Proline uptake promotes activation of lymphoid tissue inducer cells to maintain gut homeostasis.

Metabolic regulation is integral to the proper functioning of innate lymphoid cells, yet the underlying mechanisms remain elusive. Here, we show that disruption of exogenous proline uptake, either through dietary restriction or by deficiency of the proline transporter Slc6a7, in lymphoid tissue inducer (LTi) cells, impairs LTi activation and aggravates dextran sodium sulfate-induced colitis in mice. With an integrative transcriptomic and metabolomic analysis, we profile the metabolic characteristics of various innate lymphoid cell subsets and reveal a notable enrichment of proline metabolism in LTi cells. Mechanistically, defective proline uptake diminishes the generation of reactive oxygen species, previously known to facilitate LTi activation. Additionally, LTi cells deficient in Slc6a7 display downregulation of Cebpb and Kdm6b, resulting in compromised transcriptional and epigenetic regulation of interleukin-22. Furthermore, our study uncovers the therapeutic potential of proline supplementation in alleviating colitis. Therefore, these findings shed light on the role of proline in facilitating LTi activation and ultimately contributing to gut homeostasis.

KLF5­mediated expression of CARD11 promotes the progression of gastric cancer.

Caspase recruitment domain-containing protein 11 (CARD11) has been reported as functioning in multiple types of cancers. In the present study, the role and mechanism of CARD11 in gastric cancer was investigated. First, CARD11 expression in gastric cancer tissues and the association of CARD11 with overall survival were analyzed by the encyclopedia of RNA interactomes database. CARD11 expression in gastric cancer cells was detected by western blotting and reverse transcription-quantitative PCR analyses. After CARD11 silencing, cell proliferation was evaluated by Cell Counting Kit-8 assay, 5-ethynyl-2'-deoxyuridine staining and flow cytometry analysis. Wound healing and Transwell assays were used to measure the capacities of cell migration and invasion. Additionally, the expression levels of epithelial-mesenchymal transition (EMT)-related proteins and mTOR-related proteins were detected by western blot analysis. HumanTFDB predicted the binding of the transcription factor Krüppel-like factor 5 (KLF5) to the CARD11 promoter, which was confirmed by dual luciferase reporter and chromatin immunoprecipitation assays. To explore the regulatory effects between KLF5 and CARD11, KLF5 was overexpressed to perform the rescue experiments in gastric cancer cells with CARD11 silencing. Results revealed that CARD11 was highly expressed in gastric cancer and was associated with poor prognosis. CARD11 interference inhibited the proliferation of gastric cancer cells and induced cell cycle arrest. Additionally, CARD11 silencing suppressed the migration, invasion and EMT of gastric cancer cells, accompanied by upregulated E-cadherin expression and downregulated N-cadherin and vimentin expression. Moreover, the transcription factor KLF5 positively regulated the transcription of CARD11 in gastric cancer. KLF5 overexpression reversed the effects of interference of CARD11 expression in gastric cancer cells to promote their proliferation, migration, invasion and EMT. KLF5 overexpression also led to a reduction in cell cycle arrest. Finally, interference of CARD11 expression suppressed the mTOR pathway, which was activated by KLF5. In conclusion, KLF5-mediated CARD11 promoted the proliferation, migration and invasion of gastric cancer cells.

Two-Year Clinical and Radiologic Outcomes Following Surgical Treatment of Insertional Achilles Tendinopathy Using a Central Tendon-Splitting Approach.

BACKGROUND: If conservative treatment of insertional Achilles tendinopathy (IAT) fails, surgery is often considered. Various surgical approaches have been used including the central Achilles tendon splitting approach. This study aimed to report the 2-year clinical and radiologic outcomes after surgical treatment of IAT with a central tendon-splitting approach. METHODS: Seventy-five cases of IAT treated surgically via the open central tendon-splitting approach were analyzed. Clinical outcomes included the American Orthopaedic Foot & Ankle Society (AOFAS) hindfoot score, visual analog scale (VAS), 36-Item Short Form Health Survey (SF-36) physical (PCS) and mental (MCS) component summary scores, all measured at baseline and 6 and 24 months postoperatively. Radiologic parameters measured included the Fowler-Philip angle (FPA) and parallel pitch lines (PPL). RESULTS: Three patients had clinically significant wound issues but healed completely by 3 weeks. Mean AOFAS score improved from 45.63 preoperatively to 94.71 at 24 months. Mean VAS score improved from 6.73 preoperatively to 0.55, mean SF-36 PCS from 35.98 to 48.74, and mean SF-36 MCS from 53.04 to 55.43 at 24 months. Satisfaction at 2 years was 94.3%. Mean FPA decreased from 62.0 degrees preoperatively to 34.0 degrees postoperatively. PPL was positive in 82.7% (62 of 75) of cases preoperatively, decreasing to 1.3% (1 of 75) postoperatively. Increasing age and higher preoperative VAS and SF-36 MCS scores were significantly associated with improvements in postoperative AOFAS, SF-36 PCS, and MCS scores. CONCLUSION: Surgical treatment of IAT via the central tendon-splitting approach achieved substantial improvements in all patient-reported outcome measures measured. These excellent clinical outcomes continued to show improvement 2 years postoperatively. LEVEL OF EVIDENCE: Level III, retrospective cohort study.

The Analysis of the Glycosyltransferase Gene Function From a Novel Granaticin Producer, Streptomyces Vilmorinianum. YP1.

Glycosylation is common among the synthesis of natural product and imparts the bioactivity for natural product. As for granaticin, a natural product with great bioactivity, glycosylation is an unusual sugar attachment and remains enigmatic. Orf14 in the gra cluster is the predicted glycosyltransferase but without being identified. Recently, we isolated and identified a novel granaticin producer Streptomyces vilmorinianum YP1. Orf14 gene in gra cluster of YP1 is knocked out and complemented. The instrumental analysis of the blue product synthesized by orf14-deficient mutant exhibits the none-granaticin detection and deglycosylated intermediates accumulation. The bioactivity and stability test suggests the weaker or none antibacterial activity and cytotoxicity of this blue product with greater ultraviolet stability and thermostability than granaticin and derivatives produced by YP1. All the result indicates that orf14 encodes glycosyltransferase and glycosylation played an important role in the bioactivity of granaticin. Meanwhile, the blue pigment, deglycosylated intermediates, has favorable processing characteristics. Our finding supplies the function of orf14 and glycosylation, but also indicates a promising candidate of edible blue pigment applicated in food industry.

PD-1 signaling facilitates activation of lymphoid tissue inducer cells by restraining fatty acid oxidation.

Anti-programmed death-1 (PD-1) immunotherapy that aims to restore T cell activity in cancer patients frequently leads to immune-related adverse events such as colitis. However, the underlying mechanism is still elusive. Here, we find that Pdcd1-deficient mice exhibit disrupted gut microbiota and aggravated dextran sulfate sodium (DSS)-induced colitis. In addition to T cells, PD-1 is also substantially expressed in colonic lymphoid tissue inducer (LTi) cells. During DSS-induced colitis, LTi cell activation is accompanied by increased PD-1 expression, whereas PD-1 deficiency results in reduced interleukin-22 (IL-22) production by LTi cells and exacerbated inflammation. Mechanistically, activated LTi cells reprogram their metabolism toward carbohydrate metabolism and fatty acid synthesis, while fatty acid oxidation (FAO) is unchanged. However, PD-1 deficiency leads to significantly elevated FAO in LTi cells, which in turn attenuates their activation and IL-22 production. Consistently, FAO suppression efficiently restores IL-22 production in Pdcd1-/- LTi cells. Thus, our study provides unforeseen mechanistic insight into colitis occurrence during anti-PD-1 immunotherapy through LTi cell metabolic reconfiguration.

Association of Elevated Body Mass Index and Outcomes of Arthroscopic Treatment for Osteochondral Lesions of the Talus.

Background: Osteochondral lesions of the talus (OLTs) are a common condition found in patients with chronic ankle pain after previous ankle sprains. Surgical management is indicated after conservative management has failed. Hypothesis/Purpose: This study evaluates the influence of body mass index (BMI) on the early clinical outcomes of arthroscopic debridement and microfracture of OLTs. Methods: A total of 252 patients with symptomatic OLTs who failed conservative management underwent arthroscopic debridement and microfracture of OLTs over the affected ankle between 2007 and 2017. Patients from this cohort were divided into 2 groups based on BMI: the normal BMI group (NB Group) (BMI 18.5-25.0) and overweight and obese BMI group (OB Group) (BMI ≥25). Visual analogue scale (VAS), American Orthopaedic Foot & Ankle Society (AOFAS) hindfoot score, and the physical and mental component summaries of the 36-Item Short-Form Health Survey (PCS and MCS, respectively) were prospectively collected from the cohort during their standard postoperative outpatient follow-up. Results: The NB Group (n=105) and OB Group (n=147) were well matched demographically. The operative duration was significantly shorter for the NB Group compared to the OB Group. Patients from both groups had significant improvements in VAS, AOFAS, and PCS scores postoperatively at 6 and 24 months after surgery (P < .05). Between both groups, patients had comparable VAS, AOFAS, and PCS scores at preoperation, 6 months postoperation, and 24 months postoperation (P > .05). However, MCS in the OB Group was lower at 24 months postoperatively compared with the NB Group (P < .05). The OB Group reported better satisfaction scores (82.4% vs 72.6%, P < .05), and a greater proportion had their expectations met (88.2% vs 77.9%, P < .05). Conclusion: A BMI ≥25 was not associated with worse postoperative pain and functional outcomes, but rather was found to be associated with greater satisfaction and fulfillment. However, patients with BMI ≥25 required longer procedure duration and had poorer MCS scores at 24 months after surgery.Level of Evidence: Level III, retrospective cohort study.

Folic Acid/Peptides Modified PLGA-PEI-PEG Polymeric Vectors as Efficient Gene Delivery Vehicles: Synthesis, Characterization and Their Biological Performance.

Polymeric vectors are safer alternatives for gene delivery owing to their advantages as compared to viral vectors. To improve the stability and transfection efficiency of poly(lactic-co-glycolic acid) (PLGA)- and poly(ethylenimine) (PEI)-based vectors, poly(ethylene glycol) (PEG), folic acid (FA), arginylglycylaspartic acid (RGD) peptides and isoleucine-lysine-valine-alanine-valine (IKVAV) peptides were employed and PLGA-PEI-PEG-FA and PLGA-PEI-PEG-RGD copolymers were synthesized. PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA nanocomplexes (NCs) were formed through bulk mixing. The structure and properties, including morphology, particle size, surface charge and DNA encapsulation, of NCs were studied. Robust NCs with spherical shape, uniform size distribution and slightly positive charge were able to completely bind DNA above their respective N/P ratios. The critical N/P ratio for PLGA-PEI-PEG-FA/DNA, PLGA-PEI-PEG-RGD/DNA and PLGA-PEI-PEG-RGD/IKVAV/DNA NCs was identified to be 12:1, 8:1 and 10:1, respectively. The covalent modification of PEI through a combination of biodegradable PLGA, hydrophilic PEG and targeting motifs significantly decreased the cytotoxicity of PEI. The developed NCs showed both N/P ratio and cell type-dependent transfection efficiency. An increase in N/P ratio resulted in increased transfection efficiency, and much improved transfection efficiency of NCs was observed above their respective critical N/P ratios. This study provides a promising means to produce polymeric vectors for gene delivery.

Diagnostic value and prognostic significance of LI-cadherin and miR-378e in colorectal cancer.

Expression levels of LI-cadherin and miR-378e in the serum of patients with colorectal cancer, and the diagnostic value and prognostic significance in colorectal cancer were investigated. A total of 110 patients who were diagnosed with colorectal cancer in Weihai Central Hospital, from January 2012 to November 2014, were selected and enrolled in the experimental group, and 90 healthy subjects who underwent physical examination were enrolled in the control group. The expression level of miR-378e in serum was detected by reverse transcription-quantitative PCR and the expression of LI-cadherin in serum was detected by ELISA. ROC curves of LI-cadherin and miR-378e were drawn and the sensitivity and specificity of the diagnosis were estimated. The association of the expression levels of LI-cadherin and miR-378e with the survival of the patients was analyzed. LI-cadherin and miR-378e expression levels were significantly higher in the control group than those in the experimental group (P<0.001). LI-cadherin was significantly associated with the pathogenic site, the lymphatic metastasis, depth of infiltration, degree of differentiation and clinical stage (P<0.05). The sensitivity and specificity of the LI-cadherin combined with miR-378e detection were respectively 86 and 94%; the sensitivity of miR-378e detection was the highest, as well as the specificity of the combined detection. At the end of the follow-up period, the survival rates of the patients in the LI-cadherin high-expression group and miR-378e high-expression group were significantly higher than those in the low-expression groups (P<0.05). There was a significant positive correlation between the LI-cadherin and miR-378e expression levels in both the experimental and control group (r=0.5845 and 0.6356, respectively; P<0.05). In conclusion, LI-cadherin and miR-378e are expressed at low levels in colorectal cancer, suggesting that they have a good diagnostic value for colorectal cancer and can be used as biomarkers for colorectal cancer prognosis.

Mu-KRAS attenuates Hippo signaling pathway through PKCι to sustain the growth of pancreatic cancer.

The atypical protein kinase C isoform ι (PKCι) is upregulated, which cooperates with mutated KRAS (mu-KRAS) to promote the development of pancreatic cancers. However, the exact role of PKCι in KRAS-mediated pancreatic tumorigenesis is not fully defined. In the present study, we demonstrate that mu-KRAS upregulates and activates PKCι, accompanied by dephosphorylation of large tumor suppressor (LATS), a key member of the growth-inhibiting Hippo signaling pathway. As a result, Yes-associated protein 1 (YAP1; a transcriptional coactivator) is dephosphorylated and translocates to the nucleus, which promotes transcription of downstream target genes to sustain the transformed growth of pancreatic cancer cells. In contrast, when PKCι is suppressed by the chemical inhibitor or small-hairpin RNA, the levels of phosphorylated LATS and YAP1 are elevated and YAP1 is excluded from the nucleus, which enhances the susceptibility of pancreatic cancer cells harboring mu-KRAS to apoptosis. These findings shed new light on the mechanisms underlying the pancreatic tumorigenesis initiated by mu-KRAS, and suggest that the PKCι-YAP1 signaling may potentially be therapeutically targeted for restricting the growth and inducing apoptosis in pancreatic tumors expressing mu-KRAS.

[The Effects of PKCι on Anti-tumor Activity of Cytokine-induced Killer Cells Against Pancreatic Cancer Cells and the Possible Underlying Mechanisms].

OBJECTIVE: To study the impact of atypical protein kinase Cι (PKCι) isoform PKC on the pancreatic cancer cells towards the tumoricidal effect of cytokine-induced killer (CIK) cells and explore its mechanisms. METHODS: CIK cells were prepared by inducing mononuclear cells isolated from the peripheral blood of healthy people with interleukin-2 (IL-2), interferon (IFN) and CD3 mAb and subsequently co-cultured with pancreatic epithelial cell HPDE6-C7, pancreatic cancer cells MiaPaCa and PANC-1 with or without PKC inhibitor named sodium thiomalate (ATM). All cells were divided into control group, ATM group, co-culture group with CIK and co-culture group with CIK+ATM. Cell count was used to detect the growth of each group from 1 to 8 d. Flow cytometry was used to detect the death rate of the cell lines after 48 h cell culture in each group. The small hairpin RNA (shRNA) was used for PKCι knockdown and the recombinant plasmid transfection was for PKCι overexpression in pancreatic cancer cells. Western blot and real-time fluorescent quantitative PCR (qRT-PCR) were utilized to determine the expression of PKCι protein and the impact on gene expression of transforming growth factor-ß (TGF-ß), a downstream effector modulated by PKC. Different mass concentrations of TGF-ß (1, 10, 20 ng/mL) were added into the co-culture of MiaPaCa and PANC-1 with CIK. The cell death rate was detected by flow cytometry 48 h later, so as to explore the possible mechanisms of the impact of PKCι on the tumoricidal effects of CIK cells. RESULTS: ATM and CIK were shown to suppress the growth and induce apoptosis or death of pancreatic cancer cells, meanwhile, ATM can enhance the tumoricidal effect of CIK on pancreatic cancer cells. Moreover, we found that PKCι knockdown in pancreatic cancer cells can down-regulate the gene expression of TGF-ß. In return, PKCι overexpression in pancreatic cancer cells can increase the gene expression of TGF-ß. The death rate of cancer cells with 10, 20 ng/mL TGF-ß was lower compared with the control group (P < 0.05). CONCLUSIONS: PKCι knockdown in pancreatic cancer cells can not only inhibit the growth of pancreatic cancer cells, but also enhance the tumoricidal effects of CIK on cancer cells. The possible mechanism of PKCι is to affect the immune escape of tumor cells by regulating the expression of TGF-ß.

Spatio-temporal release of NGF and GDNF from multi-layered nanofibrous bicomponent electrospun scaffolds.

Scaffolds capable of providing dual neurotrophic factor (NTF) delivery with different release kinetics, spatial delivery of NTFs at different loci and topographical guidance are promising for enhanced peripheral nerve regeneration. In this study, we have designed and fabricated multi-layered aligned-fiber scaffolds through combining emulsion electrospinning, sequential electrospinning and high-speed electrospinning (HS-ES) to modulate the release behavior of glial cell line-derived growth factor(GDNF) and nerve growth factor (NGF). GDNF and NGF were incorporated into poly(lactic-co-glycolic acid) (PLGA) fibers and poly(D,L-lactic acid) (PDLLA) fibers, respectively. Aligned fibers were obtained in each layer of multi-layered scaffolds and relatively thick tri-layered and tetra-layered scaffolds with controlled layer thickness were obtained. Their morphology, structure, properties, and the in vitro release of growth factors were examined. Dual and spatio-temporal release of GDNF and NGF with different release kinetics from multi-layered scaffolds was successfully demonstrated. High separation efficiency by PDLLA fibrous barrier layer for spatial neurotrophic factor delivery from both tri-layered scaffolds and tetra-layered scaffolds was achieved.

Radiological outcomes of pinless navigation in total knee arthroplasty: a randomized controlled trial.

PURPOSE: This study aimed to investigate the accuracy of pinless navigation (BrainLAB(®) VectorVision(®) Knee 2.5 Navigation System) as an intra-operative alignment guide in total knee arthroplasty (TKA). The authors hypothesized that pinless navigation would reduce the proportion of outliers in conventional TKA, without a significant increase in the duration of surgery. METHODS: Between 2011 and 2012, 100 patients scheduled for a unilateral primary TKA were randomized into two groups: pinless navigation and conventional surgery. All TKAs were performed with the surgical aim of achieving neutral coronal alignment with a 180° mechanical axis. The primary outcomes of this study were post-operative radiographic assessment of lower limb alignment using hip-knee-ankle angle (HKA) and components placement using coronal femoral-component angle (CFA) and coronal tibia-component angle (CTA). RESULTS: There was a smaller proportion of outliers for HKA, CFA and CTA at 10, 2 and 2 % respectively, in the pinless navigation group, compared to 32, 16 and 16 %, respectively, in the conventional group (p = 0.013, p = 0.032 and p = 0.032, respectively). The mean CFA was also more accurate at 90° in the pinless navigation group compared to 91° in the conventional group (p = 0.002). There was no difference in the duration of surgery between the two groups (n.s.). CONCLUSIONS: Pinless navigation improves lower limb alignment and components placement without a significant increase in the duration of surgery. The authors recommend the use of pinless navigation to verify the coronal alignments of conventional cutting blocks in TKA before the bone cuts are made. LEVEL OF EVIDENCE: I.

No association of single nucleotide polymorphisms involved in GHRL and GHSR with cancer risk: a meta-analysis.

BACKGROUND: Ghrelin was associated with several of cancers. The conflict results of SNPs with GHRL and GHSR gene were demonstrated in different studies. Thus, this meta-analysis is to evaluate the associations. METHODS: Systematic literature search was done on PubMed database up to October 2013. We used odds ratios (ORs) with 95% confidence intervals (CIs) to assess the strength of the association by a fixed-effect model and a random-effect model. RESULTS: A total of 7 studies, which included 3 studies for breast cancer, 2 for colorectal cancer, 1 for hepatocellular carcinoma, 1 for esophageal cancer and 1 for Non-Hodgkin lymphoma. When analyzed all the GHRL SNPs with all kinds of cancers, there was significantly difference with cancer patients compared with controls (Recessive model: OR 0.938, 95% CI 0.890-0.989, p=0.017), while no significant difference was existed in the additive model (OR 0.9903, 95% CI 0.957-1.024, p=0.558) and dominant model (OR 1.014, 95% CI 0.970-1.061, p=0.536). When analyzed all the GHSR SNPs with all kinds of cancers, no significant difference was observed. CONCLUSION: Our results suggest that the SNP with GHRL and GHSR might be weaker association with cancer risk, especially with breast cancer risk.