Acetylation coordinates the crosstalk between carbon metabolism and ammonium assimilation in Salmonella enterica.

Enteric bacteria use up to 15% of their cellular energy for ammonium assimilation via glutamine synthetase (GS)/glutamate synthase (GOGAT) and glutamate dehydrogenase (GDH) in response to varying ammonium availability. However, the sensory mechanisms for effective and appropriate coordination between carbon metabolism and ammonium assimilation have not been fully elucidated. Here, we report that in Salmonella enterica, carbon metabolism coordinates the activities of GS/GDH via functionally reversible protein lysine acetylation. Glucose promotes Pat acetyltransferase-mediated acetylation and activation of adenylylated GS. Simultaneously, glucose induces GDH acetylation to inactivate the enzyme by impeding its catalytic centre, which is reversed upon GDH deacetylation by deacetylase CobB. Molecular dynamics (MD) simulations indicate that adenylylation is required for acetylation-dependent activation of GS. We show that acetylation and deacetylation occur within minutes of "glucose shock" to promptly adapt to ammonium/carbon variation and finely balance glutamine/glutamate synthesis. Finally, in a mouse infection model, reduced S. enterica growth caused by the expression of adenylylation-mimetic GS is rescued by acetylation-mimicking mutations. Thus, glucose-driven acetylation integrates signals from ammonium assimilation and carbon metabolism to fine-tune bacterial growth control.

Tryptophanylation of insulin receptor by WARS attenuates insulin signaling.

Increased circulating amino acid levels have been linked to insulin resistance and development of type 2 diabetes (T2D), but the underlying mechanism remains largely unknown. Herein, we show that tryptophan modifies insulin receptor (IR) to attenuate insulin signaling and impair glucose uptake. Mice fed with tryptophan-rich chow developed insulin resistance. Excessive tryptophan promoted tryptophanyl-tRNA synthetase (WARS) to tryptophanylate lysine 1209 of IR (W-K1209), which induced insulin resistance by inhibiting the insulin-stimulated phosphorylation of IR, AKT, and AS160. SIRT1, but not other sirtuins, detryptophanylated IRW-K1209 to increase the insulin sensitivity. Collectively, we unveiled the mechanisms of how tryptophan impaired insulin signaling, and our data suggested that WARS might be a target to attenuate insulin resistance in T2D patients.

Monocarboxylate transporter 4 inhibition potentiates hepatocellular carcinoma immunotherapy through enhancing T cell infiltration and immune attack.

BACKGROUND AND AIMS: Monocarboxylate transporter (MCT) 4 is a high-affinity lactate transporter that is primarily involved in the maintenance of intracellular pH homeostasis and highly expressed in different tumors. However, the role of MCT4 in modulating immune responses against HCC remains unknown. APPROACH AND RESULTS: In this study, we demonstrated that MCT4 was overexpressed in HCC, which was associated with poor prognosis in patients. Genetic or pharmacological inhibition of MCT4 using VB124 (a highly potent MCT4 inhibitor) suppressed HCC tumor growth in immunocompetent mice model by enhancing CD8 + T cell infiltration and cytotoxicity. Such improved immunotherapy response by MCT4 targeting was due to combined consequences characterized by the alleviated acidification of tumor microenvironment and elevated the chemokine (C-X-C motif) ligand (CXCL) 9/CXCL10 secretion induced by reactive oxygen species/NF-κB signaling pathway. Combining MCT4 inhibition improved the therapeutic benefit of anti-programmed cell death 1 immunotherapy in HCC and prolonged mice survival. Moreover, higher MCT4 expression was observed in tumor tissues from nonresponder patients with HCC receiving neoadjuvant therapy with toripalimab. CONCLUSIONS: Our results revealed that lactate exportation by MCT4 has a tumor-intrinsic function in generating an immunosuppressive HCC environment and demonstrated the proof of the concept of targeting MCT4 in tailoring HCC immunotherapeutic approaches.

SULF1 regulates malignant progression of colorectal cancer by modulating ARSH via FAK/PI3K/AKT/mTOR signaling.

BACKGROUND: Colorectal cancer (CRC) has the third highest incidence and second mortality rate of malignant tumors globally, highlighting the urgency to explore the mechanisms underlying CRC progression for refined treatment of this patient population. METHODS: R Studio was used for data sorting and analysis. Cell apoptosis and cell cycle detection were performed by flow cytometry. Quantitative real-time PCR (qRT-PCR) was used to explore mRNA expression levels. Western blotting was used to explore protein expression levels. CCK8, EdU, and colony formation assays were performed to explore the proliferation capacity of CRC cells. Transwell invasion and migration assays, along with the wound healing assay, were used to explore the invasive and migratory abilities of CRC cells. Subcutaneous Xenograft Assay was utilized to evaluate the tumorigenic capacity of CRC cells in vivo. RESULTS: SULF1 was highly expressed in CRC samples and cell lines. The knockdown of SULF1 inhibited the proliferation, invasion, and migration of CRC and increased the rate of cell apoptosis. Meanwhile, we demonstrated that SULF1 could negatively regulate ARSH through the FAK/PI3K/AKT/mTOR pathway. CONCLUSION: We demonstrated that SULF1 could promote CRC progression by regulating ARSH. The SULF1/ARSH/FAK/PI3K/AKT/mTOR signaling pathway represents a promising target for the treatment of this patient population. Colorectal cancer (CRC) has the third highest incidence and second mortality rate of malignant tumors globally. Sulfatase 1 (SULF1) belongs to the sulfatase family, The function of SULF1 in CRC remains elusive. Our study demonstrated that the knockdown of SULF1 could inhibit the proliferation, invasion, and migration of CRC. Meanwhile, our findings indicated that SULF1 could interact with Arylsulfatase Family Member H (ARSH) to regulate the proliferation, invasion, and migration of CRC via the FAK/PI3K/AKT/mTOR signaling pathway. Taken together, our findings suggest that SULF1 might be a new therapeutic target in CRC.

Initial [18F]DCFPyL PET/CT in treatment-naïve prostate cancer: correlation with post-ADT PSA outcomes and recurrence.

PURPOSE: Positron emission tomography (PET) with prostate-specific membrane antigen (PSMA) targeting tracers has emerged as a valuable diagnostic tool for prostate cancer (PCa), androgen deprivation therapy (ADT) stands as the cornerstone treatment for advanced PCa, yet forecasting the response to hormonal therapy poses a significant clinical hurdle. METHODS: In a prospective cohort of 86 PCa patients undergoing short-term ADT, this study evaluated the prognostic potential of [18F]DCFPyL PET/CT scans. Comprehensive data encompassing clinical profiles, baseline prostate-specific antigen (PSA) levels, and imaging metrics were assessed. We developed predictive models for assessing decreases in PSA levels (PSA50 and PSA70) based on a combination of PET-related parameters and clinical factors. Kaplan-Meier survival analysis was utilized to ascertain the prognostic value of PET-based metrics. RESULTS: In this study, elevated [18F]DCFPyL uptake within the primary tumor, as indicated by a SUV ≥ 6.78 (p = 0.0024), and a reduction in the tumor volume (TV) of primary PSMA-avid tumor with PSMA-TV < 41.96 cm3 (p = 0.038), as well as an increased burden of metastatic PSMA-avid tumor, with PSMA-TV (PSMA-TV ≥ 71.39 cm3) (p = 0.012) were identified in association with diminished progression-free survival (PFS). PET and clinical parameters demonstrated constrained predictive capacity for PSA50 response as indicated by an area under the curve (AUC) of 0.442. CONCLUSION: Our study revealed that pretreatment [18F]DCFPyL uptake in primary or metastatic tumor sites is prognostically relevant in high-risk PCa patients undergoing ADT. Further research is needed to develop robust predictive models in this multifaceted landscape of PCa management.

OTU deubiquitinase, ubiquitin aldehyde binding 2 (OTUB2) modulates the stemness feature, chemoresistance, and epithelial-mesenchymal transition of colon cancer via regulating GINS complex subunit 1 (GINS1) expression.

BACKGROUND: Colon cancer is one of the most prevalent tumors in the digestive tract, and its stemness feature significantly contribute to chemoresistance, promote the epithelial-mesenchymal transition (EMT) process, and ultimately lead to tumor metastasis. Therefore, it is imperative for researchers to elucidate the molecular mechanisms underlying the enhancement of stemness feature, chemoresistance, and EMT in colon cancer. METHODS: Sphere-formation and western blotting assays were conducted to assess the stemness feature. Edu, flow cytometry, and cell viability assays were employed to evaluate the chemoresistance. Immunofluorescence and western blotting assays were utilized to detect EMT. Immunoprecipitation, ubiquitination, agarose gel electrophoresis, chromatin immunoprecipitation followed by quantitative PCR (chip-qPCR), and dual luciferase reporter gene assays were employed for mechanistic investigations. RESULTS: We demonstrated a markedly higher expression level of OTUB2 in colon cancer tissues compared to adjacent tissues. Furthermore, elevated OTUB2 expression was closely associated with poor prognosis and distant tumor metastasis. Functional experiments revealed that knockdown of OTUB2 attenuated stemness feature of colon cancer, enhanced its sensitivity to oxaliplatin, inhibited its EMT process, ultimately reduced the ability of tumor metastasis. Conversely, overexpression of OTUB2 exerted opposite effects. Mechanistically, we identified OTUB2 as a deubiquitinase for SP1 protein which bound specifically to SP1 protein, thereby inhibiting K48 ubiquitination of SP1 protein. The SP1 protein functioned as a transcription factor for the GINS1, exerting its regulatory effect by binding to the 1822-1830 region of the GINS1 promoter and enhancing its transcriptional activity. Ultimately, alterations in GINS1 expression directly regulated stemness feature, chemosensitivity, and EMT progression in colon cancer. CONCLUSION: Collectively, the OTUB2/SP1/GINS1 axis played a pivotal role in driving stemness feature, chemoresistance, and EMT in colon cancer. These results shed new light on understanding chemoresistance and metastasis mechanisms involved in colon cancer.

ULK1/2 Constitute a Bifurcate Node Controlling Glucose Metabolic Fluxes in Addition to Autophagy.

Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.

Outcome of regenerative endodontic procedures in delayed replanted immature permanent teeth with apical periodontitis: A retrospective study.

BACKGROUND: Regenerative endodontic procedures (REPs) is effective for treating young permanent teeth with pulp necrosis. However, its efficacy on delayed replanted avulsed teeth is unclear. AIM: This retrospective study aimed to assess the efficacy of REPs in treating delayed replanted immature permanent teeth with apical periodontitis. DESIGN: Avulsed teeth receiving REPs were systematically screened based on predetermined criteria. This study assessed the REP outcomes, postoperative periodontal healing, and overall treatment efficacy. Samples were grouped by REP outcomes and root development stage, with Fisher's exact tests used to compare outcomes among different groups. RESULTS: Among the included 17 teeth, 47.1% exhibited successful REPs and periodontal healing. Another 47.1%, due to replacement resorption or REP failure, were categorized as tooth survival. Healing of periapical lesions was observed in 88.2% of the cases, but only 41.2% demonstrated continued root development. Although differences were not significant (p = 0.05), teeth with continued root development had a higher rate of functional healing (85.7%) compared to those without (30%). CONCLUSION: Within the limitations of this study, REPs presented reliable outcomes for treating delayed replanted immature permanent teeth with apical periodontitis mainly in periapical lesion healing. Teeth with continued root development after REPs exhibited a higher rate of functional healing. Further investigation is required to explore potential synergies between REP outcomes and periodontal healing.

A retrospective study on the outcome of pulpotomy with iRoot BP plus in primary molars and its relationship with hemostasis time.

BACKGROUND: Pulpotomy is a crucial method to preserve primary teeth until natural exfoliation. This study aimed to evaluate the clinical and radiographic outcomes of pulpotomy with iRoot BP Plus in primary molars and to explore the association between hemostasis time and these outcomes. METHODS: Primary molars that underwent iRoot BP Plus pulpotomy and were followed for at least 12 months were selected for this study. Clinical and radiographic data were collected, and the success rate was analyzed in relation to factors such as hemostasis time, tooth type, and arch type. The tests of significance used were the chi-square test, Fisher's exact test, or Kruskal-Wallis test. Statistical significance was set at P < 0.05. RESULTS: A total of 183 teeth in 106 patients were included in the analysis. The follow-up period fell into a range of 1-3 years, with a mean of 1.6 years. The clinical and radiographic success rates were 96.7% and 92.9%, respectively. The earliest time to observe the radiographic failures was half a year after the treatment, and the latest time was two years after the treatment. Among all the teeth, 130 were recorded with hemostasis time before the application of iRoot BP Plus. Compared to teeth with a hemostasis time of 5 min or less, teeth with a hemostasis time exceeding 5 min showed no significant differences in clinical and radiographic success (P = 1.000 and 0.879). Additionally, neither arch nor teeth type showed a relationship with the pulpotomy success rate (P > 0.05). CONCLUSIONS: Pulpotomy using iRoot BP Plus in primary molars achieved favorable results. The hemostasis time may not significantly impact the outcomes of pulpotomy using iRoot BP Plus in primary molars.

The evaluation of effective rate and pain intensity of root canal treatment in primary teeth-a retrospective study.

Two-visit root canal treatment for children reduce the time of visits and the by-chair time in comparison with the three-visit root canal treatment. However, it is not clear whether two-visit root canal treatment increase the risk of complications. This study aimed to evaluate the clinical effects and post-operative pain intensity after the root canal treatment between two-visit and three-visit groups in primary molars from children.106 patients were screened for eligibility, of which 74 went back to the preservation visit. Therefore, 74 primary molars from 74 children that diagnosed with chronic pulp and periodontal tissue diseases in the clinics of pediatric dentistry were retrospectively analyzed, in which 37 in the two-visit group and 37 in the three-visit group. The total effective rate and postoperative pain intensity were assessed after treatment and all statistical data were carried out with SPSS software.The average age of children in the two-visit and three-visit groups was 6.4 and 7.0, respectively, with no significant difference (p = 0.056). The two-visit group consisted of 59.5% male and 40.5% female children, while the three-visit group consisted of 56.8% male children and 43.2% female children (p = 0.813). Two months after treatment, the total effective rate in the three-visit group was 97.30%, a little higher than that in the two-visit group (94.59%), but with no significant difference (p = 0.201). Besides, there was also no significant difference in pain intensity between the two-visit and three-visit groups (p = 0.692). Therefore, there were no significant difference of total effective rate and pain intensity in root canal treatment between the two-visit and three-visit groups in primary molars from children.

Substrate selectivity APPLies to Akt.

The protein kinase Akt occupies a central position in multiple signaling pathways. Although numerous Akt substrates have been identified, less is known about the factors that regulate specific cellular responses to Akt signaling. In this issue, Schenck et al. (2008) demonstrate that the endosomal protein Appl1 modulates Akt's substrate selectivity to promote cell survival during zebrafish development.

NADP(+)-IDH Mutations Promote Hypersuccinylation that Impairs Mitochondria Respiration and Induces Apoptosis Resistance.

Elucidating the tumorigenic mechanism of R-2-hydroxyglutarate (R-2HG) is critical for determining how NADP(+)-IDH mutations cause cancer. Here we report that R-2HG induces cancerous metabolism and apoptosis resistance through promoting hypersuccinylation. By competitive inhibition of the mitochondrial tricarboxylic acid cycle enzyme succinate dehydrogenase (SDH), R-2HG preferentially induced succinyl-CoA accumulation and hypersuccinylation in the mitochondria. IDH1 mutation-bearing glioma samples and cells were hypersuccinylated in the mitochondria. IDH1 mutation or SDH inactivation resulted in hypersuccinylation, causing respiration inhibition and inducing cancerous metabolism and mitochondrial depolarization. These mitochondrial dysfunctions induced BCL-2 accumulation at the mitochondrial membrane, leading to apoptosis resistance of hypersuccinylated cells. Relief of hypersuccinylation by overexpressing the desuccinylase SIRT5 or supplementing glycine rescued mitochondrial dysfunctions, reversed BCL-2 accumulation, and slowed the oncogenic growth of hypersuccinylated IDH1(R132C)-harboring HT1080 cells. Thus, R-2HG-induced hypersuccinylation contributes to the tumorigenicity of NADP(+)-IDH mutations, suggesting the potential of hypersuccinylation inhibition as an intervention for hypersuccinylation-related tumors.

IL-27 deficiency inhibits proliferation and invasion of trophoblasts via the SFRP2/Wnt/ß-catenin pathway in fetal growth restriction.

Background: Fetal growth restriction (FGR) is characterized by restricted fetal growth and dysregulated placental development. The etiology and pathogenesis still remain elusive. IL-27 shows multiple roles in regulating various biological processes, however, how IL-27 involves in placentation in FGR pregnancy hasn't been demonstrated. Methods: The levels of IL-27 and IL-27RA in FGR and normal placentae were determined by immunohistochemistry, western blot and RT-PCR. HTR-8/SVneo cells and Il27ra-/- murine models have been adopted to evaluate the effects of IL-27 on the bio-functions of trophoblast cells. GO enrichment and GSEA analysis were performed to explore the underlying mechanism. Findings: IL-27 and IL-27RA was lowly expressed in FGR placentae and administration of IL-27 on HTR-8/SVneo could promote its proliferation, migration and invasion. Comparing with wildtypes, Il27ra-/- embryos were smaller and lighter, and the placentae from which were poorly developed. In mechanism, the molecules of canonical Wnt/ß-catenin pathway (CCND1, CMYC, SOX9) were downregulated in Il27ra-/- placentae. In contrast, the expression of SFRP2 (negative regulator of Wnt) was increased. Overexpression of SFRP2 in vitro could impair trophoblast migration and invasion capacity. Interpretation: IL-27/IL-27RA negatively regulates SFRP2 to activate Wnt/ß-catenin, and thus promotes migration and invasion of trophoblasts during pregnancy. However, IL-27 deficiency may contribute to the development of FGR by restricting the Wnt activity.

Oral health-related quality of life and associated factors among a sample from East China with severe early childhood caries: a cross-sectional study.

BACKGROUND: To investigate the oral health-related quality of life (OHRQoL) and associated factors among a sample from East China with severe early childhood caries (S-ECC). METHODS: A total of 316 children with S-ECC and their parents were recruited to participate in a cross-sectional study. Children were examined for caries status using criteria proposed by World Health Organization (WHO). The accompanying parent was required to provide demographic information and complete two validated questionnaires in Chinese: the early childhood oral health impact scale (ECOHIS) and the 5-item oral health impact profile (OHIP). RESULTS: The study had a 98.1% response rate. Finally, the data of 300 children and their parents were analyzed. Mothers cared for their children far more than fathers in the included family (78.7% mother, 21.3% father). The mean age of children was 4.1 ± 0.7 years, ranging from 3 to 5. The mean dmft score was 13.8 ± 3.8. Few (13.7%) children never had a toothache. ECOHIS scores ranged from 0 to 38, with a mean score of 16.2 ± 7.2. The mean OHIP score was 2.9 ± 2.7. The parental age, family income, residence, history of pain, the dmft scores and parents' OHIP showed associations with ECOHIS scores or domain scores (P < 0.05). The multiple regression analysis showed that the history of pain, accompanying parents' OHIP, and the dmft scores were mainly associated with ECOHIS and child impact (P < 0.05); parental age was associated with family impact (P = 0.024). CONCLUSIONS: The parent's OHRQoL was associated with the children's OHRQoL, indicating that policymakers and clinical practitioners should improve both children's and their parents' oral health. Furthermore, the caries severity and the history of dental pain impacted children's OHRQoL.

Bi-allelic Mutations in TTC21A Induce Asthenoteratospermia in Humans and Mice.

Male infertility is a major concern affecting human reproductive health. Asthenoteratospermia can cause male infertility through reduced motility and abnormal morphology of spermatozoa. Several genes, including DNAH1 and some CFAP family members, are involved in multiple morphological abnormalities of the sperm flagella (MMAF). However, these known genes only account for approximately 60% of human MMAF cases. Here, we conducted further genetic analyses by using whole-exome sequencing in a cohort of 65 Han Chinese men with MMAF. Intriguingly, bi-allelic mutations of TTC21A (tetratricopeptide repeat domain 21A) were identified in three (5%) unrelated, MMAF-affected men, including two with homozygous stop-gain mutations and one with compound heterozygous mutations of TTC21A. Notably, these men consistently presented with MMAF and additional abnormalities of sperm head-tail conjunction. Furthermore, a homozygous TTC21A splicing mutation was identified in two Tunisian cases from an independent MMAF cohort. TTC21A is preferentially expressed in the testis and encodes an intraflagellar transport (IFT)-associated protein that possesses several tetratricopeptide repeat domains that perform functions crucial for ciliary function. To further investigate the potential roles of TTC21A in spermatogenesis, we generated Ttc21a mutant mice by using CRISPR-Cas9 technology and revealed sperm structural defects of the flagella and the connecting piece. Our consistent observations across human populations and in the mouse model strongly support the notion that bi-allelic mutations in TTC21A can induce asthenoteratospermia with defects of the sperm flagella and head-tail conjunction.

Management of radicular cyst associated with primary teeth using decompression: a retrospective study.

BACKGROUND: Radicular cysts arising from primary teeth are rare. Enucleation and marsupialization or decompression are treatment approach to odontogenic cysts. Decompression known to achieve good results in various cysts is widely used in clinic. This study aims to evaluate the efficiency of decompression in reducing radicular cysts associated with primary teeth in children. METHODS: Cases of radicular cysts associated with primary teeth treated by decompression were reviewed in the present study. Clinical information and radiologic data of pre and post decompression were measured and analyzed. RESULTS: Twenty-three patients treated for 25 cysts were included. All lesions with mean initial area 3.66 ± 2.00 cm2 were reduced after decompression time ranging 2 to 10 months. Mean rate of reduction was 0.77 ± 0.44 cm2/mo and large lesions (> 3.5 cm2) had a significantly higher reduction rate compared to smaller ones (< 3.5 cm2) (P < 0.00). All effected succedaneous teeth erupted after treatment at follow-up while 12 (46%) of them had root development problems. CONCLUSIONS: Decompression represents superiority as an effective and less invasive treatment in radicular cysts associated with primary teeth. TRIAL REGISTRATION: This study was retrospectively registered in the Ethics Committee of Ninth People's Hospital Affiliated with Shanghai JiaoTong University School of Medicine (No.SH9H-2022-T158-1).

Faecalibacterium prausnitzii produces butyrate to decrease c-Myc-related metabolism and Th17 differentiation by inhibiting histone deacetylase 3.

Decreased levels of Faecalibacterium prausnitzii (F. prausnitzii), whose supernatant plays an anti-inflammatory effect, are frequently found in inflammatory bowel disease (IBD) patients. However, the anti-inflammatory products in F. prausnitzii supernatant and the mechanism have not been fully investigated. Here we found that F. prausnitzii and F. prausnitzii-derived butyrate were decreased in the intestines of IBD patients. Supplementation with F. prausnitzii supernatant and butyrate could ameliorate colitis in an animal model. Butyrate, but not other substances produced by F. prausnitzii, exerted an anti-inflammatory effect by inhibiting the differentiation of T helper 17 (Th17) cells. The mechanism underlying the anti-inflammatory effects of the butyrate produced by F. prausnitzii involved the enhancement of the acetylation-promoted degradation of c-Myc through histone deacetylase 3 (HDAC3) inhibition. In conclusion, F. prausnitzii produced butyrate to decrease Th17 differentiation and attenuate colitis through inhibiting HDAC3 and c-Myc-related metabolism in T cells. The use of F. prausnitzii may be an effective new approach to decrease the level of Th17 cells in the treatment of inflammatory diseases.

DNA-PK facilitates piggyBac transposition by promoting paired-end complex formation.

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components Ku70, Ku80, and DNA-PKcs Overexpression or knockdown of DNA-PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA-PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA-PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA-PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.

Dysregulation of INF2-mediated mitochondrial fission in SPOP-mutated prostate cancer.

Next-generation sequencing of the exome and genome of prostate cancers has identified numerous genetic alternations. SPOP (Speckle-type POZ Protein) was one of the most frequently mutated genes in primary prostate cancer, suggesting SPOP is a potential driver of prostate cancer development and progression. However, how SPOP mutations contribute to prostate cancer pathogenesis remains poorly understood. SPOP acts as an adaptor protein of the CUL3-RBX1 E3 ubiquitin ligase complex that generally recruits substrates for ubiquitination and subsequent degradation. ER-localized isoform of the formin protein inverted formin 2 (INF2) mediates actin polymerization at ER-mitochondria intersections and facilitates DRP1 recruitment to mitochondria, which is a critical step in mitochondrial fission. Here, we revealed that SPOP recognizes a Ser/Thr (S/T)-rich motif in the C-terminal region of INF2 and triggers atypical polyubiquitination of INF2. These ubiquitination modifications do not lead to INF2 instability, but rather reduces INF2 localization in ER and mitochondrially associated DRP1 puncta formation, therefore abrogates its ability to facilitate mitochondrial fission. INF2 mutant escaping from SPOP-mediated ubiquitination is more potent in prompting mitochondrial fission. Moreover, prostate cancer-associated SPOP mutants increase INF2 localization in ER and promote mitochondrial fission, probably through a dominant-negative effect to inhibit endogenous SPOP. Moreover, INF2 is important for SPOP inactivation-induced prostate cancer cell migration and invasion. These findings reveal novel molecular events underlying the regulation of INF2 function and localization, and provided insights in understanding the relationship between SPOP mutations and dysregulation of mitochondrial dynamics in prostate cancer.

The expression of thioredoxin-1 and inflammatory cytokines in patients with sepsis.

Background: Sepsis is life-threatening organ dysfunction caused by a dysregulated host response to infection. Inflammatory response and oxidative stress play an important role in the pathophysiological process of sepsis. Thioredoxin-1 (Trx-1) is a small ubiquitous thiol protein with redox/inflammation modulatory properties relevant to the pathogenesis of sepsis. We therefore investigated the expression level and significance of Trx-1, inflammatory factors and oxidative stress in peripheral blood of sepsis patients, and to explore Trx-1 relationship with inflammatory factors and oxidative stress.Methods: Plasma samples were collected from patients with sepsis and those with healthy control. Enzyme-linked immunosorbent assays (ELISA) were used to detect for interleukin (IL-1ß), IL-6, tumor necrosis factor (TNF-α), E-selectin, endothelin-1 (ET-1), thioredoxin-1, C-reactive protein (CRP), procalcitonin (PCT) for human plasma samples; RT-PCR detection of Trx-1 and thioredoxin-interacting protein (TXNIP) mRNA levels. Colorimetric assay for glutathione (GSH) and malondialdehyde (MDA) expression level in peripheral blood of patients with sepsis; Disease severity was assessed as APACHE II.Results: The expression levels of Trx-1, inflammatory factors and oxidative stress in plasma of patients with sepsis were significantly increased, TXNIP opposite.Conclusion: Our results show that Trx-1 play important role in inflammation and oxidative stress in sepsis patients. Trx-1 may be a potential therapeutic target in sepsis.