, BACH2 plays a key role in stabilizing the phenotype and suppressive function of transforming growth factor-beta (TGF-β)-derived human forkhead box protein P3 (FOXP3) + inducible regulatory T cells (iTregs), a cell type that holds great clinical potential as a cell therapeutic for diverse inflammatory conditions. As such, BACH2 potentially could be targeted to overcome the instability of the iTreg phenotype and suppressive function that has hampered their clinical application. In this review, we focus on the role of BACH2 in T cell fate and iTreg function and stability. We suggest approaches to modulate BACH2 function that may lead to more stable and efficacious Treg cell therapies.

Recently, viruses have been shown to regulate selective autophagy for productive infections. For instance, human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV), activates selective autophagy of mitochondria, termed mitophagy, thereby inhibiting antiviral innate immune responses during lytic infection in host cells. We previously demonstrated that HHV-8 viral interferon regulatory factor 1 (vIRF-1) plays a crucial role in lytic replication-activated mitophagy by interacting with cellular mitophagic proteins, including NIX and TUFM. However, the precise molecular mechanisms by which these interactions lead to mitophagy activation remain to be determined. Here, we show that vIRF-1 binds directly to mammalian autophagy-related gene 8 (ATG8) proteins, preferentially GABARAPL1 in infected cells, in an LC3-interacting region (LIR)-independent manner. Accordingly, we identified key residues in vIRF-1 and GABARAPL1 required for mutual interaction and demonstrated that the interaction is essential for mitophagy activation and HHV-8 productive replication. Interestingly, the mitophagy receptor NIX promotes vIRF-1-GABARAPL1 interaction, and NIX/vIRF-1-induced mitophagy is significantly inhibited in GABARAPL1-deficient cells. Moreover, a vIRF-1 variant defective in GABARAPL1 binding substantially loses the ability to induce vIRF-1/NIX-induced mitophagy. These results suggest that NIX supports vIRF-1 activity as a mitophagy mediator. In addition, we found that NIX promotes vIRF-1 aggregation and stabilizes aggregated vIRF-1. Together, these findings indicate that vIRF-1 plays a role as a viral mitophagy mediator that can be activated by a cellular mitophagy receptor., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Vo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Viral control of mitochondria via mitophagy has a dampening effect on mitochondrion-mediated innate immune responses. We previously found that human herpesvirus 8 (HHV-8) could activate mitophagy via its lytic gene product vIRF-1 (viral interferon regulatory factor 1). Mechanistically, we previously demonstrated that vIRF-1 interacts with the mitophagic proteins BNIP3L (BCL2 interacting protein 3 like) and TUFM (Tu translation elongation factor, mitochondrial). Despite these significant findings, however, the precise molecular mechanisms underlying vIRF-1-activated mitophagy, particularly with core components of the autophagy machinery, remained to be fully elucidated. We recently reported that vIRF-1 binds preferentially and directly to GABARAPL1 (GABA type A receptor associated protein like 1) in a noncanonical manner, and this interaction is essential for virus-productive replication. Furthermore, we found that BNIP3L is a crucial factor that promotes vIRF-1 oligomerization and associated mitophagy activation, including GABARAPL1 interaction with vIRF-1 and TUFM dimerization. Together, our findings deepen our understanding of lytic infection-induced mitophagy and provide the key protein-protein interactions involved in vIRF-1-mediated mitophagy., Competing Interests: Disclosure statement All authors declare no conflict of interest.

TAX1BP1 is a selective macroautophagy/autophagy receptor that plays a central role in host defense to pathogens and in regulating the innate immune system. TAX1BP1 facilitates the xenophagic clearance of pathogenic bacteria such as Salmonella typhimurium and Mycobacterium tuberculosis and regulates TLR3 (toll-like receptor 3)-TLR4 and DDX58/RIG-I-like receptor (RLR) signaling by targeting TICAM1 and MAVS for autophagic degradation respectively. In addition to these canonical autophagy receptor functions, TAX1BP1 can also exert multiple accessory functions that influence the biogenesis and maturation of autophagosomes. In this review, we will discuss and integrate recent findings related to the autophagy function of TAX1BP1 and highlight outstanding questions regarding its functions in autophagy and regulation of innate immunity and host defense. Abbreviations: ATG: autophagy related; CALCOCO: calcium binding and coiled-coil domain; CC: coiled-coil; CHUK/IKKα: conserved helix-loop-helix ubiquitous kinase; CLIR: noncanonical LC3-interacting region; GABARAP: gamma-aminobutyric acid receptor associated protein; HTLV-1: human T-lymphotropic virus 1; IFN: interferon; IL1B/IL1β: interleukin 1 beta; LIR: LC3-interacting region; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MAPK/JNK: mitogen-activated protein kinase; mATG8: mammalian Atg8 homolog; MAVS: mitochondrial antiviral signaling protein; MEF: mouse embryonic fibroblast; MTB: Mycobacterium tuberculosis ; MYD88: myeloid differentiation primary response gene 88; NBR1: NBR1, autophagy cargo receptor; NFKB/NF-κB: nuclear factor of kappa light polypeptide gene enhancer in B cells; OPTN: optineurin; Poly(I:C): polyinosinic:polycytidylic acid; PTM: post-translational modification; RB1CC1: RB1-inducible coiled-coil 1; RIPK: receptor (TNFRSF)-interacting serine-threonine kinase; RLR: DDX58/RIG-I-like receptor; RSV: respiratory syncytia virus; SKICH: SKIP carboxyl homology; SLR: SQSTM1 like receptor; SQSTM1: sequestosome 1; TAX1BP1: Tax1 (human T cell leukemia virus type I) binding protein 1; TBK1: TANK-binding kinase 1; TICAM1: toll-like receptor adaptor molecule 1; TLR: toll-like receptor; TNF: tumor necrosis factor; TNFAIP3: TNF alpha induced protein 3; TNFR: tumor necrosis factor receptor; TOM1: target of myb1 trafficking protein; TRAF: TNF receptor-associated factor; TRIM32: tripartite motif-containing 32; UBD: ubiquitin binding domain; ZF: zinc finger.

Background: Nannizzia incurvata, a species belonging to the Nannizzia gypsea complex, is considered a neglected pathogen., Objective: To detected N. incurvata isolates from dermatophytosis patients in Hue city - Viet Nam, and test the antifungal susceptibility of this species. Moreover, fungal capability to produce hydrolytic enzymes was evaluated., Methods: Patients' samples were collected and cultured on Sabouraud-chloramphenicol-cycloheximide medium. Dermatophytes isolates were initially macroscopically and microscopically identified. ITS PCR-RFLP and ITS rDNA sequences were performed to determine and confirm species. An ITS Neighbor-Joining phylogenetic tree evaluated the genetic relationship among isolates. Fungal hydrolytic enzymes were examined, including lipase, phospholipase and protease. Antifungal susceptibility testing was carried out by the disk diffusion method. MICs of itraconazole, voriconazole, and terbinafine against these isolates were determined by the broth microdilution method., Results: Twelve isolates of N. gypsea complex were preliminary morphologically identified. PCR-RFLP and ITS-rDNA sequencing identified and confirmed dermatophytes as N. incurvata strains, respectively. An evident polymorphism among isolates was highlighted in the phylogenetic tree. All isolates showed the activity of lipase, phospholipase, and protease production. Overall, all N. incurvata isolates were susceptible to itraconazole, voriconazole, clotrimazole, miconazole, and terbinafine. Few isolates were susceptible to griseofulvin, and none of them were susceptible to fluconazole., Conclusions: There was a presence of polyclonal N. incurvata isolates in dermatophytosis patients from Hue city, identified by PCR-RLFP and confirmed by ITS sequencing. We confirmed PCR-RLFP as a reliable technique to identify this species. Azole and terbinafine are the optimal choices for N. incurvata treatment except for fluconazole., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2022 SFMM. Published by Elsevier Masson SAS. All rights reserved.)

Mitochondria support multiple cell functions, but an accumulation of dysfunctional or excessive mitochondria is detrimental to cells. We previously demonstrated that a defect in the autophagic removal of mitochondria, termed mitophagy, leads to the acceleration of apoptosis induced by herpesvirus productive infection. However, the exact molecular mechanisms underlying activation of mitophagy and regulation of apoptosis remain poorly understood despite the identification of various mitophagy-associated proteins. Here, we report that the mitochondrial translation elongation factor Tu, a mitophagy-associated protein encoded by the TUFM gene, locates in part on the outer membrane of mitochondria (OMM) where it acts as an inhibitor of altered mitochondria-induced apoptosis through its autophagic function. Inducible depletion of TUFM potentiated caspase-8-mediated apoptosis in virus-infected cells with accumulation of altered mitochondria. In addition, TUFM depletion promoted caspase-8 activation induced by treatment with TNF-related apoptosis-inducing ligand in cancer cells, potentially via dysregulation of mitochondrial dynamics and mitophagy. Importantly, we revealed the existence of and structural requirements for autophagy-competent TUFM on the OMM; the GxxxG motif within the N-terminal mitochondrial targeting sequences of TUFM was required for self-dimerization and mitophagy. Furthermore, we found that autophagy-competent TUFM was subject to ubiquitin-proteasome-mediated degradation but stabilized upon mitophagy or autophagy activation. Moreover, overexpression of autophagy-competent TUFM could inhibit caspase-8 activation. These studies extend our knowledge of mitophagy regulation of apoptosis and could provide a novel strategic basis for targeted therapy of cancer and viral diseases., (© 2021. The Author(s).)

Recently, plant-derived anti-inflammatory products have received an increasing attention from researchers due to their excellent in vivo activity with limited side effects. Therefore, the extraction of natural active compounds from the plant with high purity for use in anti-inflammatory formulations is required. In this study, oily Capsicum oleoresin (OCO) was extracted from Capsicum frutescens L. in ethanol by the ultrasound-assisted extraction technique, followed by a centrifugation step for a high purity OCO extract, which can be applied to develop anti-inflammatory formulations. The impact of various conditions (ethanol concentration, sonicating temperature, extraction time, solvent-to-sample ratio, and extraction repetition) on the efficiency of the extraction process was investigated. The results showed that the optimized conditions for the high yield of OCO were 95% ethanol, 50-60°C, 60 minutes, solvent-to-sample ratio of 5 : 1 ml/g, and one extraction repetition, followed by centrifuging at 5000 rpm in 2 hours. Then, the purity and in vivo anti-inflammatory activities of the obtained OCO was then determined by using the HPLC method and carrageenan-induced mice paw edema model, respectively. The purity of OCO was determined as 3.408 mg capsaicin per gram of Capsicum powder; meanwhile, its anti-inflammatory effect value was approximate to that of the commercial drug diclofenac after 48 hours of treatment. The high purity OCO prepared by this low-cost and ecofriendly extraction process would be a promising material for anti-inflammatory formulations., Competing Interests: The authors declare that there are no conflicts of interest., (Copyright © 2021 Dinh Tien Dung Nguyen et al.)

Background: Residual/reconverted red bone marrow (RBM) in adult knees is occasionally observed on routine knee magnetic resonance imaging (MRI). We aimed to identify its prevalence, distribution, and associations with lifestyle factors, knee structural abnormalities, and knee symptoms in young adults., Methods: Participants ( n = 327; aged = 31-41 years) were selected from the Childhood Determinants of Adult Health (CDAH) knee study. They underwent T1-weighted and proton-density-weighted fat-suppressed MRI scans of knees. Residual/reconverted RBM in distal femur and proximal tibia were graded semi-quantitatively (grades: 0-3) based on the percentage area occupied. Knee structural abnormalities were graded semi-quantitatively using previously published MRI scoring systems. Knee symptoms (pain, stiffness, and dysfunction) were assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scale during CDAH knee study (year: 2008-2010) and at 6-9-year follow-up during the CDAH-3 study (year: 2014-2019). Associations between definite RBM (grade ≥ 2) and lifestyle factors, knee symptoms, and structural abnormalities were described using log-binomial regressions., Results: Definite RBM was seen in females only, in 29 out of 154 cases (18.8%), with femoral involvement preceding tibial involvement. Definite RBM was associated with increased BMI (PR = 1.09/kg/m 2 ; 95% CI: 1.03, 1.16), overweight status (PR = 2.19; 95% CI: 1.07, 4.51), and WOMAC knee pain (PR = 1.75; 95% CI: 1.11, 2.74) in cross-section analysis. However, there was no association between RBM and knee-pain after seven years (PR = 1.15; 95% CI: 0.66, 2.00). There were no associations between RBM and knee structural abnormalities., Conclusion: Presence of definite RBM in young adult knees was observed in females only. Definite RBM was associated with overweight measures, and the modest association with knee pain may not be causally related.

Selective autophagy has emerged as a key mechanism of quality and quantity control responsible for the autophagic degradation of specific subcellular organelles and materials. In addition, a specific type of selective autophagy (xenophagy) is also activated as a line of defense against invading intracellular pathogens, such as viruses. However, viruses have evolved strategies to counteract the host's antiviral defense and even to activate some proviral types of selective autophagy, such as mitophagy, for their successful infection and replication. This review discusses the current knowledge on the regulation of selective autophagy by human herpesviruses.

Objective(s): To compare psychomotor development during 5 months to 30 months of age among children who were conceived via ICSI in-vitro fertilization to those conceived naturally., Study Design: Prospective cohort study was conducted during 2017-2018 with children visiting Tu Du Hospital for scheduled health checks. We randomly recruited 935 five-to-thirty-month-old children and distributed them into two groups for comparison: a group of 426 ICSI/IVF children and a group of 509 Naturally Conceived (NC) children. A team of pediatric mental health professionals used the revised Brunet-Lézine scale to directly examine those children and assess their Developmental Quotient (DQ). Controlling confounders with Propensity Score Matching (PSM), we analyzed and compared psychomotor development in 421 IVF children conceived by intracytoplasmic sperm injection (ICSI) against 421 NC., Result(s): At DQ cut-off point of 85, study data pinpointed no difference in such single DQs as motor posture, sociability and global scores between the two cohort groups. However, there was a difference in coordination and language DQs. IVF group's low-score proportion for coordination was 2.16 times that of NC group [95 % CI: 1.11-4.21] and its low-score proportion for language was 2.15 times that of NC group [95 % CI: 1.15-4.01]., Conclusion: This study showed that IVF was not completely free from adverse effects. At the age of five to thirty months, IVF children would develop language and motor coordination more slowly than NC children, suggesting IVF should be done only when needed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Electric energy consumption forecasting is an interesting, challenging, and important issue in energy management and equipment efficiency improvement. Existing approaches are predictive models that have the ability to predict for a specific profile, i.e., a time series of a whole building or an individual household in a smart building. In practice, there are many profiles in each smart building, which leads to time-consuming and expensive system resources. Therefore, this study develops a robust framework for the Multiple Electric Energy Consumption forecasting (MEC) of a smart building using Transfer Learning and Long Short-Term Memory (TLL), the so-called MEC-TLL framework. In this framework, we first employ a k-means clustering algorithm to cluster the daily load demand of many profiles in the training set. In this phase, we also perform Silhouette analysis to specify the optimal number of clusters for the experimental datasets. Next, this study develops the MEC training algorithm, which utilizes a cluster-based strategy for transfer learning the Long Short-Term Memory models to reduce the computational time. Finally, extensive experiments are conducted to compare the computational time and different performance metrics for multiple electric energy consumption forecasting on two smart buildings in South Korea. The experimental results indicate that our proposed approach is capable of economical overheads while achieving superior performances. Therefore, the proposed approach can be applied effectively for intelligent energy management in smart buildings.

Malignant metastatic melanoma (MM) is the most lethal of all skin cancers, but detailed mechanisms for regulation of melanoma metastasis are not fully understood. Here, we demonstrated that developmentally regulated GTP-binding protein 2 (DRG2) is required for the growth of primary tumors and for metastasis. DRG2 expression was significantly increased in MM compared with primary melanoma (PM) and dysplastic nevi. A correlation between DRG2 expression and poor disease-specific survival in melanoma patients was also identified. Furthermore, inhibition of DRG2 suppressed the binding of Hypoxia-inducible factor 1α to the VEGF-A promoter region, expression of vascular endothelial growth factor (VEGF)-A, and formation of endothelial cell tubes. In experimental mice, DRG2 depletion inhibited the growth of PM and lung metastases and increased survival. These results identify DRG2 as a critical regulator of VEGF-A expression and of growth of PMs and lung metastases., (© 2019 Federation of European Biochemical Societies.)

The enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases 3 (PFKFB3) catalyzes the first committed rate-limiting step of glycolysis and is upregulated in cancer cells. The mechanism of PFKFB3 expression upregulation in cancer cells has not been fully elucidated. The PFKFB3 3'-UTR is reported to contain AU-rich elements (AREs) that are important for regulating PFKFB3 mRNA stability. However, the mechanisms by which PFKFB3 mRNA stability is determined by its 3'-UTR are not well known. We demonstrated that tristetraprolin (TTP), an ARE-binding protein, has a critical function regulating PFKFB3 mRNA stability. Our results showed that PFKFB3 mRNA contains three AREs in the 3'-UTR. TTP bound to the 3rd ARE and enhanced the decay of PFKFB3 mRNA. Overexpression of TTP decreased PFKFB3 expression and ATP levels but increased GSH level in cancer cells. Overexpression of PFKFB3 cDNA without the 3'-UTR rescued ATP level and GSH level in TTP-overexpressing cells. Our results suggested that TTP post-transcriptionally downregulated PFKFB3 expression and that overexpression of TTP may contribute to suppression of glycolysis and energy production of cancer cells in part by downregulating PFKFB3 expression., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Developmentally regulated GTP-binding protein 2 (DRG2) was first identified in the central nervous system of mice. However, the physiological function of DRG2 in the brain remains largely unknown. Here, we demonstrated that knocking out DRG2 impairs the function of dopamine neurons in mice. DRG2 was strongly expressed in the neurons of the dopaminergic system such as those in the striatum (Str), ventral tegmental area (VTA), and substantia nigra (SN), and on neuronal cell bodies in high-density regions such as the hippocampus (HIP), cerebellum, and cerebral cortex in the mouse brain. DRG2 knockout (KO) mice displayed defects in motor function in motor coordination and rotarod tests and increased anxiety. However, unexpectedly, DRG2 depletion did not affect the dopamine (DA) neuron population in the SN, Str, or VTA region or dopamine synthesis in the Str region. We further demonstrated that dopamine release was significantly diminished in the Str region of DRG2 KO mice and that treatment of DRG2 KO mice with l-3,4-dihydroxyphenylalanine (L-DOPA), a dopamine precursor, rescued the behavioral motor deficiency in DRG2 KO mice as observed with the rotarod test. This is the first report to identify DRG2 as a key regulator of dopamine release from dopamine neurons in the mouse brain.

Viral control of mitochondrial quality and content has emerged as an important mechanism for counteracting the host response to virus infection. Despite the knowledge of this crucial function of some viruses, little is known about how herpesviruses regulate mitochondrial homeostasis during infection. Human herpesvirus 8 (HHV-8) is an oncogenic virus causally related to AIDS-associated malignancies. Here, we show that HHV-8-encoded viral interferon regulatory factor 1 (vIRF-1) promotes mitochondrial clearance by activating mitophagy to support virus replication. Genetic interference with vIRF-1 expression or targeting to the mitochondria inhibits HHV-8 replication-induced mitophagy and leads to an accumulation of mitochondria. Moreover, vIRF-1 binds directly to a mitophagy receptor, NIX, on the mitochondria and activates NIX-mediated mitophagy to promote mitochondrial clearance. Genetic and pharmacological interruption of vIRF-1/NIX-activated mitophagy inhibits HHV-8 productive replication. Our findings uncover an essential role of vIRF-1 in mitophagy activation and promotion of HHV-8 lytic replication via this mechanism.

Background: Primary care providers (PCPs) are often challenged to address multiple patient concerns during time-limited visits. The need for PCPs to limit the number of issues addressed may have a negative impact on discussion of patient-defined visit priorities., Methods: Using data from a recent clinical trial (Aligning Patients and Providers, ClinicalTrials.gov: NCT02707146), we examined the association between patient-defined visit priorities and subsequent provider actions taken during and after the visit. We tested the hypothesis that psychosocial concerns (eg, stress, anxiety, caregiving demands) are less likely to be addressed than traditional medical concerns., Results: We analyzed 147 patient-defined visit priorities submitted just before the visit by 109 patients (mean age, 59.0 ± 12.7 years; including 73.4% women, 47.7% non-White race/ethnicity). Nearly one quarter of patient-defined visit priorities were related to psychosocial concerns (35/147; 23.8%). In models adjusting for age, gender, race/ethnicity, and familiarity with PCP, patients' psychosocial priorities were significantly less likely than medical priorities to be addressed during the visit (63% vs. 88%; adjusted odds ratio [aOR], 0.16; 95% CI, 0.06 to 0.41; P < .001), to receive clinical action (51% vs. 82%; aOR, 0.15; 95% CI, 0.06 to 0.38; P < .001), or to receive post visit information from the primary care doctor (17% vs. 32%; aOR, 0.39; 95% CI, 0.14 to 1.08; P = .07)., Conclusions: Patient-defined psychosocial priorities are less likely to be addressed during (or immediately after) primary care visits compared with patient-defined medical priorities., Competing Interests: Conflict of interest: none declared., (© Copyright 2019 by the American Board of Family Medicine.)

Background: Most patients with diabetes do not meet all evidence-based goals of care, and many patients report poor communication and lack of involvement in decision-making during primary care visits., Objective: To test the hypothesis that a "Pre-Visit Prioritization" secure email message could improve visit communication and glycemic control among patients with type 2 diabetes., Design: We conducted a pragmatic, provider-randomized, multi-site clinical trial from March 2015 to October 2016 across 30 primary care practices within Kaiser Permanente Northern California (KPNC), a large integrated care delivery system., Participants: Eligible patients had at least 1 year of KPNC membership, type 2 diabetes with most recently measured hemoglobin A1c (HbA1c) > = 8.0%, and were registered users of the KPNC online patient portal., Interventions: Patients in the intervention arm, upon booking an appointment, received a secure email through the KPNC online portal with a link to the EHR allowing them to submit their top one or two priorities prior to the visit. Control patients received usual care., Main Measures: Glycemic control; change in HbA1c 6 and 12 months after the initial visit; patient-reported outcomes related to patient-provider communication and patient care experiences., Key Results: During the study period, 1276 patients had at least one eligible visit. In post-visit surveys (n = 457), more intervention arm patients reported preparing questions for their visit (72% vs 63%, p = 0.048) and being given treatment choices to consider (81% vs 73%, p = 0.041). Patients in both arms had similar reductions in HbA1c over the 12-month study period (0.56% ± 1.45%), with no significant differences between arms., Conclusions: A "light touch" email-based pre-visit intervention resulted in improved measures of visit interaction but did not significantly improve glycemic control relative to usual care. Improving diabetes clinical outcomes through more effective primary care visits may require more intensive approaches to patient visit preparation., Trial Registry: NCT02375932.

Hexokinase 2 (HK2) catalyzes the first step of glycolysis and is up-regulated in cancer cells. The mechanism has not been fully elucidated. Tristetraprolin (TTP) is an AU-rich element (ARE)-binding protein that inhibits the expression of ARE-containing genes by enhancing mRNA degradation. TTP expression is down-regulated in cancer cells. We demonstrated that TTP is critical for down-regulation of HK2 expression in cancer cells. HK2 mRNA contains an ARE within its 3'-UTR. TTP binds to HK2 3'-UTR and enhances degradation of HK2 mRNA. TTP overexpression decreased HK2 expression and suppressed the glycolytic capacity of cancer cells, measured as glucose uptake and production of glucose-6-phosphate, pyruvate, and lactate. TTP overexpression reduced both the extracellular acidification rate (ECAR) and the oxygen consumption rate (OCR) of cancer cells. Ectopic expression of HK2 in cancer cells attenuated the reduction in glycolytic capacity, ECAR, and OCR from TTP. Taken together, these findings suggest that TTP acts as a negative regulator of HK2 expression and glucose metabolism in cancer cells.

Purpose: Time during primary care visits is limited. We tested the hypothesis that a waiting room health information technology (IT) tool to help patients identify and voice their top visit priorities would lead to better visit interactions and improved quality of care., Methods: We designed a waiting room tool, the Visit Planner, to guide adult patients through the process of identifying their top priorities for their visit and effectively expressing these priorities to their clinician. We tested this tool in a cluster-randomized controlled trial with usual care as the control. Eligible patients had at least 1 clinical care gap (eg, overdue for cancer screening, suboptimal chronic disease risk factor control, or medication nonadherence)., Results: The study (conducted March 31, 2016 through December 31, 2017) included 750 English- or Spanish-speaking patients. Compared with usual care patients, intervention patients more often reported "definitely" preparing questions for their doctor (59.5% vs 45.1%, P <.001) and "definitely" expressing their top concerns at the beginning of the visit (91.3% vs 83.3%, P = .005). Patients in both arms reported high levels of satisfaction with their care (86.8% vs 89.9%, P = .20). With 6 months of follow-up, prevalence of clinical care gaps was reduced by a similar amount in each study arm., Conclusions: A simple waiting room-based tool significantly improved visit communication. Patients using the Visit Planner were more prepared and more likely to begin the visit by communicating their top priorities. These changes did not, however, lead to further reduction in aggregate clinical care gaps beyond the improvements seen in the usual care arm., (© 2019 Annals of Family Medicine, Inc.)

Methods of producing medium-chain-length poly-3-hydroxyalkanoate (mcl-PHA) with high content of the dominant subunit, 3-hydroxydecanoate (HD), were examined with an emphasis on a high yield of polymer from decanoic acid. High HD content was achieved by using a β-oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA-F1) or by inhibiting β-oxidation with addition of acrylic acid (Aa) to wild type P. putida KT2440 in carbon-limited, fed-batch fermentations. At a substrate feed ratio of decanoic acid and acetic acid to glucose (DAA:G) of 6:4 g/g, P. putida DBA-F1 accumulated significantly higher HD (97 mol%), but much lower biomass (8.5 g/L) and PHA (42% of dry biomass) than the wild type. Both biomass and PHA concentrations were improved by decreasing the ratio of DAA:G to 4:6. Moreover, when the substrate feed ratio was further decreased to 2:8, 18 g/L biomass containing 59% mcl-PHA consisting of 100 mol% HD was achieved. The yield of PHA from decanoic acid was 1.24 (g/g) indicating that de novo synthesis had contributed to production. Yeast extract and tryptone (YET) addition allowed the mutant strain to accumulate 74% mcl-PHA by weight with 97 mol% HD at a production rate of 0.41 g/L/hr, at least twice that of published data for any β-oxidation knock-out mutant. Higher biomass concentration was achieved with Aa inhibition of β-oxidation in the wild type but the HD content (84 mol%) was less than that of the mutant. A carbon balance showed a marked increase in supernantant organic carbon for the mutant indicating overflow metabolism. Increasing the dominant monomer content (HD) greatly increased melting point, crystallinity, and rate of crystallization., (© 2017 Wiley Periodicals, Inc.)

Mitochondrial dynamics play critical roles in maintaining mitochondrial functions. Here, we report a novel mechanism for regulation of mitochondrial dynamics mediated by tristetraprolin (TTP), an AU-rich element (ARE)-binding protein. Overexpression of TTP resulted in elongated mitochondria, down-regulation of mitochondrial oxidative phosphorylation, reduced membrane potential, cytochrome c release, and increased apoptotic cell death in cancer cells. TTP overexpression inhibited the expression of α-Synuclein (α-Syn). TTP bound to the ARE within the mRNA 3'-untranslated regions (3'-UTRs) of α-Syn and enhanced the decay of α-Syn mRNA. Overexpression of α-Syn without the 3'-UTR restored TTP-induced defects in mitochondrial morphology, mitochondrial oxidative phosphorylation, membrane potential, and apoptotic cell death. Taken together, our data demonstrate that TTP acts as a regulator of mitochondrial dynamics through enhancing degradation of α-Syn mRNA in cancer cells. This finding will increase understanding of the molecular basis of mitochondrial dynamics.

Mitochondrial dynamics, including constant fusion and fission, play critical roles in maintaining mitochondrial morphology and function. Here, we report that developmentally regulated GTP-binding protein 2 (DRG2) regulates mitochondrial morphology by modulating the expression of the mitochondrial fission gene dynamin-related protein 1 (Drp1). shRNA-mediated silencing of DRG2 induced mitochondrial swelling, whereas expression of an shRNA-resistant version of DRG2 decreased mitochondrial swelling in DRG2-depleted cells. Analysis of the expression levels of genes involved in mitochondrial fusion and fission revealed that DRG2 depletion significantly decreased the level of Drp1. Overexpression of Drp1 rescued the defect in mitochondrial morphology induced by DRG2 depletion. DRG2 depletion reduced the mitochondrial membrane potential, oxygen consumption rate (OCR), and amount of mitochondrial DNA (mtDNA), whereas it increased reactive oxygen species (ROS) production and apoptosis. Taken together, our data demonstrate that DRG2 acts as a regulator of mitochondrial fission by controlling the expression of Drp1., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: False-positive infectious transfusion screening results remain a challenge with continued loss of both donors and blood products. We sought to identify associations between donor demographic characteristics (age, race, sex, education, first-time donor status) and testing false positive for viruses during routine blood donation screening. In addition the study assessed the prevalence of high-risk behaviors in false-positive donors., Study Design and Methods: Blood Systems, Inc. donors with allogeneic donations between January 1, 2011, and December 31, 2012, were compared in a case-control study. Those with a false-positive donation for one of four viruses (human immunodeficiency virus [HIV], human T-lymphotropic virus [HTLV], hepatitis B virus [HBV], and hepatitis C virus [HCV]) were included as cases. Those with negative test results were controls. For a subset of cases, infectious risk factors were evaluated., Results: Black race and Hispanic ethnicity were associated with HCV and HTLV false-positive results. Male sex and lower education were associated with HCV false positivity, and age 25 to 44 was associated with HTLV false positivity. First-time donors were more likely to be HCV false positive although less likely to be HBV and HTLV false positive. No significant associations between donor demographics and HIV false positivity were observed. A questionnaire for false-positive donors showed low levels of high-risk behaviors., Conclusion: Demographic associations with HCV and HTLV false-positive results overlap with those of true infection. While true infection is unlikely given current testing algorithms and risk factor evaluation, the findings suggest nonrandom association. Further investigation into biologic mechanisms is warranted., (© 2015 AABB.)

A medium-chain-length poly-3-hydroxyalkanote (MCL-PHA) depolymerase knockout mutant of Pseudomonas putida KT2440 was produced by double homologous recombination. A carbon-limited shake-flask study confirmed that depolymerase activity was eliminated. Lysis of both mutant and wild-type strains occurred under these conditions. In carbon-limited, fed-batch culture, the yield of unsaturated monomers from unsaturated substrate averaged only 0.62 mol mol(-1) for the phaZ minus strain compared to 0.72 mol mol(-1) for the wild type. The mutant strain also produced more CO2 and less residual biomass from the same amount of carbon substrate. However, most results indicated that elimination of PHA depolymerase activity had little impact on the overall yield of biomass and PHA.

Interleukin 23 (IL-23) is an inflammatory cytokine that plays an important role in tumor promotion. Expression of IL-23 is increased in cancer cells and correlates with tumor progression. However, the mechanisms regulating IL-23 expression in cancer cells are still unclear. Here we report that tristetraprolin (TTP), an AU-rich element (ARE)-binding protein, inhibits IL-23 production in CT26 mouse colon cancer cells. Overexpression of TTP decreased the stability of IL-23 mRNA and the expression level of IL-23 in CT26 cells. Conversely, inhibition of TTP by siRNA increased IL-23 production. TTP destabilized a luciferase mRNA reporter containing the IL-23 mRNA 3'UTR, which contains five AREs. Analyses of deletion and point mutants of the IL-23 mRNA 3'UTR demonstrated that the ARE cluster between the third and fifth AREs was responsible for TTP-mediated destabilization of IL-23 mRNA. A RNA electrophoretic mobility shift assay confirmed that TTP binds to this ARE cluster. Taken together, these results demonstrate that TTP acts as a negative regulator of IL-23 gene expression in mouse colon cancer cells and suggest its potential application as a novel therapeutic target to control IL-23-mediated tumor promotion.

The proviral integration site for Moloney murine leukemia virus 1 (Pim-1) is an oncogenic serine/threonine kinase that is up-regulated in several human cancers, facilitates cell cycle progression, and suppresses apoptosis. Previously, it has been reported that the Pim-1 3'-UTR plays important roles in the regulation of Pim-1 mRNA stability. However, the mechanisms explaining how Pim-1 mRNA stability is determined by its 3'-UTR are not well known. Here, we demonstrate that tristetraprolin (TTP) plays a critical role in the regulation of Pim-1 mRNA stability. Our results show that the level of Pim-1 expression is inversely correlated with TTP expression in human cancer cells. Pim-1 mRNA contains two AU-rich elements (ARE1 and ARE2) in the 3'-UTR. TTP bound to ARE2 and enhanced the decay of Pim-1 mRNA. Overexpression of TTP decreased Pim-1 expression and p21 and p27 phosphorylation and inhibited cell growth. Overexpression of Pim-1 cDNA without the 3'-UTR attenuated the inhibitory effects of TTP on p21 phosphorylation and cell growth. In addition, inhibition of p21 by siRNA attenuated the inhibitory effect of TTP on cell growth. Our results suggest that TTP post-transcriptionally down-regulates Pim-1 expression and that the overexpression of TTP may contribute to tumor suppression in part by down-regulating Pim-1 expression.

Tristetraprolin (TTP) is a AU-rich element (ARE) binding protein and exhibits suppressive effects on cell growth through down-regulation of ARE-containing oncogenes. The let-7 microRNA has emerged as a significant factor in tumor suppression. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. In this work, an unexpected link between TTP and let-7 has been found in human cancer cells. TTP promotes an increase in expression of mature let-7, which leads to the inhibition of let-7 target gene CDC34 expression and suppresses cell growth. This event is associated with TTP-mediated inhibition of Lin28, which has emerged as a negative modulator of let-7. Lin28 mRNA contains ARE within its 3'-UTR and TTP enhances the decay of Lin28 mRNA through binding to its 3'-UTR. This suggests that the TTP-mediated down-regulation of Lin28 plays a key role in let-7 miRNA biogenesis in cancer cells.

An excess of interleukin 17 (IL-17) may contribute to chronic inflammatory disorders, but mechanisms that regulate IL-17 in immune cells are unclear. Here we report that tristetraprolin (TTP) inhibits IL-17 production in human T cell lines. Overexpression of TTP decreased the expression of IL-17. Conversely, TTP inhibition by siRNA increased IL-17 production. IL-17 mRNA contains eight AREs within its 3'UTR. TTP bound directly to the IL-17 mRNA 3'UTR at a location between the fourth and seventh AREs and enhanced decay of IL-17 transcripts. These results suggest that TTP could control IL-17-mediated inflammation., (Copyright © 2011. Published by Elsevier B.V.)

Tristetraprolin (TTP) is an AU-rich element-binding protein that regulates mRNA stability. We previously showed that TTP acts as a negative regulator of VEGF gene expression in colon cancer cells. The p38 MAPK pathway is known to suppress the TTP activity. However, until now the signaling pathway to enhance TTP function is not well known. Here, we show that casein kinase 2 (CK2) enhances the TTP function in the regulation of the VEGF expression in colon cancer cells. CK2 increased TTP protein levels and enhanced VEGF mRNA decaying activity of TTP. TTP was not a direct target of CK2. Instead, CK2 increased the phosphorylation of MKP-1, which led to a decrease in the phosphorylation of p38 MAPK. Inhibition of MKP-1 by siRNA attenuated the increase in TTP function and the decrease of p38 phosphorylation induced by CK2α overexpression. TGF-β1 increased the expressions of CK2 and TTP and the TTP function. The siRNA against CK2α or TTP reversed TGF-β1-induced increases in the expression of CK2 and TTP and the TTP function. Our data suggest that CK2 enhances the protein level and activity of TTP via the modulation of the MKP-1-p38 MAPK signaling pathway and that TGF-β1 enhances the activity of CK2.

Background and Purpose: Beyond the medical history, the clinical exam and lab findings, non-invasive ultrasound parameters such as kidney size and Doppler values (e.g. the resistive index) are important tools assisting clinical decision making in the monitoring of renal allografts. The gold standard for the diagnosis of renal allograft dysfunction remains the renal biopsy; while an invasive procedure, the justifiable necessity for this derives from its definitive nature a requirement beyond the synopses of all non-invasive tools. "Acoustic Radiation Force Impulse Imaging"(ARFI)-quantification is a novel ultrasound-based technology measuring tissue elasticity properties. So far experience related to this new method has not been reported in renal transplant follow-up. The purpose of this study was to evaluate changes in ARFI-measurements between clinically stable renal allografts and biopsy-proven transplant dysfunction., Methods: We employed "Virtual Touch™ tissue quantification" (Siemens Acuson, S2000) for the quantitative measurement of tissue stiffness in the cortex of transplant kidneys. We performed initial baseline and later disease-evaluative ultrasound examinations in 8 renal transplant patients in a prospective study design. Patients were first examined during stable allograft function with a routine post-transplant renal ultrasound protocol. A second follow-up examination was carried out on subsequent presentation with transplant dysfunction prior to allograft biopsy and histological evaluation. All patiens were examined using ARFI-quantification (15 measurements/kidney). Resistive indices (RI) were calculated using pulsed-wave Doppler ultrasound, and transplant kidney size was measured on B-mode ultrasound images. All biopsies were evaluated histologically by a reference nephropathologist unaware of the results of the ultrasound studies. Histopathological diagnoses were based on biopsy results, taking clinical and laboratory findings into account. Finally we calculated the relative changes in ARFI-quantification, resistive indices and the absolute change of kidney size on a percentage basis at these defined assessment times and compared the results with the final pathologic diagnosis., Results: Histological results enumerated five cases of acute T-cell-mediated rejection, one case of calcineurin inhibitor toxicity and two cases of acute tubular necrosis. Calcineurin inhibitor toxicity and acute tubular necrosis were subsumed as "other pathologies". Mean ARFI-values showed an average increase of more than 15% percent in transplants with histologically proven acute rejection whereas no increase was seen in transplants with other pathologies. Mean RI-values showed no increase either in the diagnostic group of acute rejection, nor in the group with other pathologies. Kidney size showed a mean absolute increase of 0.5 centimetres in allografts with acute rejection, whereas a mean decrease of 0.17 centimetres was seen in the group with other pathologies., Conclusion: As shown before in other studies, RI values and kidney size are of doubtful utility in the evaluation of kidney allograft dysfunction. ARFI-based elasticity measurement shows promise as a complementary non-invasive parameter in follow-on diagnosis of renal allograft rejection.

cIAP2 is a key regulator of programmed cell death and the NF-κB pathway. Here, we investigated the post-transcriptional regulation of cIAP2 expression by tristetraprolin (TTP). Our results showed that overexpression of TTP reduced the stability of cIAP2 mRNA and the expression level of cIAP2. In addition, TTP destabilized a luciferase mRNA containing cIAP2 mRNA 3'UTR. cIAP2 mRNA 3'UTR contains four AU-rich elements (AREs) and the 2nd ARE was responsible for the TTP-mediated destabilization of the cIAP2 mRNA. RNA EMSA revealed that TTP directly bound to 42 nucleotides from the 3'UTR of cIAP2 mRNA containing the 2nd ARE. However, the 42 nucleotides did not promote TTP-dependent destabilization of mRNA and did not recruit the decapping enzyme Dcp2 and the 5'-3' exonuclease Xrn1. When we used a 52 nucleotide sequence containing an additional 5 nucleotides from cIAP2 mRNA 3'UTR at both ends, this long nucleotide sequences recruited Dcp2 and Xrn1 and promoted TTP-dependent destabilization of mRNA. Collectively, our results suggest that TTP can bind to the 2nd ARE of cIAP2 mRNA 3'UTR and destabilize cIAP2 mRNA by forming complexes with Dcp2 and Xrn1. However, while a short nucleotide sequence containing the 2nd ARE of cIAP2 mRNA can recruit the TTP binding, this cannot recruit Dcp2 and Xrn1 and cannot induce TTP-mediated destabilize the mRNA. Instead, additional nucleotide sequences are required to recruit Dcp2 and Xrn1 and to destabilize mRNA., (Copyright © 2010 Elsevier Inc. All rights reserved.)

LATS2 is a tumor suppressor gene implicated in the control of cell growth and the cell cycle. Here, we investigated the post-transcriptional regulation of LATS2 expression by tristetraprolin (TTP). Our results show that the expression level of LATS2 is inversely correlated with TTP expression in human cancer cell lines. Overexpression of TTP reduced the expression level of LATS2. Conversely, treatment with small interfering RNA against TTP increased the expression level of LATS2 through stabilization of LATS2 mRNA and suppressed the proliferation of A549 human lung cancer cells. LATS2 mRNA contains AU-rich elements (AREs) within the 3'-untranslated region, and TTP destabilized a luciferase mRNA containing LATS2 ARE. In addition, RNA electrophoretic mobility shift assay revealed that TTP directly bound to the ARE of LATS2 mRNA. These results establish LATS2 mRNA as a physiological target of TTP and suggest the possibility that TTP controls cell growth through regulation of LATS2 mRNA stability.

phaJ and fabG genes encoding (R)-specific 2-enoyl-CoA hydratase and 3-ketoacyl-acyl carrier protein reductase, respectively, were cloned from Pseudomonas putida KCTC1639 and amplified in the parent strain to achieve oversupplementation of (R)-3-hydroxyalkanoate monomer units for the biosynthesis of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The comparative effect of the overexpressed fabG and phaJ genes in P. putida KCTC1639 on the biosynthesis of mcl-PHA and the cell growth were elucidated. Overexpression of phaJ enhanced the biosynthesis of mcl-PHA, increasing its content and concentration from 18% to 27% and 0.38 to 0.51 g/l, respectively. Conversely, fabG overexpression tended to depress the biosynthesis of mcl-PHA, possibly due to the reversible conversion of (R)-3-hydroxyalkanoate monomer units into 3-ketoacyl-CoA.

The fadBA operon in the fatty acid beta-oxidation pathway of P. putida KCTC1639 was blocked to induce a metabolic flux of the intermediates to the biosynthesis of medium chain-length PHA (mcl-PHA). Succinate at 150 mg l(-1) stimulated cell growth and also the biosynthesis of medium chain-length-polyhydroxyalkanoate. pH-stat fed-batch cultivation of the fadA knockout mutant P. putida KCTC1639 was carried out for 60 h, in which mcl-PHA reached 8 g l(-1) with a cell dry weight of 10.3 g l(-1).

Two PHA synthase phaC1 and phaC2 genes cloned from the new strain Pseudomonas putida KCTC1639 were metabolically engineered for the overproduction of medium-chain-length polyhydroxyalkanoate (mcl-PHA). The overexpressed phaC1 and phaC2 genes in P. putida KCTC1639 were compared in terms of the biosynthesis of mcl-PHA, fatty acid assimilation, distribution of 3-hydroxylacyl monomer units, granular morphology, and thermophysical properties of the accumulated mcl-PHA. The biosynthesis of mcl-PHA was enhanced only by the overexpressed phaC1 gene up to 2.86-fold, in contrast, the phaC2 gene did not activate the biosynthesis of mcl-PHA. The overexpressed phaC1 gene tended to form enlarged, high molecular weight, and lower crystalline mcl-PHA granules, whereas the amplified phaC2 gene induced the fragmentation of mcl-PHA into a few small-sized granules. The transformant P. putida KCTC1639 overexpressing the phaC1 gene encoding PHA synthase I was cultivated by pH-stat fed-batch cultivation, and the concentration and content of mcl-PHA increased up to 8.91 g L-1 and 70.5%, respectively.