Elevated expression of LCN13 through FXR activation ameliorates hepatocellular lipid accumulation and inflammation.

BACKGROUND: Lipocalin 13 (LCN13) is a member of the lipocalin family that consists of numerous secretory proteins. LCN13 high-expression has been reported to possess anti-obesity and anti-diabetic effects. Although metabolic dysfunction-associated steatotic liver diseases (MASLD) including metabolic dysfunction-associated steatohepatitis (MASH) are frequently associated with obesity and insulin resistance, the functional role of endogenous LCN13 and the therapeutic effect of LCN13 in MASH and related metabolic deterioration have not been evaluated. METHODS: We employed a methionine-choline deficient diet model and MASH cell models to investigate the role of LCN13 in MASH development. We sought to explore the effects of LCN13 on lipid metabolism and inflammation in hepatocytes under PA/OA exposure using Western blotting, real-time RT-PCR, enzyme-linked immunosorbent assay, hematoxylin and eosin staining, oil red O staining. Using RNA sequencing, chromatin immunoprecipitation assay, and luciferase reporter assays to elucidate whether farnesoid X receptor (FXR) regulates human LCN13 transcription as a transcription factor. RESULTS: Our study found that LCN13 was down-regulated in MASH patients, MASH mouse and cell models. LCN13 overexpression in hepatocyte cells significantly inhibited lipid accumulation and inflammation in vitro. Conversely, LCN13 downregulation significantly exacerbated lipid accumulation and inflammatory responses in vivo and in vitro. Mechanistically, we provided the first evidence that LCN13 was transcriptionally activated by FXR, representing a novel direct target gene of FXR. And the key promoter region of LCN13 binds to FXR was also elucidated. We further revealed that LCN13 overexpression via FXR activation ameliorates hepatocellular lipid accumulation and inflammation in vivo and in vitro. Furthermore, LCN13-down-regulated mice exhibited aggravated MASH phenotypes, including increased hepatic lipid accumulation and inflammation. CONCLUSION: Our findings provide new insight regarding the protective role of LCN13 in MASH development and suggest an innovative therapeutic strategy for treating MASH or related metabolic disorders.

Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

Isolation and Identification of Limbal Niche Cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 °C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 °C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFRß). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFRß, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were 68%, 99%, 20%, and 3% in BMMSCs. The standard process for LNC isolation and identification could provide a reliable laboratory basis for the widespread use of LNCs.

Suspensory Device Fixation of Lisfranc Injuries in a Southeast Asian Urban Population: Patient-Reported Functional Outcomes.

Introduction Open reduction internal fixation (ORIF) and primary arthrodesis are two conventional options for the treatment of Lisfranc injuries. However, they are associated with implant-related complications. An alternative suspensory device construct using interosseous nonabsorbable sutures with endobuttons has been described with satisfactory results. This study aims to explore functional outcomes after suture button fixation of Lisfranc injuries in a Southeast Asian population. Methods This was a single-surgeon retrospective study of patients with Lisfranc injuries treated surgically using a suture button fixation technique between 2017 and 2019. Data collected included demographic information, pre-injury levels of activity, nature of injury, and type of surgery performed. The minimum postoperative follow-up was one year. The Foot and Ankle Outcome Score (FAOS) and Foot and Ankle Ability Measure (FAAM) were used to evaluate patient-reported outcomes. Scores were reported in percentage (%) with median and interquartile range. Results Twenty-nine patients with a mean age of 29 years (21-76) were recruited. Sixteen underwent suture button fixation only (SB), and 13 underwent suture button fixation with intercuneiform screw fixation and plating (SBM). The median scores for the FAOS and FAAM questionnaires were at least 80% in all domains. Twenty-eight patients (97%) were able to return to pre-injury activity level, 27 patients (93%) were able to return to sports. Only one patient was not satisfied with the outcomes of surgery. No patients had post-traumatic arthritis or hardware failure necessitating implant removal at the final follow-up. Conclusion This study has demonstrated that treatment of Lisfranc injuries with a suspensory device construct resulted in good outcomes with 97% of patients being able to return to pre-injury activity levels, and 93% of patients being able to return to sports. It may not be necessary to perform primary arthrodesis in uncomplicated Lisfranc injuries. This technique is also advantageous as implant removal is not routinely required due to the design and biomechanical properties of suspensory devices.

SHFL inhibits enterovirus A71 infection by triggering degradation of viral 3Dpol protein via the ubiquitin-proteasome pathway.

Enterovirus A71 (EV-A71) is a highly contagious virus that poses a major threat to global health, representing the primary etiological agent for hand-foot and mouth disease (HFMD) and neurological complications. It has been established that interferon signaling is critical to establishing a robust antiviral state in host cells, mainly mediated through the antiviral effects of numerous interferon-stimulated genes (ISGs). The host restriction factor SHFL is a novel ISG with broad antiviral activity against various viruses through diverse underlying molecular mechanisms. Although SHFL is widely acknowledged for its broad-spectrum antiviral activity, it remains elusive whether SHFL inhibits EV-A71. In this work, we validated that EV-A71 triggers the upregulation of SHFL both in cell lines and in a mouse model. Knockdown and overexpression of SHFL in EVA71-infected cells suggested that this factor could markedly suppress EV-A71 replication. Our findings further revealed an intriguing mechanism of SHFL that it could interact with the nonstructural proteins 3Dpol of EV-A71 and promoted the degradation of 3Dpol through the ubiquitin-proteasome pathway. Furthermore, the zinc-finger domain and the 36 amino acids (164-199) of SHFL were crucial to the interaction between SHFL and EV-A71 3Dpol . Overall, these findings broadened our understanding of the pivotal roles of SHFL in the interaction between the host and EV-A71.

Limbal Niche Cells and Three-Dimensional Matrigel-Induced Dedifferentiation of Mature Corneal Epithelial Cells.

Purpose: To investigate the phenotypic changes of mature corneal epithelial cells (MCECs) that cocultured with limbal niche cells (LNCs) in three-dimensional Matrigel (3D Matrigel) in vitro. Methods: MCECs were isolated from central corneas, and limbal epithelial progenitor cells (LEPCs) were isolated from limbal segments with Dispase II. LNCs were isolated and cultured from limbal niche using the collagenase A digestion method and identified with PCK/VIM/CD90/CD105/SCF/PDGFRß. MCECs were cultured on 3D Matrigel (50%, v/v) with or without LNCs for 10 days. Expression of CK12 and p63α and clone formation test were used to compare the progenitor phenotypic changes for MCECs before and after induction using LEPCs as control. Results: Homogeneous LNCs were isolated and identified as spindle shape and adherent to a plastic surface coated with 5% Matrigel. Double immunostaining of the fourth-passage LNCs was uniformly PCK-/VIM+/CD90+/CD105+/SCF+/PDGFRß+. Reverse transcription and quantitative real-time polymerase chain reaction (RT-qPCR) revealed the decrease of PCK expression from the second passage and elevation of Vim, CD90, CD105, SCF, and PDGFRß transcripts from the third passage, and the transcription level of Vim, CD90, CD105, SCF, and PDGFRß was elevated statistically in the fourth passage compared to the first passage (P < 0.01). Both immunofluorescence (IF) staining for cross section and cytospin cells demonstrated that MCECs expressed higher CK12 while lower p63α than LEPCs (P < 0.01). Sphere growth formation was noticed as early as 24 hours in the MCEC + LNC group, 48 hours in the LEPC group, and 72 hours in the MCEC group. The diameters of the spheres were the biggest in the MCEC + LNC group (182.24 ± 57.91 µm), smaller in the LEPC group (125.71 ± 41.20 µm), and smallest in the MCEC group (109.39 ± 34.85 µm) by the end of the 10-day culture (P < 0.01). Double immunostaining with CK12/p63α showed that cells in the sphere formed from MCECs expressed CK12 but not p63α; in contrast, some cells in the MCEC + LNC group expressed CK12, but most of them expressed p63α. RT-qPCR revealed a significant reduction of CK12 transcript but elevation of p63α, Oct4, Nanog, Sox2, and SSEA4 (P < 0.05). Holoclone composed of cubic epithelial cells could be generated in the MCEC + LNC group but not in the other two groups. Conclusions: The data shows that human MCEC cell phenotype could be induced to the dedifferentiation stage when cocultured with LNCs in 3D Matrigel that simulated the microenvironment of limbal stem cells in vitro.

Does Posterior Scoliosis Correction Improve Respiratory Function in Adolescent Idiopathic Scoliosis? A Systematic Review and Meta-analysis.

STUDY DESIGN: A systematic review and meta-analysis. OBJECTIVES: Pulmonary dysfunction is often advocated among the indications for surgical correction of adolescent idiopathic scoliosis (AIS). Previous studies have discussed the effect of scoliosis correction on respiratory function without reaching a definitive conclusion: Some showed that the respiratory function can improve after scoliosis surgery without defining the precise role of anterior, posterior, and combined approaches on this improvement; furthermore, the majority of these studies did not take normal growth into account. As a result, the role of surgery remains to be clarified. The object of the present study was to synthesize the current knowledge regarding changes in respiratory function after posterior corrective surgery for AIS. METHODS: A comprehensive systematic search was performed to identify all relevant studies in the following electronic databases: MEDLINE, EMBASE, CINAHL (EBSCO). We focused on the studies (1) that discussed posterior fusion surgery for AIS without thoracoplasty, (2) that discussed comparisons of pre- and postoperative percent-predicted values of forced vital capacity (%FVC) or forced expiratory volume (%FEV), and (3) with minimum 2-year follow-up. Forest plots were depicted and Z value was calculated as a test for overall effect. RESULTS: Ten studies (6 prospective and 4 retrospective studies) met our inclusion criteria. The overall effect showed that there was no significant difference in %FVC or %FEV between pre- and postoperative measurements (very low evidence). CONCLUSIONS: Posterior correction surgery for mild to moderate AIS patients showed no significant improvement of postoperative respiratory function measured by relative, percent-predicted values at minimum 2-year follow-up.

Observing temporal trends in cardiac rehabilitation from 1996 to 2010 in Ontario: characteristics of referred patients, programme participation and mortality rates.

OBJECTIVES: We sought to describe temporal trends in the sociodemographic and clinical characteristics of participants referred to cardiac rehabilitation (CR), and its effect on programme participation and all-cause mortality over 14 years. SETTING: A large CR centre in Toronto, Canada. PARTICIPANTS: Consecutive patients between 1996 and 2010. PRIMARY AND SECONDARY OUTCOME MEASURES: Referrals received were deterministically linked to administrative data, to complement referral form abstraction. Out-of-hospital deaths were identified using vital statistics. Patients were tracked until 2012, and mortality was ascertained. Percentage attendance at prescribed sessions was also assessed. RESULTS: There were 29,171 referrals received, of which 28,767 (98.6%) were successfully linked, of whom 22,795 (79.2%) attended an intake assessment. The age of the referred population steadily increased, with more females, less affluent and more single patients referred over time (p<0.001). More patients were referred following percutaneous coronary intervention and less following coronary artery bypass graft surgery (p<0.001). The number of comorbidities decreased (p<0.001). Hypertension increased over time (p<0.001), yet the control of cholesterol steadily improved over time. The proportion of smokers decreased over time (p<0.001). Participation in CR significantly declined, and there were no significant changes in mortality. 3-year mortality rates were less than 5%. CONCLUSIONS: Characteristics of referred patients tended to reflect broader trends in risk factors and cardiovascular disease burden. Physicians appear to be referring more sociodemographically diverse patients to CR; however, programmes may need to better adapt to engage these patients to fully participate. More complex patients should be referred, using explicit criteria-based referral processes.