Reflecting the human lip in vitro: Cleft lip skin and mucosa keratinocytes keep their identities.

OBJECTIVES: Cell models have shown great promise as tools for research, potentially providing intriguing alternatives to animal models. However, the original tissue characteristics must be maintained in culture, a fact that is often assumed, but seldom assessed. We aimed to follow the retention of the original tissue identities of cleft lip-derived skin and mucosa keratinocytes in vitro. METHODS: Cleft lip-derived keratinocytes were isolated from discarded tissue along the cleft margins during cheiloplasty. Cell identities were assessed by immunohistochemistry and quantitative real-time PCR for tissue-specific markers and compared with native lip tissue. Moreover, keratinocytes were regularly analyzed for the retention of the original tissue characteristics by the aforementioned methods as well as by differentiation assays. RESULTS: The various anatomical zones of the human lip could be distinguished using a panel of differentiation and functional-based markers. Using these markers, retention of the original tissue identities could be followed and confirmed in the corresponding primary keratinocytes in culture. CONCLUSIONS: Our findings promote patient-derived cells retaining their original identities as astonishing and clinically relevant in vitro tools. Such cells allow a better molecular understanding of various lip-associated pathologies as well as their modeling in vitro, including but not restricted to orofacial clefts.

Sec22b regulates phagosome maturation by promoting ORP8-mediated lipid exchange at endoplasmic reticulum-phagosome contact sites.

Phagosome maturation is critical for immune defense, defining whether ingested material is destroyed or converted into antigens. Sec22b regulates phagosome maturation, yet how has remained unclear. Here we show Sec22b tethers endoplasmic reticulum-phagosome membrane contact sites (MCS) independently of the known tether STIM1. Sec22b knockdown increases calcium signaling, phagolysosome fusion and antigen degradation and alters phagosomal phospholipids PI(3)P, PS and PI(4)P. Levels of PI(4)P, a lysosome docking lipid, are rescued by Sec22b re-expression and by expression of the artificial tether MAPPER but not the MCS-disrupting mutant Sec22b-P33. Moreover, Sec22b co-precipitates with the PS/PI(4)P exchange protein ORP8. Wild-type, but not mutant ORP8 rescues phagosomal PI(4)P and reduces antigen degradation. Sec22b, MAPPER and ORP8 but not P33 or mutant-ORP8 restores phagolysosome fusion in knockdown cells. These findings clarify an alternative mechanism through which Sec22b controls phagosome maturation and beg a reassessment of the relative contribution of Sec22b-mediated fusion versus tethering to phagosome biology.

Consistent downregulation of the cleft lip/palate-associated genes IRF6 and GRHL3 in carcinomas.

Interferon Regulatory Factor 6 (IRF6) and Grainyhead Like Transcription Factor 3 (GRHL3) are transcription factors that orchestrate gene regulatory networks required for the balance between keratinocyte differentiation and proliferation. Absence of either protein results in the lack of a normal stratified epidermis with keratinocytes failing to stop proliferating and to terminally differentiate. Numerous pathological variants within IRF6 and GRHL3 have been identified in orofacial cleft-affected individuals and expression of the two transcription factors has been found to be often dysregulated in cancers. However, whether orofacial cleft-associated IRF6 and GRHL3 variants in patients might also affect their cancer risk later in life, is not clear yet. The fact that the role of IRF6 and GRHL3 in cancer remains controversial makes this question even more challenging. Some studies identified IRF6 and GRHL3 as oncogenes, while others could attribute tumor suppressive functions to them. Trying to solve this apparent conundrum, we herein aimed to characterize IRF6 and GRHL3 function in various types of carcinomas. We screened multiple cancer and normal cell lines for their expression, and subsequently proceeded with functional assays in cancer cell lines. Our data uncovered consistent downregulation of IRF6 and GRHL3 in all types of carcinomas analyzed. Reduced levels of IRF6 and GRHL3 were found to be associated with several tumorigenic properties, such as enhanced cell proliferation, epithelial mesenchymal transition, migration and reduced differentiation capacity. Based on our findings, IRF6 and GRHL3 can be considered as tumor suppressor genes in various carcinomas, which makes them potential common etiological factors for cancer and CLP in a fraction of CLP-affected patients.

Two-MILP models for scheduling elective surgeries within a private healthcare facility.

This paper deals with an Integrated Elective Surgery-Scheduling Problem (IESSP) that arises in a privately operated healthcare facility. It aims to optimize the resource utilization of the entire surgery process including pre-operative, per-operative and post-operative activities. Moreover, it addresses a specific feature of private facilities where surgeons are independent service providers and may conduct their surgeries in different private healthcare facilities. Thus, the problem requires the assignment of surgery patients to hospital beds, operating rooms and recovery beds as well as their sequencing over a 1-day period while taking into account surgeons' availability constraints. We present two Mixed Integer Linear Programs (MILP) that model the IESSP as a three-stage hybrid flow-shop scheduling problem with recirculation, resource synchronization, dedicated machines, and blocking constraints. To assess the empirical performance of the proposed models, we conducted experiments on real-world data of a Tunisian private clinic: Clinique Ennasr and on randomly generated instances. Two criteria were minimised: the patients' average length of stay and the number of patients' overnight stays. The computational results show that the proposed models can solve instances with up to 44 surgical cases in a reasonable CPU time using a general-purpose MILP solver.

Longevity and outcomes of axillary valve transplantation for severe lower extremity chronic venous insufficiency.

BACKGROUND: To assess the efficacy of axillary vein transplantation in the treatment of severe chronic venous insufficiency (CVI). METHODS: Among 139 complex venous reconstructions performed between 1991 and 2007 for CVI, 18 patients underwent upper extremity to lower extremity venous valve transplantation. An upper extremity valve was transplanted to the popliteal vein in 13 cases, to the common femoral vein in six cases, and to the saphenofemoral junction in two cases for a total of 21 procedures. All patients had follow-up with duplex scanning to assess valve competency and clinical visits to assess clinical improvement. Mean follow-up period was 37 months. RESULTS: Mean patient age was 44 years, and 57% were men. Clinically, 57% of the limbs were Clincal (C) class C5-C6. The mean preoperative venous disability score was 2.95. Most of the patients (66%) had post-thrombotic valvular dysfunction. At the time of valve transplantation, there was no proximal venous obstruction documented. A successful operation was defined as a competent valve at the end of the procedure and was achieved in 20 of 21 (95%) patients. Eight patients had at least one postoperative complication, primarily bleeding. The mean postoperative venous disability score was 2.65 and this increased to 2.75 (p = not significant as compared with baseline) at the last postoperative visit. Median time to return of symptoms was 12 months, and median reflux-free survival period was 15 months. CONCLUSION: Despite initial technical and symptomatic success with venous valve transplantation, there is a poor long-term valve competency rate and symptomatic control. These data suggest that a better understanding and therapy for severe CVI associated with valvular incompetence needs to be found.