Selection of the Most Suitable Culture Medium for Patient-Derived Lung Cancer Organoids.

Introduction Patient-derived organoids have emerged as a promising in vitro model for precision medicine, particularly in cancer, but also in non-cancer-related diseases. However, the optimal culture medium for culturing patient-derived lung organoids has not yet been agreed upon. This study aimed to shed light on the optimal selection of a culture media for developing studies using patient-derived lung organoids. Methods Tumor and normal paired tissue from 71 resected non-small cell lung cancer patients were processed for organoid culture. Lung cancer organoids (LCOs) were derived from tumor tissue and normal lung organoids (LNOs) from non-neoplastic lung tissue. Three different culture media were compared: Permissive culture medium (PCM); Limited culture medium (LCM); and Minimum basal medium (MBM). We assessed their effectiveness in establishing organoid cultures, promoting organoid growth and viability, and compared their differential phenotypic characteristics. Results While PCM was associated with the highest success rate and useful for long-term expansion, MBM was the best option to avoid normal organoid overgrowth in the organoid culture. The density, size, and viability of LNOs organoids were reduced using LCM and severely affected with MBM. LNOs cultured in PCM tend to differentiate to bronchospheres, while alveolosphere differentiation can be observed in those cultured with LCM. The morphological phenotype of LCO was influenced by the culture media of election. Mesenchymal cell overgrowth was observed when LCM was used. Conclusion This work highlights the importance of considering the research objectives when selecting the most suitable culture medium for growing patient-derived lung organoids.

Viviparity is associated with larger female size and higher sexual size dimorphism in a reproductively bimodal lizard.

Squamate reptiles are central for studying phenotypic correlates of evolutionary transitions from oviparity to viviparity because these transitions are numerous, with many of them being recent. Several models of life-history theory predict that viviparity is associated with increased female size, and thus more female-biased sexual size dimorphism (SSD). Yet, the corresponding empirical evidence is overall weak and inconsistent. The lizard Zootoca vivipara, which occupies a major part of Northern Eurasia and includes four viviparous and two non-sister oviparous lineages, represents an excellent model for testing these predictions. We analysed how sex-specific body size and SSD is associated with parity mode, using body length data for nearly 14,000 adult individuals from 97 geographically distinct populations, which cover almost the entire species' range and represent all six lineages. Our analyses controlled for lineage identity, climatic seasonality (the strongest predictor of geographic body size variation in previous studies of this species) and several aspects of data heterogeneity. Parity mode, lineage and seasonality are significantly associated with female size and SSD; the first two predictors accounted for 14%-26% of the total variation each, while seasonality explained 5%-7%. Viviparous populations exhibited a larger female size than oviparous populations, with no concomitant differences in male size. The variation of male size was overall low and poorly explained by our predictors. Albeit fully expected from theory, the strong female bias of the body size differences between oviparous and viviparous populations found in Z. vivipara is not evident from available data on three other lizard systems of closely related lineages differing in parity mode. We confront this pattern with the data on female reproductive traits in the considered systems and the frequencies of evolutionary changes of parity mode in the corresponding lizard families and speculate why the life-history correlates of live-bearing in Z. vivipara are distinct. Comparing conspecific populations, our study provides the most direct evidence for the predicted effect of parity mode on adult body size but also demonstrates that the revealed pattern may not be general. This might explain why across squamates, viviparity is only weakly associated with larger size.

Design and Evaluation of a Technological Platform for Monitoring Patients with Dementia: Unifying Requirements from Mexican Day Centers.

The monitoring of patients with dementia who receive comprehensive care in day centers allows formal caregivers to make better decisions and provide better care to patients. For instance, cognitive and physical therapies can be tailored based on the current stage of disease progression. In the context of day centers of the Mexican Federation of Alzheimer, this work aims to design and evaluate Alzaid, a technological platform for assisting formal caregivers in monitoring patients with dementia. Alzaid was devised using a participatory design methodology that consisted in eliciting and validating requirements from 22 and 9 participants, respectively, which were unified to guide the construction of a high-fidelity prototype evaluated by 14 participants. The participants were formal caregivers, medical staff, and management. This work contributes a high-fidelity prototype of a technological platform for assisting formal caregivers in monitoring patients with dementia considering restrictions and requirements of four Mexican day centers. In general, the participants perceived the prototype as quite likely to be useful, usable, and relevant in the job of monitoring patients with dementia (p-value < 0.05). By evaluating and designing Alzaid that unifies requirements for monitoring patients of four day centers, this work is the first effort towards a standard monitoring process of patients with dementia in the context of the Mexican Federation of Alzheimer.

Characterization of the MicroRNA Cargo of Extracellular Vesicles Isolated from a Pulmonary Tumor-Draining Vein Identifies miR-203a-3p as a Relapse Biomarker for Resected Non-Small Cell Lung Cancer.

In resected non-small cell lung cancer (NSCLC), post-surgical recurrence occurs in around 40% of patients, highlighting the necessity to identify relapse biomarkers. An analysis of the extracellular vesicle (EV) cargo from a pulmonary tumor-draining vein (TDV) can grant biomarker identification. We studied the pulmonary TDV EV-miRNAome to identify relapse biomarkers in a two-phase study (screening and validation). In the screening phase, a 17-miRNA relapse signature was identified in 18 selected patients by small RNAseq. The most expressed miRNA from the signature (EV-miR-203a-3p) was chosen for further validation. Pulmonary TDV EV-miR-203a-3p was studied by qRT-PCR in a validation cohort of 70 patients, where it was found to be upregulated in relapsed patients (p = 0.0194) and in patients with cancer spread to nearby lymph nodes (N+ patients) (p = 0.0396). The ROC curve analysis showed that TDV EV-miR-203a-3p was able to predict relapses with a sensitivity of 88% (AUC: 0.67; p = 0.022). Moreover, patients with high TDV EV-miR-203a-3p had a shorter time to relapse than patients with low levels (43.6 vs. 97.6 months; p = 0.00703). The multivariate analysis showed that EV-miR-203a-3p was an independent, predictive and prognostic post-surgical relapse biomarker. In conclusion, pulmonary TDV EV-miR-203a-3p is a promising new relapse biomarker for resected NSCLC patients.

The tibiocalcaneal bundle of the deltoid ligament - Prevalence and variations.

INTRODUCTION: A precise understanding of the anatomy of the multiple bundles of the deltoid ankle ligament might have clinical impact. The most relevant deltoid anatomical series report a variable frequency of the tibiocalcaneal ligament, possibly the most important bundle to be reconstructed in medial ankle insufficiency. Our purpose was to access the deltoid's tibiocalcaneal ligament morphology in a large anatomical study as well as to perform a historical literature review on the reasons for its variable prevalence. MATERIALS AND METHODS: Forty-three ankle specimen were dissected to describe the prevalence of superficial and deep deltoid bundles, with special attention to the tibiocalcaneal ligament and its variants. RESULTS: All ankles had distinct deep and superficial bundles. In all 43 ankles the tibionavicular and tibiospring ligaments were clearly identified. The superficial posterior tibiotalar ligament was identified in 38 ankles (88%). The deep anterior tibiotalar bundle was identified in 35 ankles (81%). The deep posterior tibiotalar bundle was identified in all ankles. The tibiocalcaneal ligament was identified in 33 ankles (77%). In ten ankles there wasn't a direct bundle between the tibia and the sustentaculum tali. In all of these, however, we found some fibers spanning the gap between the tibiospring ligament and the sustentaculum tali. CONCLUSION: The tibiocalcaneal ligament is present in most specimens. In those in which we could not identify a direct bundle between the tibia and the calcareous we found a variant of the tibiospring ligament that connects to the sustentaculum tali.

Loss of the Immune Checkpoint CD85j/LILRB1 on Malignant Plasma Cells Contributes to Immune Escape in Multiple Myeloma.

Mechanisms of immune regulation may control proliferation of aberrant plasma cells (PCs) in patients with monoclonal gammopathy of undetermined significance (MGUS) preventing progression to active multiple myeloma (MM). We hypothesized that CD85j (LILRB1), an inhibitory immune checkpoint for B cell function, may play a role in MM pathogenesis. In this study, we report that patients with active MM had significantly lower levels of CD85j and its ligand S100A9. Decreased CD85j expression could also be detected in the premalignant condition MGUS, suggesting that loss of CD85j may be an early event promoting tumor immune escape. To gain insight into the molecular mechanisms underlying CD85j functions, we next enforced expression of CD85j in human myeloma cell lines by lentiviral transduction. Interestingly, gene expression profiling of CD85j-overexpressing cells revealed a set of downregulated genes with crucial functions in MM pathogenesis. Furthermore, in vitro functional assays demonstrated that CD85j overexpression increased susceptibility to T cell- and NK-mediated killing. Consistently, ligation of CD85j decreased the number of PCs from individuals with MGUS but not from patients with MM. In conclusion, downregulation of inhibitory immune checkpoints on malignant PCs may provide a novel mechanism of immune escape associated with myeloma pathogenesis.

Macular choroidal thickness in patients with pseudoxanthoma elasticum measured by enhanced-depth imaging spectral-domain optical coherence tomography.

BACKGROUND/OBJECTIVES: To analyze macular choroidal thickness in patients with pseudoxanthoma elasticum (PXE) by enhanced depth imaging optical coherence tomography (EDI-OCT). SUBJECTS/METHODS: This is a prospective cross-sectional study. Sixty-eight eyes of 34 patients with PXE and 68 normal eyes of 34 controls were included to study the macular area with enhanced depth imaging spectral-domain optical coherence tomography (EDI-OCT). Eyes with PXE were classified in three groups: those without choroidal neovascularization (CNV) or chorioretinal macular atrophy macular (Group 1); those with active CNV (Group 2) and those with macular atrophy secondary to inactive CNV (Group 3). RESULTS: Mean subfoveal choroidal thickness (CT) was 266.70 ± 46.93 µm in control group, 304.24 ± 65.52 µm in group 1, 198.55 ± 66.33 µm in group 2, and 119.45 ± 63.89 µm in group 3 (p = 0.00). Comparison between PXE subgroups showed that subfoveal CT was significantly decreased in group 2 and 3 compared to group 1 (p < 0.0001 for both groups). The CT in the different quadrants (superior, inferior, temporal and nasal) was significantly thinner in group 3, followed by group 2 and 1 in ascendant order. Group 1 showed significant increased thickness compared to the other groups. CONCLUSION: To the best of our knowledge, this is the first report suggesting thicker macular choroid in patients with PXE without active or inactive CNV than in normal eyes. Initial changes in Bruch membrane (MB) and choroid, in addition to the increased oxidative stress, would lead to hyperpermeability of the choroid and alterations of the barrier BM-RPE causing a thick choroid in early stages.

Impact of Autologous Stem Cell Transplantation on the Incidence and Outcome of Oligoclonal Bands in Patients with Light-Chain Amyloidosis.

The emergence of oligoclonal bands (OB) in patients with multiple myeloma achieving a complete remission (CR) after autologous stem cell transplantation (ASCT) and the use of novel agents is a well-recognized event. The presence of OB is associated with favorable outcome. However, the emergence of OB in light-chain (AL) amyloidosis has never been investigated. The aim of the study was to determine the incidence, natural history, and prognostic impact of OB in 50 patients with AL amyloidosis who achieved at least a partial response either after upfront ASCT (20 patients [40%]) or after conventional treatment in patients ineligible for transplantation (30 patients [60%]). OB were observed in 60% of the patients, with IgG-kappa (30.7%) the most frequently detected isotype. This phenomenon was more prevalent in patients achieving CR than those in other response categories (88% versus 32%, P = .0001). The landmark analysis at 1 year after diagnosis demonstrates a significantly longer progression-free survival and an improvement trend in overall survival (P = .04 and P = .06, respectively). This prognostic impact was also observed in patients who achieved CR and in patients with more advanced stage. In summary, this is the first report of OB in patients with AL amyloidosis. Although its biological meaning remains unclear, it could reflect a more robust humoral immune response.

The BET bromodomain inhibitor CPI203 improves lenalidomide and dexamethasone activity in in vitro and in vivo models of multiple myeloma by blockade of Ikaros and MYC signaling.

Most patients with multiple myeloma treated with current therapies, including immunomodulatory drugs, eventually develop relapsed/refractory disease. Clinical activity of lenalidomide relies on degradation of Ikaros and the consequent reduction in IRF4 expression, both required for myeloma cell survival and involved in the regulation of MYC transcription. Thus, we sought to determine the combinational effect of an MYC-interfering therapy with lenalidomide/dexamethasone. We analyzed the potential therapeutic effect of the combination of the BET bromodomain inhibitor CPI203 with the lenalidomide/dexamethasone regimen in myeloma cell lines. CPI203 exerted a dose-dependent cell growth inhibition in cell lines, indeed in lenalidomide/dexamethasone-resistant cells (median response at 0.5 µM: 65.4%), characterized by G1 cell cycle blockade and a concomitant inhibition of MYC and Ikaros signaling. These effects were potentiated by the addition of lenalidomide/dexamethasone. Results were validated in primary plasma cells from patients with multiple myeloma co-cultured with the mesenchymal stromal cell line stromaNKtert. Consistently, the drug combination evoked a 50% reduction in cell proliferation and correlated with basal Ikaros mRNA expression levels (P=0.04). Finally, in a SCID mouse xenotransplant model of myeloma, addition of CPI203 to lenalidomide/dexamethasone decreased tumor burden, evidenced by a lower glucose uptake and increase in the growth arrest marker GADD45B, with simultaneous downregulation of key transcription factors such as MYC, Ikaros and IRF4. Taken together, our data show that the combination of a BET bromodomain inhibitor with a lenalidomide-based regimen may represent a therapeutic approach to improve the response in relapsed/refractory patients with multiple myeloma, even in cases with suboptimal prior response to immunomodulatory drugs.

ß-Arrestin scaffolds and signaling elements essential for the obestatin/GPR39 system that determine the myogenic program in human myoblast cells.

Obestatin/GPR39 signaling stimulates skeletal muscle repair by inducing the expansion of satellite stem cells as well as myofiber hypertrophy. Here, we describe that the obestatin/GPR39 system acts as autocrine/paracrine factor on human myogenesis. Obestatin regulated multiple steps of myogenesis: myoblast proliferation, cell cycle exit, differentiation and recruitment to fuse and form multinucleated hypertrophic myotubes. Obestatin-induced mitogenic action was mediated by ERK1/2 and JunD activity, being orchestrated by a G-dependent mechanism. At a later stage of myogenesis, scaffolding proteins ß-arrestin 1 and 2 were essential for the activation of cell cycle exit and differentiation through the transactivation of the epidermal growth factor receptor (EGFR). Upon obestatin stimulus, ß-arrestins are recruited to the membrane, where they functionally interact with GPR39 leading to Src activation and signalplex formation to EGFR transactivation by matrix metalloproteinases. This signalplex regulated the mitotic arrest by p21 and p57 expression and the mid- to late stages of differentiation through JNK/c-Jun, CAMKII, Akt and p38 pathways. This finding not only provides the first functional activity for ß-arrestins in myogenesis but also identify potential targets for therapeutic approaches by triggering specific signaling arms of the GPR39 signaling involved in myogenesis.

Role of miR-200 family members in survival of colorectal cancer patients treated with fluoropyrimidines.

BACKGROUND AND OBJECTIVES: Surgery is the standard treatment for colorectal cancer (CRC), and adjuvant chemotherapy has been shown to be effective in stage III but less so in stage II. We have analyzed the expression of the miR-200 family in tissue samples from resected CRC patients and correlated our findings with survival to adjuvant treatment with fluoropyrimidines. METHODS: Tumor tissue samples were obtained from 127 surgically resected patients with stage I-III CRC. miRNA detection was performed using TaqMan MicroRNA assays. RESULTS: High levels of miR-200a and miR-200c were associated with longer overall survival, while high levels of miR-429 correlated with longer overall and disease-free survival (DFS). In the subgroup of 56 patients treated with fluoropyrimidines and in the smaller subgroup of 32 stage II patients treated with fluoropyrimidines, those with high levels of miR-200a, miR-200c, miR-141, or miR-429 had significantly longer overall and DFS. Low miR-429 levels were identified as an independent prognostic marker. High levels of miR-429 combined with 5-fluorouracil inhibited cell invasion in LOVO cells. CONCLUSIONS: miR-200a, miR-200c, miR-141, and miR-429 expression levels may identify CRC patients, including those with stage II disease, who are most likely to benefit from adjuvant chemotherapy.

Discovering genes and microRNAs involved in human lung development unveils IGFBP3/miR-34a dynamics and their relevance for alveolar differentiation.

BACKGROUND: During pseudoglandular stage of the human lung development the primitive bronchial buds are initially conformed by simple tubules lined by endoderm-derived epithelium surrounded by mesenchyme, which will progressively branch into airways and start to form distal epithelial saculles. For first time alveolar type II (AT2) pneumocytes appears. This study aims to characterize the genes and microRNAs involved in this differentiation process and decipher its role in the starting alveolar differentiation. METHODS: Gene and microRNA profiling was performed in human embryonic lungs from 7 to 12 post conception weeks (pcw). Protein expression location of candidate genes were analyzed by immunofluorescense in embryonic lung tissue sections. mRNA/miRNA target pairs were identified using computational approaches and their expression was studied in purified epithelial/mesenchymal cell populations and in isolated tips and stalks from the bronchial tree. Additionally, silencing experiments in human embryonic lung mesenchymal cells and in human embryonic tip-derived lung organoids were performed, as well as organoid differentiation studies. AT2 cell markers were studied by qRT-PCR and by immunofluorescence. The TGFB-ß phosphorylated pathways was analyzed with membrane protein arrays. Lung explants were cultured in air/liquid interface with/without peptides. RESULTS: We identified 88 differentially expressed genes, including IGFBP3. Although IGFBP3 mRNA was detected in both epithelial and mesenchymal populations, the protein was restricted to the epithelium, indicating post-transcriptional regulation preventing IGFBP3 protein expression in the mesenchyme. MicroRNA profiling identified miR-34a as an IGFBP3 regulator. miR-34a was up-regulated in mesenchymal cells, and its silencing in human embryonic lung mesenchymal cells increased IGFBP3 levels. Additionally, IGFBP3 expression showed a marked downregulation from 7 to 12 pcw, suggesting its involvement in the differentiation process. The differentiation of human tip-derived lung embryonic organoids showed a drastic reduction in IGFBP3, supported by the scRNAseq data. IGFBP3 silencing in organoids activated an alveolar-like differentiation process characterized by stem cell markers downregulation and upregulation of AT2 markers. This process was mediated by TGFß signalling inhibition and BMP pathway activation. CONCLUSIONS: The IGFBP3/miR-34a axis restricts IGFBP3 expression in the embryonic undifferentiated lung epithelium, and the progressive downregulation of IGFBP3 during the pseudoglandular stage is required for alveolar differentiation.

Increased PVR Expression on Bone Marrow Macrophages May Promote Resistance to TIGIT Blockade in Multiple Myeloma.

PURPOSE: TIGIT blockade in our ex vivo model of bone marrow (BM) reduced the number of malignant plasma cells (PC) in only half of patients with multiple myeloma. Here, we wanted to investigate whether increased expression of TIGIT ligands may inhibit T-cell immune response promoting resistance to TIGIT blockade. EXPERIMENTAL DESIGN: We first characterized the number and phenotype of BM macrophages in different stages of the disease by multiparameter flow cytometry. We assessed the effect of TIGIT ligands on PC survival by performing experiments in the ex vivo BM model and analyzed changes in gene expression by using NanoString technology and real-time PCR. RESULTS: The frequency of BM macrophages was significantly decreased in multiple myeloma, which was accompanied by changes in their immunophenotype. Moreover, we found a higher number of malignant PC in ex vivo BM cells cultured onto the poliovirus receptor (PVR) and nectin-2 compared with control, suggesting that both ligands may support PC survival. In addition, the presence of PVR, but not nectin-2, overcame the therapeutic effect of TIGIT blockade or exogenous IL2. Furthermore, exogenous IL2 increased TIGIT expression on both CD4+ and CD8+ T cells and, indirectly, PVR on BM macrophages. Consistently, PVR reduced the number of cytotoxic T cells and promoted a gene signature with reduced effector molecules. CONCLUSIONS: IL2 induced TIGIT on T cells in the BM, in which increased PVR expression resulted in cytotoxic T-cell inhibition, promoting PC survival and resistance to TIGIT blockade.

Protocol to characterize mouse intestinal epithelial cell lineage using Opal multiplex immunofluorescence.

Applying Opal multiplex immunofluorescence (OMI) to characterize intestinal tissues of genetically engineered mouse models provides an excellent tool for studying complex processes. However, detecting appropriate signals from multiple target molecules is challenging. Here, we present a protocol to characterize mouse intestinal epithelial cell lineage using OMI. We describe steps for processing small intestine and colonic mouse tissues and designing and optimizing panels for OMI in mouse intestinal tissues. We then detail procedures for performing a quantitative evaluation of acquired images. For complete details on the use and execution of this protocol, please refer to Kinoshita et al.1.

Epithelial aPKC deficiency leads to stem cell loss preceding metaplasia in colorectal cancer initiation.

The early mechanisms of spontaneous tumor initiation that precede malignancy are largely unknown. We show that reduced aPKC levels correlate with stem cell loss and the induction of revival and metaplastic programs in serrated- and conventional-initiated premalignant lesions, which is perpetuated in colorectal cancers (CRCs). Acute inactivation of PKCλ/ι in vivo and in mouse organoids is sufficient to stimulate JNK in non-transformed intestinal epithelial cells (IECs), which promotes cell death and the rapid loss of the intestinal stem cells (ISCs), including those that are LGR5+. This is followed by the accumulation of revival stem cells (RSCs) at the bottom of the crypt and fetal-metaplastic cells (FMCs) at the top, creating two spatiotemporally distinct cell populations that depend on JNK-induced AP-1 and YAP. These cell lineage changes are maintained during cancer initiation and progression and determine the aggressive phenotype of human CRC, irrespective of their serrated or conventional origin.

Low miR-145 and high miR-367 are associated with unfavourable prognosis in resected nonsmall cell lung cancer.

The transcription factors SRY-related HMG box (SOX)2 and octamer-binding transcription factor (OCT)4 regulate the expression of the miR-302-367 cluster. miR-145 regulates SOX2 and OCT4 translation and p53 regulates miR-145 expression. We analysed the expression of the miR-302-367 cluster and miR-145 and the mutational status of p53 in resected nonsmall cell lung cancer (NSCLC) patients and correlated results with time to relapse (TTR). Tumour and paired normal tissue samples were obtained from 70 NSCLC patients. MicroRNA expression was assessed with TaqMan MicroRNA Assays. p53 exons 5 to 8 were sequenced. miR-145 was downregulated (p<0.0001) and miR-367 was upregulated (p<0.0001) in tumour compared with normal tissue. Mean TTR was 18.4 months for patients with low miR-145 levels and 28.2 months for those with high levels (p=0.015). Mean TTR was 29.1 months for patients with low miR-367 levels and 23.4 months for those with high levels (p=0.048). TTR was shorter for patients with both unfavourable variables (p=0.009). Low miR-145 expression (p=0.049), the combination of unfavourable microRNA levels (p=0.02) and the combination of low miR-145 with p53 mutations (p=0.011) were independent markers of shorter TTR. In conclusion, miR-145 and miR-367 expression could be novel markers for relapse in surgically treated NSCLC. p53 may play a role in modulating miR-145 expression in NSCLC.

Anatomy of the Ankle and Subtalar Joint Ligaments: What We Do Not Know About It?

Understanding of the ankle and subtalar joint ligaments is essential to recognize and manage foot and ankle disorders. The stability of both joints relies on the integrity of its ligaments. The ankle joint is stabilized by the lateral and medial ligamentous complexes while the subtalar joint is stabilized by its extrinsic and intrinsic ligaments. Most injuries to these ligaments are linked with ankle sprains. Inversion or eversion mechanics affect the ligamentous complexes. A profound knowledge of the ligament's anatomy allows orthopedic surgeons to further understand anatomic or nonanatomic reconstructions.

Outcomes of Combined Posterior Tibial Tendon Tendoscopy and Medializing Calcaneal Osteotomy for Stage IA Progressive Collapsing Foot Deformity.

BACKGROUND: Posterior tibial tendon (PTT) tendoscopy and medializing calcaneal osteotomy (MCO) are among the available techniques for patients presenting with symptomatic flexible hindfoot valgus (stage IA) progressive collapsing foot deformity (PCFD). The aim of this study was to determine clinical and radiographic outcomes of combined PTT tendoscopy and MCO for patients presenting with symptomatic stage IA PCFD. METHODS: A retrospective cohort study was performed in order to determine clinical and radiographic outcomes of 30 combined PTT tendoscopies and MCO on 27 patients presenting with symptomatic stage IA PCFD, with a minimum follow-up of 24 months. Patient satisfaction was assessed at last available follow-up as very satisfied, satisfied, and unsatisfied. Clinical assessment was performed evaluating preoperative and last available follow-up visual analog scale for pain (VAS-P), Foot and Ankle Outcome Score (FAOS), and the 36-Item Short Form Health Survey (SF-36). Magnetic resonance imaging (MRI) was performed preoperatively on all patients. Standard weightbearing anteroposterior, lateral, and long axial view radiographs of the foot and ankle were taken preoperatively, immediate postoperatively, at 6 weeks, 3 months, 6 months, 1 year postoperatively, and last follow-up evaluation available for each patient. RESULTS: The mean follow-up was 38.6 (range, 26-62) months. We registered 27 very satisfied, 1 satisfied, and 2 unsatisfied patients. There was statistically significant improvement on all clinical scores (VAS-P, FAOS and SF-36), as well as on lateral talo-first metatarsal and hindfoot alignment angles. We found low-grade PTT tears in 5 patients (16.67%) in whom preoperative MRI documented PTT tenosynovitis alone. CONCLUSION: We found that combined PTT tendoscopy and MCO provide significant clinical and radiographic improvement for patients presenting with symptomatic stage IAB PCFD. PTT tendoscopy should be considered in the treatment of all surgically addressed flexible valgus feet as it detects tendon tears which are frequently missed on an MRI. LEVEL OF EVIDENCE: Level IV, retrospective case series.

Whole-mount staining of mouse colorectal cancer organoids and fibroblast-organoid co-cultures.

Imaging organoid culture provides an excellent tool for studying complex diseases such as cancer. However, retaining the morphology of intact organoids for immunolabeling has been challenging. Here, we describe a protocol for immunofluorescence staining in intact colorectal cancer organoids derived from mice. We also describe additional steps for co-culture with mouse fibroblasts to enable the study of interactions with other cellular components of the tissue microenvironment. For complete details on the use and execution of this protocol, please refer to Martinez-Ordoñez et al. (2023).1.

Enhanced SREBP2-driven cholesterol biosynthesis by PKCλ/ι deficiency in intestinal epithelial cells promotes aggressive serrated tumorigenesis.

The metabolic and signaling pathways regulating aggressive mesenchymal colorectal cancer (CRC) initiation and progression through the serrated route are largely unknown. Although relatively well characterized as BRAF mutant cancers, their poor response to current targeted therapy, difficult preneoplastic detection, and challenging endoscopic resection make the identification of their metabolic requirements a priority. Here, we demonstrate that the phosphorylation of SCAP by the atypical PKC (aPKC), PKCλ/ι promotes its degradation and inhibits the processing and activation of SREBP2, the master regulator of cholesterol biosynthesis. We show that the upregulation of SREBP2 and cholesterol by reduced aPKC levels is essential for controlling metaplasia and generating the most aggressive cell subpopulation in serrated tumors in mice and humans. Since these alterations are also detected prior to neoplastic transformation, together with the sensitivity of these tumors to cholesterol metabolism inhibitors, our data indicate that targeting cholesterol biosynthesis is a potential mechanism for serrated chemoprevention.