Longitudinal Comparative Analysis of Circulating Tumor DNA and Matched Tumor Tissue DNA in Patients with Metastatic Colorectal Cancer Receiving Palliative First-Line Systemic Anti-Cancer Therapy.

Purpose: This study aimed to compare tumor tissue DNA (ttDNA) and circulating tumor DNA (ctDNA) to explore the clinical applicability of ctDNA and to better understand clonal evolution in patients with metastatic colorectal cancer undergoing palliative first-line systemic therapy. Materials and Methods: We performed targeted sequencing analysis of 88 cancer-associated genes using germline DNA, ctDNA at baseline (baseline-ctDNA), and ctDNA at progressive disease (PD-ctDNA). The results were compared with ttDNA data. Results: Among 208 consecutively enrolled patients, we selected 84 (41 males; median age 59, range 35 to 90) with all four sample types available. A total of 202 driver mutations were found in 34 genes. ttDNA exhibited the highest mutation frequency (n=232), followed by baseline-ctDNA (n=155) and PD-ctDNA (n=117). Sequencing ctDNA alongside ttDNA revealed additional mutations in 40 patients (47.6%). PD-ctDNA detected 13 novel mutations in 10 patients (11.9%) compared to ttDNA and baseline-ctDNA. Notably, 7 mutations in 5 patients (6.0%) were missense or nonsense mutations in APC, TP53, SMAD4, and CDH1 genes. In baseline-ctDNA, higher maximal variant allele frequency (VAF) values (p=0.010) and higher VAF values of APC (p=0.012), TP53 (p=0.012), and KRAS (p=0.005) mutations were significantly associated with worse overall survival. Conclusion: While ttDNA remains more sensitive than ctDNA, our ctDNA platform demonstrated validity and potential value when ttDNA was unavailable. Post-treatment analysis of PD-ctDNA unveiled new pathogenic mutations, signifying cancer's clonal evolution. Additionally, baseline-ctDNA's VAF values were prognostic after treatment.

More than 3 mm of preoperative medial meniscal extrusion is identified as a key risk factor for varus progression in limb alignment after arthroscopic repair of medial meniscus posterior root tear.

PURPOSE: To investigate the risk factors for varus progression after arthroscopic medial meniscal posterior root tear (MMPRT) repair and to compare the clinical outcomes between two groups: one with more varus progression and the other with less varus progression. METHODS: Patients who underwent isolated arthroscopic repair of MMPRT between 2015 and 2020 were enroled, and 2-year follow-up data were collected. Participants were categorized into two groups based on preoperative values of the weight-bearing line (WBL) ratio: group A with <5.9% increase and group B with ≥5.9% increase. Various factors, including demographic features and radiological findings, were analysed and compared between the two groups. Intra-meniscal signal intensity, meniscal healing, medial meniscal extrusion (MME), and articular cartilage grade were assessed preoperatively and 1-year postoperatively using coronal magnetic resonance imaging. RESULTS: The final cohort consisted of 34 patients in group A and 46 in group B, with a mean age of 55.8 ± 11.2 and 59.8 ± 6.6 years, respectively. Preoperative WBL ratio and cartilage lesions in the medial compartment did not differ between the groups. Preoperative MME were significantly lower in group A than those in group B (2.6 ± 0.6 mm in group A and 3.5 ± 0.7 mm in group B, p < 0.05). Patient-reported outcomes at the 2-year follow-up did not differ between the two groups (n. s.). In a logistic analysis, the odds ratio of MME was 2.1 (p < 0.05), and the cutoff value of MME was 3.02 mm. CONCLUSION: Preoperative MME is a risk factor for varus progression. However, no differences in patient-reported outcomes were observed at 2-year follow-up, even in the group with greater varus progression. LEVEL OF EVIDENCE: Level IV.

A Comparison of Results in Older, Middle-aged, and Younger Patients after Primary Anterior Cruciate Ligament Reconstruction: Minimum 10-Year Follow-up.

Background: Anterior cruciate ligament (ACL) reconstruction is commonly performed to prevent decreased knee function and restore stability in middle-aged and even older patients. However, few studies have compared the long-term clinical outcomes of ACL reconstruction between older, younger, and middle-aged patients. The purpose of this study was to compare the long-term clinical outcomes of ACL reconstruction in older patients with those in younger and middle-aged patients. Methods: A total of 352 patients who underwent primary ACL reconstruction between January 2003 and March 2008 were retrospectively reviewed and classified into three groups (group A: 246 [age, 20-29 years], group B: 72 [age, 40-49 years], group C: 34 [age, 50-65 years]). The mean follow-up period was 14.2 ± 1.6 years. Clinical outcomes were evaluated and compared between groups. Results: The differences in the range of motion, clinical scores, and stability tests were not statistically significant among the three groups. The difference in the graft failure rate among the three groups was significant (group A: 16 [6.5%], group B: 7 [9.7%], group C: 6 [17.6%]; p = 0.040). In particular, when compared between the two groups, there was a significant difference between group A and group C (p = 0.036). The 10-year survival rates were 93.5%, 90.3%, and 82.4% for groups A, B, and C, respectively (p = 0.048). Conclusions: Although graft failure rates were higher in older patients than younger and middle-aged patients, clinical outcomes of ACL reconstruction in older patients were comparable to those of younger and middle-aged patients in terms of the range of motion, clinical scores, and stability tests at a minimum follow-up of 10 years.

MPFL reconstruction with proximal rather than distal femoral tunnel position leads to less favorable short-term results.

BACKGROUND: This study aimed to compare the clinical and radiological outcomes of medial patellofemoral ligament (MPFL) reconstruction (MPFLR) between anatomic femoral tunnel positions: proximal (near adductor tubercle [AT]) and distal (near medial epicondyle [ME]). HYPOTHESIS: MPFLR with the proximal femoral tunnel position has worse clinical and radiological outcomes than those with the distal femoral tunnel position. PATIENTS AND METHODS: Fifty-five patients who underwent isolated MPFLR with proximal or distal femoral tunnels with at least 2 years of follow-up were retrospectively analyzed. Based on postoperative CT images, 28 patients were classified as group AT and the remaining 27 patients were classified as group ME. The International Knee Documentation Committee, Lysholm, Tegner, Kujala scores, and complications were evaluated. Radiologically, the Caton-Deschamps Index (CDI), patellar tilt angle, patellofemoral osteoarthritis (PFOA), patellofemoral cartilage status by the International Cartilage Repair Society (ICRS) grade, bone contusion, and MPFL graft signal intensity were evaluated. RESULTS: All clinical scores significantly improved in both groups (p<0.01). No statistically significant difference was noted between the two groups in regards to their preoperative demographic data, postoperative clinical scores, complications, or radiological findings (CDI, patellar tilt angle, PFOA, bone contusion, and graft signal intensity). The group AT had worse cartilage status on the medial facet of the patella (p=0.02). The ICRS grade for the medial facet of the patella statistically progressed in group AT compared to group ME (p=0.04) as well. DISCUSSION: Both groups showed significantly improved clinical outcomes. However, for the medial facet of the patella, MPFLR with the proximal femoral tunnel position had worse cartilage status and ICRS grade progression than those with the distal femoral tunnel position. LEVEL OF EVIDENCE: III; retrospective comparative study.

Similar Midterm Outcomes of Total Knee Arthroplasties with Anterior and Posterior Tibial Slopes Performed on Paired Knees at a Minimum Follow-up of 5 Years.

A small posterior tibial slope (PTS) is generally recommended in posterior stabilized (PS) total knee arthroplasty (TKA). An unwanted anterior tibial slope (ATS), which can affect postoperative results, may be created in PS TKA because of the inaccuracy of surgical instruments and techniques, as well as high interpatient variability. We compared midterm clinical and radiographic results of PS TKAs with ATS and PTS performed on paired knees using the same prosthesis. One-hundred-twenty-four patients who underwent TKAs with ATS and PTS on paired knees using ATTUNE posterior-stabilized prostheses were retrospectively reviewed after a minimum follow-up period of 5 years. The mean follow-up period was 5.4 years. The Knee Society Knee and Function scores, Western Ontario and McMaster Universities Osteoarthritis Index, Feller and Kujalar scores, and range of motion (ROM) were evaluated. The preferred TKA out of ATS and PTS was also investigated. The hip-knee-ankle angle, component positions, tibial slope, posterior femoral offset, Insall-Salvati ratio, and knee sagittal angle were measured by radiography. There were no significant differences in the clinical results, including ROM, between TKAs with ATS and PTS preoperatively and at the last follow-up. Regarding patient preference, 58 patients (46.8%) were satisfied with bilateral knees, 30 (24.2%) preferred knees with ATS, and 36 (29%) preferred knees with PTS. There was no significant difference in the rate of preference between TKAs with ATS and PTS (p = 0.539). Except for the postoperative tibial slope (-1.8 vs. 2.5 degrees, p < 0.001), there were also no significant differences in the radiographic results, including the knee sagittal angle, preoperatively and at the last follow-up. The midterm outcomes were similar between PS TKAs with ATS and PTS performed on paired knees at a minimum of 5 years of follow-up. Nonsevere ATS did not affect midterm outcomes in PS TKA with proper soft tissue balancing and the current prosthesis of improved design. However, a long-term follow-up study is required to confirm the safety of nonsevere ATS in PS TKA. LEVEL OF EVIDENCE:: III.

Costal chondrocyte-derived pellet-type scaffold-free autologous chondrocyte implantation provided acceptable mid-term outcomes in osteochondral defects with up to 10-mm depth.

PURPOSE: To evaluate clinical, radiographic, and magnetic resonance (MR) results of costal chondrocyte-derived pellet-type scaffold-free autologous chondrocyte implantation (CCP-ACI) in osteochondral defects (ODs) up to 10-mm depth during 5 years of follow-up. METHODS: Ten patients with CCP-ACI performed in ODs with depth up to 10 mm were retrospectively analyzed. The minimum follow-up period was 5 years. The median age was 36.5 (range 20-55) years. The median size and the depth of the OD lesion were 4.25 cm2 (range 2-6) and 7.0 mm (6-9), respectively. Clinically, the International Knee Documentation Committee, Lysholm, and visual analog scale pain scores were evaluated. Radiographically, the hip‒knee‒ankle (HKA) angle and the Kellgren‒Lawrence (K‒L) grade were assessed. On MR imaging, the magnetic resonance observation of cartilage repair tissue (MOCART) 2.0 score and the defect depth were evaluated. RESULTS: All average clinical scores improved significantly by 1, 2, and 5 years postoperatively. The average HKA angle and the proportion of K‒L grade did not change significantly within 5 years. The median total MOCART scores were 50 (range 45-65), 50 (35-90), 57.5 (40-90), and 65 (50-85) at 6 months, 1 year, 2 years, and 5 years postoperatively, respectively (p = 0.001), with significant improvement at 2 years compared to that at 6 months postoperatively. The signal intensity of the repair tissue and subchondral change significantly improved from 10 (range 10-10) to 12.5 (10-15) (p = 0.036), and from 10 (10-10) to 17.5 (0-20) (p = 0.017), respectively. Significant improvements were seen at 5 years postoperatively for the former and at 2 years postoperatively for the latter. The average depths on MR imaging were 6.7, 6.7, 6.8, 6.6, and 6.6 mm preoperatively and at 6 months, 1 year, 2 years, and 5 years postoperatively with no significant changes (n.s). CONCLUSION: CCP-ACI provided acceptable mid-term outcomes in ODs up to 10-mm in depth without bone grafting despite of no scaffold. The procedure can be one of minimally invasive treatment options for ODs without scaffold-related problems. LEVEL OF EVIDENCE: IV.

Aseptic survival of the 1.5-stage exchange arthroplasty for periprosthetic joint infection was acceptable when using an autoclaved femoral component and a new polyethylene insert.

PURPOSE: To investigate the aseptic survival of 1.5-stage exchange arthroplasty for periprosthetic joint infection (PJI) after total knee arthroplasty (TKA). METHODS: Eighty-eight cases of 1.5-stage exchange arthroplasty for PJI without reinfection were retrospectively analysed. The autoclaved femoral component and new polyethylene insert (PE) were implanted using antibiotic mixed cement. The explanted tibial component was not reinserted. The Western Ontario and McMaster Universities Osteoarthritis Index and range of motion were clinically evaluated preoperatively and at the last follow-up (the last time for the implant in situ). Radiographically, hip-knee-ankle angle (HKA) and component positions were measured preoperatively, postoperatively (1 month after the 1.5-stage exchange arthroplasty), and at the last follow-up. The survival rate was analysed using the Kaplan-Meier method, in which failure was defined as reoperation due to aseptic failure. Mean period to failure and failure site were analysed. Factors affecting survival were investigated in terms of demographics and inappropriateness of the postoperative HKA (HKA > 0 ± 3°) and component positions (α angle > 95 ± 3°, ß angle > 90 ± 3°, γ angle > 3 ± 3°, and δ angle > 87 ± 3°). RESULTS: The spacer in-situ time was 3.7 years (0.2-6.4). The clinical results improved hip-knee-ankle significantly at the last follow-up. Radiographically, the average HKA was valgus 0.1° postoperatively. The average α, ß, γ, and δ angles of the postoperative component positions were 95.9°, 90.4°, 3.8°, and 86.7°, respectively. The 1-, 2-, and 5-year postoperative survival rates were 90.9%, 86.4%, and 80.6%, respectively. The mean period to failure was 2.0 years (0.2-5.3). There were 18 cases of aseptic loosening (20.8%), occurring on both the femur and tibial sides in 1 knee, and only on the tibial side in 17 knees. Inappropriate coronal position of the PE (ß angle > 90 ± 3°) was a significant factor affecting survival (odds ratio = 5.491; p = 0.011). CONCLUSION: The aseptic survival of the 1.5-stage exchange arthroplasty was acceptable when using an autoclaved femoral component and new PE. The appropriate coronal position of the PE helps ensure favourable survival of 1.5-stage exchange arthroplasty. LEVEL OF EVIDENCE: IV.

Additional anteromedial staple fixation prevents changes in the posterior tibial slope in retrotuberosity bi-planar open-wedge high tibial osteotomy.

PURPOSE: To compare the changes in posterior tibial slope (PTS) between retrotuberosity biplane open-wedge high tibial osteotomies (RT-OWHTOs) with and without additional anteromedial staple fixation. METHODS: Seventy-nine and 77 cases of RT-OWHTOs without (Group N) and with (Group S) additional staple fixation, respectively, were retrospectively reviewed. All procedures were performed using a locking spacer plate. Demographics and preoperative knee condition were similar between the groups. Clinically, the Western Ontario and McMaster Universities Arthritis Index and range of motion were evaluated preoperatively and 2 years postoperatively. Radiographically, the mechanical axis (MA), medial proximal tibial angle (MPTA), and PTS were evaluated preoperatively and within 2 years postoperatively. Hinge fractures were investigated using computed tomography at 2 weeks postoperatively. PTS loss was defined as the difference between the corresponding values at 2 weeks and 2 years postoperatively. The incidence of PTS failure (PTS loss ≥ ± 3°) was also investigated. RESULTS: The clinical results were not significantly different between groups N and S preoperatively and 2 years postoperatively. There were no significant differences in the MA, MPTA, and PTS between the groups preoperatively and 2 weeks postoperatively; changes in these variables did not differ significantly between the groups. The incidence of hinge fractures, all of which were categorized as Takeuchi type 1, did not differ significantly. PTS loss within 2 years postoperatively was significantly greater in group N than in group S (1.0° vs. 0.1°; p < 0.01). The incidence of the PTS failure was 16.5% (13/79) and 2.6% (2/77) in groups N and S, respectively (p < 0.01). CONCLUSION: Additional anteromedial staple fixation could prevent changes in the PTS in RT-OWHTO. It is a simple method for preventing an increase in the PTS after RT-OWHTO. LEVEL OF EVIDENCE: III.

Oleic acid from cancer-associated fibroblast promotes cancer cell stemness by stearoyl-CoA desaturase under glucose-deficient condition.

BACKGROUND: Cancer-associated fibroblasts (CAFs) coordinate the malignancy of cancer cells via secretory materials. Reprogrammed lipid metabolism and signaling play critical roles in cancer biology. Oleic acid (OA) serves as a source of energy under glucose-deficient conditions, but its function in cancer progression remains unclear. The present study investigated that CAFs in xenografted tumors had higher amounts of fatty acids, particularly OA, compared to normal fibroblasts, and promoted the cancer cell stemness in lung adenocarcinoma cells under glucose-deficient condition. METHODS: Xenografts were established in immunodeficient mice by injection of NCI-H460 (H460) cells. Lipids and fatty acids were evaluated using the BODIPY staining and fatty-acid methyl esters analysis. The expression levels of markers for lipid metabolism and cancer stemness were determined by western blot, flow cytometry, and real-time PCR. Cancer cell subclones against stearoyl-CoA desaturase (SCD) were produced by lentiviral vector and CRISPR/cas9 systems. The expression of SCD was examined immunochemically in human adenocarcinoma tissues, and its clinical relevance to survival rate in lung adenocarcinoma patients was assessed by Kaplan-Meier analysis. RESULTS: Transferred CAF-derived OA through lipid transporter upregulated SCD in cancer cells under glucose-deficient conditions, resulting in enhanced lipid metabolism and autophagosome maturation. By OA treatment under glucose deficient condition, cancer cell stemness was significantly enhanced through sequential activation of SCD, F-actin polymerization and nuclear translocation of yes-associated protein. These findings were confirmed by experiments using chemical inhibitors, SCD-overexpressing cells and SCD-knockout (KO) cells. When xenografted, SCD-overexpressing cells produced larger tumors compared with parental cells, while SCD-KO cells generated much smaller tumors. Analysis of tumor tissue microarray from lung adenocarcinoma patients revealed that SCD expression was the marker for poor prognosis involving tumor grade, clinical stage and survival rate. CONCLUSION: Our data indicate that CAFs-derived OA activated lipid metabolism in lung adenocarcinoma cells under glucose-deficient conditions, subsequently enhancing stemness and progression toward malignancy.

Use of Grafts on an Open Gap Is Advantageous for Preventing Correction Loss in 1-Week Staged Bilateral Open-Wedge High Tibial Osteotomies.

One-week staged bilateral open-wedge high tibial osteotomies (OWHTOs) can be a safe procedure, with the added advantage of fast functional recovery, cost saving, and reduced hospital stay. However, there can be concerns about correction loss after 1-week staged OWHTOs because high loading is inevitably applied to osteotomy sites during postoperative weight bearing. Although leaving the osteotomy site with no grafts is possible in OWHTOs, the use of grafts can provide additional stability to the osteotomy site and prevent correction loss. We compared the amount and incidence of correction loss between 1-week staged bilateral OWHTOs with and without allogenic bone grafts. Seventy-five patients who underwent 1-week staged bilateral OWHTOs with a locking spacer plate (Nowmedipia, Seoul, Korea) by a single surgeon were retrospectively reviewed. Allogenic cancellous bone grafts were applied in 53 patients (group G; 106 knees, operated consecutively between 2012 and 2017) but not in 22 patients (group N; 44 knees, operated consecutively between 2017 and 2019). Demographics were similar between the groups. Radiographically, the mechanical axis (MA), medial proximal tibial angle (MPTA), and posterior tibial slope (PTS) were evaluated preoperatively and within 1 year postoperatively. Unstable hinge fracture was investigated using computed tomography in all cases. The incidence of correction loss (MPTA loss ≥ 3 degrees) was determined. There were no significant differences in the MA, MPTA, and PTS between the groups preoperatively and 2 weeks postoperatively. The incidence of unstable hinge fractures did not differ. The losses in MA, MPTA, and PTS during the first postoperative year were significantly greater in group N than in group G (MA, -5.5 vs. -2.3 degrees; MPTA, -3.0 vs. 0 degrees; PTS, -2.0 vs. -0.7 degrees; p < 0.05 on all parameters). The correction loss incidence was 6.6% (7/106) and 31.8% (14/44) in groups G and N, respectively (p < 0.001). Appropriate treatment is necessary to prevent correction loss in 1-week staged bilateral OWHTOs. Grafting, which provides additional stability to the osteotomy site, is a recommended method. Level of evidence is IV.

Subungual Pigmented Squamous Cell Carcinoma in a Dog.

An 11-year-old spayed female Miniature Schnauzer dog was presented with loss of a claw caused by a nail bed mass. Histopathological evaluation revealed that the mass comprised neoplastic squamous cells with abundant cytoplasmic melanin pigment. Immunohistochemically, the neoplastic cells were positive for cytokeratin and negative for vimentin and ionized calcium-binding adaptor molecule 1, supporting a diagnosis of pigmented squamous cell carcinoma. To our knowledge, this is the first report of subungual pigmented squamous cell carcinoma in animals.

Calcifying aponeurotic fibroma on the paw in a dog.

A 12-year-old, spayed female, Maltese dog with a round and firm mass on the dorsal part of the left rear paw and a cervical mass was brought to the clinic. The paw mass was contiguous to the adjacent tendon; it was composed of neoplastic mesenchymal cells and had scattered foci of calcification with chondroid differentiation microscopically. The neoplastic cells were positive for vimentin and S100, but negative for desmin and smooth muscle actin. Microscopic features and immunohistochemistry results were consistent with calcifying aponeurotic fibroma (CAF). The cervical mass was composed of polygonal cells forming acini with marked anisocytosis and anisokaryosis and diagnosed as thyroid follicular carcinoma. No recurrence or metastasis occurred during follow-up. To the best of our knowledge, this is the first case of canine CAF with features identical to its human counterparts. Key clinical message: This report describes the rare case of calcifying aponeurotic fibroma on the paw in a dog. This is apparently the first case in the veterinary literature with identical clinical and pathological features to the human counterpart.

Fibrome aponévrotique calcifiant sur la patte chez un chien. Une chienne maltaise stérilisée âgée de 12 ans avec une masse ronde et ferme sur la partie dorsale de la patte arrière gauche et une masse cervicale a été amenée à la clinique. La masse de la patte était contiguë au tendon adjacent; il était composé de cellules mésenchymateuses néoplasiques et présentait des foyers de calcification dispersés avec une différenciation chondroïde au microscope. Les cellules néoplasiques étaient positives pour la vimentine et le S100, mais négatives pour la desmine et l'actine des muscles lisses. Les caractéristiques microscopiques et les résultats d'immunohistochimie étaient compatibles avec un fibrome aponévrotique calcifiant (CAF). La masse cervicale était composée de cellules polygonales formant des acini avec une anisocytose et une anisocaryose marquées et diagnostiquée comme un carcinome folliculaire de la thyroïde. Aucune récidive ou métastase n'est survenue au cours du suivi. À notre connaissance, il s'agit du premier cas de CAF canin avec des caractéristiques identiques à ses homologues humains.Message clinique clé :Ce rapport décrit le cas rare de fibrome aponévrotique calcifiant sur la patte chez un chien. C'est apparemment le premier cas dans la littérature vétérinaire avec des caractéristiques cliniques et pathologiques identiques à son homologue humain.(Traduit par Dr Serge Messier).

Temozolomide abrogates the aggressiveness of urothelial carcinoma cells by enhancing senescence and depleting the side population.

Patients with advanced urothelial carcinoma (UC) generally have poor prognoses due to therapeutic resistance. Furthermore, there are limited treatment options for advanced UC. Therefore, novel or effective chemotherapeutic agents are needed to improve patient survival. The present study was conducted to investigate the effect of temozolomide (TMZ) on UC cells so as to identify a potential method to overcome therapeutic resistance. TMZ is an alkylating agent with a target different from that of other anticancer drugs used to treat UC, such as cisplatin. TMZ enhanced the autophagic response and senescence, which was mediated via the p53 and p21 pathways. Inhibiting the autophagic response using chloroquine synergistically augmented the cytotoxic effect of TMZ on UC cells. TMZ significantly reduced the invasiveness of UC cells. Notably, the abundance of side population fraction was also significantly reduced following TMZ treatment. Considering that side population fraction is known to confer therapeutic resistance, it is noteworthy that the TMZ treatment markedly decreased side population fraction. Altogether, TMZ may have the potential to be applied as a part of an alternative treatment strategy to reduce the malignancy of UC cells.

Heterogeneous response of cancer-associated fibroblasts to the glucose deprivation through mitochondrial calcium uniporter.

Cancer-associated fibroblasts (CAFs) are an abundant component of the tumor microenvironment and have distinct features from normal fibroblasts (NFs). However, the discriminative nature of heterogeneous CAFs under glucose starvation remains unknown. In this study, we investigated the changes in the mitochondrial calcium concentration and relevant intracellular machinery in CAFs under glucose-deficient conditions. Xenografted tumor masses were dissected into multiple pieces and subjected to the CAF isolation using magnetically activated cell sorting (MACS). NFs were separated from the normal lung and skin. Under glucose starvation, CAFs from the tumor mass exhibited heterogeneity in cell proliferation, ATP production and calcium concentration. Compared to NFs, mitochondrial calcium concentration was significantly higher in glucose-starved CAFs with upregulation of mitochondrial calcium uniporter (MCU) that led to enhancement of ATP production and cell growth. Intriguingly, treatment of glucose-starved CAFs with oligomycin increased apoptosis by disrupted calcium homeostasis following overactivation of the mPTP. Moreover, oligomycin-induced apoptosis was mitigated by calcium chelation. This study demonstrated that the discriminative calcium influx to mitochondria through MCU coordinated cell growth and apoptosis in glucose-starved CAFs but not in NFs.

Reduction in mitochondrial oxidative stress mediates hypoxia-induced resistance to cisplatin in human transitional cell carcinoma cells.

Tumor hypoxia is known to promote the acquisition of more aggressive phenotypes in human transitional cell carcinoma (TCC), including drug resistance. Accumulating evidence suggests that mitochondria play a central role in the chemoresistance of TCC. However, the role of mitochondria in the hypoxia-induced drug resistance in TCC remains elusive. The present study investigated the function of mitochondria in the drug resistance using a TCC cell line under hypoxic conditions. In vitro hypoxia (0.1% O2, 48 h) was achieved by incubating TCC cells in air chamber. Mitochondrial events involving hypoxia-induced drug resistance were assessed. Hypoxia significantly reduced the cisplatin-induced apoptosis of TCC cells. Additionally, hypoxia substantially decreased the level of mitochondrial reactive oxygen species (ROS) generated by cisplatin treatment. Analogously, elimination of mitochondrial ROS significantly rescued cells from cisplatin-induced apoptosis. Hypoxia enhanced mitochondrial hyperpolarization, which was not related to ATP production or the reversal of ATP synthase activity. The mitochondrial DNA (mtDNA) amplification efficiency data illustrated that hypoxia significantly prevented oxidative damage to the mitogenome. Moreover, transmission electron microscopy revealed that cisplatin-induced disruption of the mitochondrial ultrastructure was abated under hypoxic conditions. Notably, depletion of mtDNA by ethidium bromide abrogated hypoxia-induced resistance to cisplatin. Taken together, the present study demonstrated that TCC cells exposed to hypoxic conditions rendered mitochondria less sensitive to oxidative stress induced by cisplatin treatment, leading to enhanced drug resistance.

Thyroglobulin as a negative marker for malignancy in canine and human breast tumors.

Canine mammary gland tumors (CMTs) are the most common tumor type in female dogs. This study evaluated the expression pattern and role of thyroglobulin (Tg) in CMT and in human breast cancer (HBC). CMT samples were subjected to fine-needle aspiration, primary cell culture, and histopathology. The expression level of Tg was higher in benign CMT than in malignant CMT (mCMT) primary cells, particularly in the epithelial lineage. Moreover, treatment with Tg enhanced the sensitivity of doxorubicin in mCMT epithelial cells and mitigated proinflammatory response by increasing nuclear factor erythroid 2-related factor 2 (Nrf2). The proximal region of the Tg promoter was hypermethylated in mCMT epithelial cells, silencing Tg expression with concurrent downregulation of Nrf2-mediated antioxidant signaling. An identical pattern of Tg expression was observed in cytological and tissue samples. Tissue microarray analysis showed that Tg was highly expressed in normal and benign tissues when compared with their malignant counterparts, which was diminished along with higher histological grades. The survival rate was significantly higher in HBC patients with high Tg expression than those with low Tg expression. This study also showed that the progression of HBC is accompanied by the reduction of Tg expression along with augmentation of proinflammatory signaling. Our data suggested that Tg could be a negative indicator of malignancy in canine and human breast neoplasia.

Synthesis of DNA-templated copper nanoparticles with enhanced fluorescence stability for cellular imaging.

Fluorescence of DNA-templated copper nanoparticles (DNA-CuNPs) is not stable over time which limits applications in cellular imaging. This is due to the presence of oxygen during synthesis which oxidizes Cu(0) to Cu(II) and also produces the free hydroxyl radical. The authors have prepared DNA-CuNPs with enhanced temporal stability of fluorescence by optimizing the reaction conditions so as to minimize the deleterious effects of oxygen. The operational lifetime of DNA-CuNPs was increased from 25 min to 200 min. Fluorescence spectra of DNA-CuNPs in optimized condition show an emission peak at 650 nm when excited at 340 nm. DNA-CuNPs synthesized in this manner were used for cell imaging. As a proof of concept, the nucleus of a human colon cell line (HCT116) was stained. The method does not involve any chemicals other that copper sulfate and ascorbate. This new approach for generating DNA-CuNPs improves imaging of biological processes and provides a basis for developing other types of DNA-templated nanomaterials. Graphical abstract Schematic presentation of the formation of fluorescent DNA-templated copper nanoparticles (DNA-CuNPs). A large amount of ascorbate provides long operational lifetime for cellular imaging under the condition exposed to oxygen. *Asc- and **DHA stand for ascorbate and dehydroascorbic acid.

Fluorescence polarization-based detection of cancer-related mutations using target-initiated rolling circle amplification.

We devised a new method to detect cancer-related mutations based on target-initiated rolling circle amplification in combination with fluorescence polarization. We then applied this method to identify the presence of KRAS G13D and G12D, two of the most frequent mutations found in colorectal cancer patients, demonstrating high sensitivity and specificity.

Glucose starvation induces resistance to metformin through the elevation of mitochondrial multidrug resistance protein 1.

Metformin, a drug for type 2 diabetes mellitus, has shown therapeutic effects for various cancers. However, it had no beneficial effects on the survival rate of human malignant mesothelioma (HMM) patients. The present study was performed to elucidate the underlying mechanism of metformin resistance in HMM cells. Glucose-starved HMM cells had enhanced resistance to metformin, demonstrated by decreased apoptosis and autophagy and increased cell survival. These cells showed abnormalities in mitochondria, such as decreased ATP synthesis, morphological elongation, altered mitochondrial permeability transition pore and hyperpolarization of mitochondrial membrane potential (MMP). Intriguingly, Mdr1 was significantly upregulated in mitochondria but not in cell membrane. The upregulated mitochondrial Mdr1 was reversed by treatment with carbonyl cyanide m-chlorophenyl hydrazone, an MMP depolarization inducer. Furthermore, apoptosis and autophagy were increased in multidrug resistance protein 1 knockout HMM cells cultured under glucose starvation with metformin treatment. The data suggest that mitochondrial Mdr1 plays a critical role in the chemoresistance to metformin in HMM cells, which could be a potential target for improving its therapeutic efficacy.

Factors Affecting the Extent of Graft Tendon Synovialization after Double-Bundle Anterior Cruciate Ligament Reconstruction: Based on Second-Look Arthroscopic Findings.

BACKGROUND: We aimed to examine the factors that influence synovialization of the grafted tendon after double-bundle anterior cruciate ligament (ACL) reconstruction based on second-look arthroscopic findings. METHODS: Out of 205 knees that were treated between August 2008 and May 2016 with double-bundle ACL reconstruction using bio-absorbable cross-pins and Endobuttons for femoral tunnel fixation, we enrolled 65 knees (64 patients) that underwent second-look arthroscopy with hardware removal at 1 year postoperatively. Measured clinical outcomes included the Lysholm score and Tegner activity score that were evaluated preoperatively and during the final follow-up. We analyzed the relationship between synovial coverage and patient age, length of the preserved remnant tissue on the tibial side, type of bundle (anteromedial or posterolateral), type of graft (autograft or allograft), and time from injury to surgery. RESULTS: The area of synovial coverage showed a significant statistical correlation with patient age and the length of the preserved remnant tissue on the tibial side. The average synovial coverage was significantly better for the anteromedial bundle than for the posterolateral bundle, better for the autograft than for the allograft reconstruction, and better when treated in the acute stage than in the chronic stage. However, synovialization of grafted tendon did not correlate to clinical outcomes. CONCLUSIONS: While we were able to identify several factors influencing synovialization of the grafted tendon after double-bundle ACL reconstruction, including patient age, length of preserved remnant tissue of the torn ACL, type of bundle, type of graft, and time from injury to surgery, we found no evidence that increased synovialization improves clinical outcomes at 1 year postoperatively.