Persistence and enrichment of dominant T cell clonotypes in expanded tumor-infiltrating lymphocytes of breast cancer.

BACKGROUND: Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown promising results in cancer treatment, including breast cancer. However, clonal dynamics and clinical significance of TIL expansion ex vivo remain poorly understood. METHODS: We investigated T cell receptor (TCR) repertoire changes in expanded TILs from 19 patients with breast cancer. We compared TCR repertoire of TILs at different stages of expansion, including initial (2W TILs) and rapid expansion (REP TILs), and their overlap with formalin fixed paraffin embedded (FFPE) and peripheral blood. Additionally, we examined differences in TCR repertoire between CD4+ and CD8+ REP TILs. RESULTS: In descending order of proportion, average of 60% of the top 10% clonotypes of FFPE was retained in 2W TIL (60% in TRB, 64.7% in TRA). Among the overlapped clonotypes between 2W TILs and REP TILs, 69.9% was placed in top 30% of 2W TIL. The proportion of clonotypes in 2W TIL and REP TIL showed a significant positive correlation. CD4+ and CD8+ T cells show similar results in diversity and CDR3 length. CONCLUSIONS: Our study traces the changes in TILs repertoire from FFPE to 2W TIL and REP TIL and confirmed that clonotypes with high frequencies in TILs have a high likelihood of maintaining their priority throughout culture process.

Predicting disease recurrence in limited disease small cell lung cancer using cell-free DNA-based mutation and fragmentome analyses.

Background: Limited disease (LD) small cell lung cancer (SCLC) treated with definitive concurrent chemoradiotherapy (CCRT) potentially experience disease recurrence. We investigated the feasibility of circulating-tumor DNA (ctDNA)-based genomic and fragmentome analyses to assess the risk of recurrence. Methods: Targeted sequencing was conducted using pre-treatment and on-treatment blood samples from definitive CCRT-treated patients with LD-SCLC (n=50). Based on 12-month recurrence-free survival (RFS), patients were categorized into persistent-response (PeR, n=29) and non-PeR (n=21) groups. Fragmentome analysis was conducted using ctDNA fragments of different lengths: P1 (100-155 bp) and P2 (160-180 bp). Results: Patients with TP53 (n=15) and RB1 (n=11) mutation in on-treatment samples demonstrated significantly shorter RFS than patients with wild-type (WT) (P=0.05, P=0.0014, respectively). Fragmentome analysis of all available on-treatment samples (n=26) revealed that the non-PeR group (n=10) had a significantly higher P1 range (P=0.003) and lower P2 range (P=0.002). The areas under the curves for P1, P2, and the fragmentation ratio (P1/P2) in distinguishing the PeR and non-PeR were 0.850, 0.725, and 0.900, respectively. Using optimal cut-off, longer RFSs were found with the low-fragmentation-ratio group than with the high-fragmentation-ratio group (not reached vs. 7.6 months, P=0.002). Patients with both WT RB1 and a low-fragmentation-ratio (n=10) showed better outcomes than patients with both mutated RB1 and a high-fragmentation-ratio (n=10; hazard ratio, 7.55; 95% confidence interval: 2.14-26.6; P=0.002). Conclusions: RB1 mutations and high fragmentation ratios correlated with early disease recurrence. Analyzing ctDNA could help in predicting early treatment failure and making clinical decisions for high-risk patients.

Quantitative and qualitative mutational impact of ionizing radiation on normal cells.

The comprehensive genomic impact of ionizing radiation (IR), a carcinogen, on healthy somatic cells remains unclear. Using large-scale whole-genome sequencing (WGS) of clones expanded from irradiated murine and human single cells, we revealed that IR induces a characteristic spectrum of short insertions or deletions (indels) and structural variations (SVs), including balanced inversions, translocations, composite SVs (deletion-insertion, deletion-inversion, and deletion-translocation composites), and complex genomic rearrangements (CGRs), including chromoplexy, chromothripsis, and SV by breakage-fusion-bridge cycles. Our findings suggest that 1 Gy IR exposure causes an average of 2.33 mutational events per Gb genome, comprising 2.15 indels, 0.17 SVs, and 0.01 CGRs, despite a high level of inter-cellular stochasticity. The mutational burden was dependent on total irradiation dose, regardless of dose rate or cell type. The findings were further validated in IR-induced secondary cancers and single cells without clonalization. Overall, our study highlights a comprehensive and clear picture of IR effects on normal mammalian genomes.

Comparison of the Effect of Landmark-Based Midline and Paramedian Approaches on Spinal Anesthesia-Related Complications in Adult Patients: A Meta-Analysis of Randomized Controlled Trials.

Background and Objectives: Spinal anesthesia is widely used in various types of surgery. However, several complications can occur afterward. This study aimed to identify differences in the incidence of anesthesia-related complications according to the approach methods (midline versus paramedian) for landmark-based spinal anesthesia. Materials and Methods: We searched electronic databases, including PubMed, EMBASE, CENTRAL, Scopus, and Web of Science, for eligible randomized controlled trials. The primary outcome was post-dural puncture headache (PDPH) incidence, and secondary outcomes were low back pain (LBP) incidence and success rate in the first trial of spinal anesthesia. We estimated the odds ratio (OR) with 95% confidence intervals (CI) using a random-effects model. Results: In total, 2280 patients from 13 randomized controlled trials were included in the final analysis. The incidence rates of PDPH were 5.9% and 10.4% in the paramedian and midline approach groups, respectively. The pooled effect size revealed that the incidence of PDPH (OR: 0.43, 95% CI [0.22-0.83]; p = 0.01; I2 = 53%) and LBP (OR: 0.27, 95% CI [0.16-0.44]; p < 0.001; I2 = 16%) decreased, and the success rate in the first attempt was higher (OR: 2.30, 95% CI [1.36-3.87]; p = 0.002; I2 = 35%) with the paramedian than with the midline approach. Conclusions: Paramedian spinal anesthesia reduced PDPH and LBP and increased the success rate of the first attempt.

Reduced side effects and improved pain management by continuous ketorolac infusion with patient-controlled fentanyl injection compared with single fentanyl administration in pelviscopic gynecologic surgery: a randomized, double-blind, controlled study.

BACKGROUND: A combination of opioids and adjunctive drugs can be used for intravenous patient-controlled analgesia (PCA) to minimize opioid-related side effects. We investigated whether two different analgesics administered separately via a dual-chamber PCA have fewer side effects with adequate analgesia than a single fentanyl PCA in gynecologic pelviscopic surgery. METHODS: This prospective, double-blind, randomized, and controlled study included 68 patients who underwent pelviscopic gynecological surgery. Patients were allocated to either the dual (ketorolac and fentanyl delivered by a dual-chamber PCA) or the single (fentanyl alone) group. Postoperative nausea and vomiting (PONV) and analgesic quality were compared between the two groups at 2, 6, 12, and 24 h postoperatively. RESULTS: The dual group showed a significantly lower incidence of PONV during postoperative 2-6 h (P = 0.011) and 6-12 h (P = 0.009). Finally, only two patients (5.7%) in the dual group and 18 (54.5%) in the single group experienced PONV during the entire postoperative 24 h and could not maintain intravenous PCA (odds ratio: 0.056, 95% CI [0.007, 0.229], P < 0.001). Despite the administration of less fentanyl via intravenous PCA during the postoperative 24 h in the dual group than in the single group (66.0 ± 77.8 vs. 383.6 ± 70.1 µg, P < 0.001), postoperative pain had no significant intergroup difference. CONCLUSIONS: Two different analgesics, continuous ketorolac and intermittent fentanyl bolus, administered via dual-chamber intravenous PCA, showed fewer side effects with adequate analgesia than conventional intravenous fentanyl PCA in gynecologic patients undergoing pelviscopic surgery.

The Impact of Regional Nerve Blocks on Postoperative Delirium or Cognitive Dysfunction following Thoracic Surgery: A Systematic Review and Meta-Analysis.

Regional nerve blocks (NBs) mitigate the occurrence of postoperative cognitive dysfunction (POCD) and postoperative delirium (POD) in adult patients undergoing thoracic surgery. This study aimed to determine the exact effect of NBs on POCD and POD. Electronic databases, including PubMed, EMBASE, CINAHL, Scopus, and Web of Science, were searched for studies. The primary outcome was the incidence of POD or POCD. The secondary outcome was pain scores assessed 24 and 48 h postoperatively. We calculated the log odds ratio (LOR) and standardized mean difference (SMD) with 95% confidence intervals (CIs). The LOR was converted to an odds ratio (OR). In the analysis of 1010 patients from seven randomized controlled trials, POD and POCD rates were 14.1% and 16.7%, respectively, in the NB group, and higher, at 27.3% and 35.2%, in the control group. NBs reduced the incidence of POD (OR, 0.44; 95%CI 0.30 to 0.64; p < 0.001; I2 = 0.00%) and POCD (OR, 0.43; 95%CI 0.24 to 0.76; p < 0.001; I2 = 0.00%). NBs reduced pain scores at 24 h (SMD, -2.60; 95%CI -3.90 to -1.30, p < 0.001; I2 = 97.68%) and 48 h (SMD, -1.80; 95%CI -3.18 to -0.41, p = 0.01; I2 = 98.14%) postoperatively. NBs mitigated the occurrence of POD and POCD in adult patients after thoracic surgery.

Cancer signature ensemble integrating cfDNA methylation, copy number, and fragmentation facilitates multi-cancer early detection.

Cell-free DNA (cfDNA) sequencing has demonstrated great potential for early cancer detection. However, most large-scale studies have focused only on either targeted methylation sites or whole-genome sequencing, limiting comprehensive analysis that integrates both epigenetic and genetic signatures. In this study, we present a platform that enables simultaneous analysis of whole-genome methylation, copy number, and fragmentomic patterns of cfDNA in a single assay. Using a total of 950 plasma (361 healthy and 589 cancer) and 240 tissue samples, we demonstrate that a multifeature cancer signature ensemble (CSE) classifier integrating all features outperforms single-feature classifiers. At 95.2% specificity, the cancer detection sensitivity with methylation, copy number, and fragmentomic models was 77.2%, 61.4%, and 60.5%, respectively, but sensitivity was significantly increased to 88.9% with the CSE classifier (p value < 0.0001). For tissue of origin, the CSE classifier enhanced the accuracy beyond the methylation classifier, from 74.3% to 76.4%. Overall, this work proves the utility of a signature ensemble integrating epigenetic and genetic information for accurate cancer detection.

Protective effect of hydrangenol on lipopolysaccharide-induced endotoxemia by suppressing intestinal inflammation.

Hydrangenol, a dihydroisocoumarin, isolated from the leaves of Hydrangea serrata, possesses anti-inflammatory, anti-obesity, and anti-photoaging activities. In this study, we investigated the protective effects of hydrangenol (HG) against lipopolysaccharide (LPS)-induced endotoxemia and elucidated the underlying molecular mechanisms of action in C57BL/6 mice. Oral administration of HG (20 or 40 mg/kg) significantly restored the survival rate and population of macrophages, T helper cells (CD3+/CD4+), and Th17 cells (CD3+/CD4+/CCR6+) in the spleens of mice with LPS-induced endotoxemia. HG suppressed the expression of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1ß, and Interferon (IFN)-γ and the mRNA and protein expressions of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) in the intestine and lung of LPS-treated mice. Molecular data showed that HG ameliorated the activation of nuclear factor kappa B (NF-κB) p65, signal transducers and activators of transcription 3 (STAT3), and c-Fos and c-Jun (AP-1 subunits) via the myeloid differentiation primary response 88 (MyD88) dependent toll-like receptor 4 (TLR4) signaling pathway in the LPS-treated mouse intestines. HG treatment caused the recovery of LPS-induced impaired tight junction (occludin and claudin-2) protein and mRNA expressions. Furthermore, HG improved LPS-induced gut dysbiosis in mice. Taken together, our results suggest that HG protects against LPS-induced endotoxemia by restoring immune cells and the capacity of the intestinal barrier, reducing intestinal inflammation, and improving the composition of the gut microbiota.

Hydrangenol, an active constituent of Hydrangea serrata (Thunb.) Ser., ameliorates colitis through suppression of macrophage-mediated inflammation in dextran sulfate sodium-treated mice.

Ulcerative colitis (UC) is a chronic disease of the colon characterized by mucosal damage and relapsing gastrointestinal inflammation. Hydrangea serrata (Thunb.) Ser. and its bioactive compound, hydrangenol, are reported to have anti-inflammatory effects, but few studies have investigated the effects of hydrangenol in colitis. In the present study, we evaluated for the first time the anti-colitic effects and molecular mechanisms of hydrangenol in a dextran sodium sulfate (DSS)-induced mouse colitis model. To investigate the anti-colitic effects of hydrangenol, DSS-induced colitis mice, HT-29 colonic epithelial cells treated with supernatant from LPS-inflamed THP-1 macrophages, and LPS-induced RAW264.7 macrophages were used. In addition, to clarify the molecular mechanisms of this study, quantitative real time-PCR, western blot analysis, TUNEL assay, and annexin V-FITC/PI double staining analysis were conducted. Oral administration of hydrangenol (15 or 30 mg kg-1) significantly alleviated DSS-induced colitis by preventing DAI scores, shortening colon length, and colonic structural damage. F4/80+ macrophage numbers in mesenteric lymph nodes and macrophage infiltration in colonic tissues were significantly suppressed following hydrangenol treatment in DSS-exposed mice. Hydrangenol significantly attenuated DSS-induced destruction of the colonic epithelial cell layer through regulation of pro-caspase-3, occludin, and claudin-1 protein expression. Moreover, hydrangenol ameliorated abnormal tight junction protein expression and apoptosis in HT-29 colonic epithelial cells treated with supernatant from LPS-inflamed THP-1 macrophages. Hydrangenol suppressed the expression of pro-inflammatory mediators, such as iNOS, COX-2, TNF-α, IL-6, and IL-1ß through NF-κB, AP-1, and STAT1/3 inactivation in DSS-induced colon tissue and LPS-induced RAW264.7 macrophages. Taken together, our findings suggest that hydrangenol recovers the tight junction proteins and down-regulates the expression of the pro-inflammatory mediators by interfering with the macrophage infiltration in DSS-induced colitis. Our study provides compelling evidence that hydrangenol may be a candidate for inflammatory bowel disease therapy.

Personalised circulating tumour DNA assay with large-scale mutation coverage for sensitive minimal residual disease detection in colorectal cancer.

BACKGROUND: Postoperative minimal residual disease (MRD) detection using circulating-tumour DNA (ctDNA) requires a highly sensitive analysis platform. We have developed a tumour-informed, hybrid-capture ctDNA sequencing MRD assay. METHODS: Personalised target-capture panels for ctDNA detection were designed using individual variants identified in tumour whole-exome sequencing of each patient. MRD status was determined using ultra-high-depth sequencing data of plasma cell-free DNA. The MRD positivity and its association with clinical outcome were analysed in Stage II or III colorectal cancer (CRC). RESULTS: In 98 CRC patients, personalised panels for ctDNA sequencing were built from tumour data, including a median of 185 variants per patient. In silico simulation showed that increasing the number of target variants increases MRD detection sensitivity in low fractions (<0.01%). At postoperative 3-week, 21.4% of patients were positive for MRD by ctDNA. Postoperative positive MRD was strongly associated with poor disease-free survival (DFS) (adjusted hazard ratio 8.40, 95% confidence interval 3.49-20.2). Patients with a negative conversion of MRD after adjuvant therapy showed significantly better DFS (P < 0.001). CONCLUSION: Tumour-informed, hybrid-capture-based ctDNA assay monitoring a large number of patient-specific mutations is a sensitive strategy for MRD detection to predict recurrence in CRC.

The effect of intranasal dexmedetomidine administration on emergence agitation or delirium in pediatric patients after general anesthesia: A meta-analysis of randomized controlled trials.

BACKGROUND: Emergence agitation or delirium can occur in pediatric patients after anesthesia. Dexmedetomidine is known to reduce the impairment of postoperative cognitive function. AIMS: This study aimed to identify the role of intranasal administration of dexmedetomidine in lowering the development of emergence agitation or emergence delirium in pediatric patients after general anesthesia. METHODS: Electronic databases, including PubMed, EMBASE, CENTRAL, Scopus, and Web of Science, were searched to identify studies. The primary outcome was the proportion of patients who underwent emergence agitation or emergence delirium after the surgery. Secondary outcomes included emergence time and incidence of postoperative nausea and/or vomiting. We estimated the odds ratio and mean difference with 95% confidence intervals for the determination of effect size using a random-effects model. RESULTS: In total, 2103 pediatric patients from 20 randomized controlled trials were included in the final analysis. The incidence of emergence agitation or emergence delirium was 13.6% in the dexmedetomidine group and 33.2% in the control group. The pooled effect size revealed that intranasal dexmedetomidine administration significantly reduced the incidence of postoperative emergence agitation or emergence delirium in pediatric patients undergoing surgery under general anesthesia (odds ratio 0.25, 95% confidence interval 0.18-0.34; p = .0000; I2 = 37.74%). Additionally, significant difference was observed in emergence time between the two groups (mean difference 2.42, 95% confidence interval 0.37-4.46; p = .021; I2 = 98.40%). Children in the dexmedetomidine group had a significantly lower incidence of postoperative nausea and/or vomiting than those in the control group (odds ratio 0.39, 95% confidence interval 0.24-0.64; p = .0002; I2 = 0.00%). CONCLUSIONS: Intranasal dexmedetomidine reduced the incidence of emergence agitation or emergence delirium in pediatric patients after general anesthesia.

Anticancer Effect of Cold Atmospheric Plasma in Syngeneic Mouse Models of Melanoma and Colon Cancer.

Cold atmospheric plasma (CAP) may have applications in treating various types of malignant tumors. This study assessed the anticancer effects of CAP using melanoma and colon cancer cell lines. CAP treatment significantly reduced the in vitro viability of melanoma and colon cancer cell lines and had a negligible effect on the viability of normal human melanocytes. Additionally, CAP and epidermal growth factor receptor (EGFR) inhibitor had an additive anticancer effect in a CAP-resistant melanoma cell line. Reactive oxygen and nitrogen species known to be generated by CAP enhanced the anticancer effects of CAP and EGFR inhibitors. The in vivo anticancer activities of CAP were evaluated by testing its effects against syngeneic tumors induced in mice by melanoma and colon cancer cells. CAP treatment reduced tumor volume and weight in both cancer models, with the extent of tumor reduction dependent on the duration and number of CAP treatments. Histologic examination also revealed the tumoricidal effects of CAP in both tumor models. In conclusion, CAP inhibits the growth of mouse melanoma and colon cancer cell lines in vitro and shows tumoricidal effects against mouse models of melanoma and colon cancer in vivo.

The Effect of Peripheral Nerve Block on Postoperative Delirium in Older Adults Undergoing Hip Surgery: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

This meta-analysis aimed to determine whether peripheral nerve blocks (PNB) reduce postoperative delirium (POD) in elderly patients undergoing hip surgery. This study was registered in the International Prospective Register of Systematic Reviews (PROSPERO; CRD42022328320). The PubMed, EMBASE, Web of Science, and Cochrane Library databases were searched for randomized controlled trials (RCTs) on 26 April 2022. A total of 19 RCTs with 1977 participants were included. Perioperative PNB lowered the POD incidence on the third postoperative day (OR: 0.59, 95% CI [0.40 to 0.87], p = 0.007, I2 = 35%), in patients without underlying cognitive impairment (OR: 0.47, 95% CI [0.30 to 0.74], p = 0.001, I2 = 30%), and when a fascia iliaca compartment block (OR: 0.58, 95% CI [0.37 to 0.91], p = 0.02, I2 = 0%) or a femoral nerve block (OR: 0.33, 95% CI [0.11 to 0.99], p = 0.05, I2 = 66%) were performed. The pain score was also reduced (SMD: -0.83, 95% CI [-1.36 to -0.30], p = 0.002, I2 = 95%) after PNB. Perioperative PNB can lower the POD incidence and pain scores up to the third postoperative day. However, considering the wide variety of PNBs performed, more trials are needed to identify the effects of each PNB on POD.

Two Case Reports of Newly Diagnosed Crohn's Disease after COVID-19 in Pediatric Patients.

Since the coronavirus disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome-coronavirus-2 virus (SARS-CoV-2), various complications have been reported. Although most COVID-19 cases exhibited flu-like symptoms, COVID-19 may dysregulate the immune response and promote overwhelming levels of inflammation in some patients. Inflammatory bowel disease (IBD) is caused by dysregulated or inappropriate immune responses to environmental factors in a genetically susceptible host, and a SARS-CoV-2 infection may act as a possible cause of IBD. This paper describes two pediatric patients who developed Crohn's disease following a SARS-CoV-2 infection. They were previously healthy before the SARS-CoV-2 infection. On the other hand, they started to develop fever and gastrointestinal symptoms several weeks after recovery from the infection. They were diagnosed with Crohn's disease by imaging and endoscopic studies, and their symptoms improved after treatment with steroids and azathioprine. This paper suggests that a SARS-CoV-2 infection may trigger IBD in predisposed patients.

Circulating Tumor DNA Dynamics and Treatment Outcome of Regorafenib in Metastatic Colorectal Cancer.

PURPOSE: Circulating tumor DNA (ctDNA) is emerging as a valuable non-invasive tool to identify tumor heterogeneity and tumor burden. This study investigated ctDNA dynamics in metastatic colorectal cancer patients treated with regorafenib. Materials and Methods: In this prospective biomarker study, plasma cell-free DNA (cfDNA) samples obtained at baseline, at the first response evaluation after 2 cycles of treatment, and at the time of progressive disease were sequenced using a targeted next-generation sequencing platform which included 106 genes. RESULTS: A total of 285 blood samples from 110 patients were analyzed. Higher baseline cfDNA concentration was associated with worse progression-free survival (PFS) and overall survival (OS). After 2 cycles of treatment, variant allele frequency (VAF) in the majority of ctDNA mutations decreased with a mean relative change of -31.6%. Decreases in the VAF of TP53, APC, TCF7L2, and ROS1 after 2 cycles of regorafenib were associated with longer PFS. We used the sum of VAF at each time point as a surrogate for the overall ctDNA burden. A reduction in sum (VAF) of ≥ 50% after 2 cycles was associated with longer PFS (6.1 vs. 2.7 months, p=0.002), OS (11.3 vs. 5.9 months, p=0.001), and higher disease control rate (86.3% vs. 51.1%, p < 0.001). VAF of the majority of the ctDNA mutations increased at the time of disease progression, and VAF of BRAF increased markedly. CONCLUSION: Reduction in ctDNA burden as estimated by sum (VAF) could be used to predict treatment outcome of regorafenib.

In Vitro Expansion of Vδ1+ T Cells from Cord Blood by Using Artificial Antigen-Presenting Cells and Anti-CD3 Antibody.

γδ T cells have the potential for adoptive immunotherapy since they respond to bacteria, viruses, and tumors. However, these cells represent a small fraction of the peripheral T-cell pool and require activation and proliferation for clinical benefits. In cord blood, there are some γδ T cells, which exhibit a naïve phenotype, and mostly include Vδ1+ T cells. In this study, we investigated the effect of CD3 signaling on cord blood γδ T-cell proliferation using K562-based artificial antigen presenting cells expressing costimulatory molecules. There were significantly more Vδ1+ T cells in the group stimulated with anti-CD3 antibody than in the group without. In cultured Vδ1+ T cells, DNAM-1 and NKG2D were highly expressed, but NKp30 and NKp44 showed low expression. Among various target cells, Vδ1+ T cells showed the highest cytotoxicity against U937 cells, but Daudi and Raji cells were not susceptible to Vδ1+ T cells. The major cytokines secreted by Vδ1+ T cells responding to U937 cells were Granzyme B, IFN-γ, and sFasL. Cytotoxicity by Vδ1+ T cells correlated with the expression level of PVR and Nectin of DNAM-1 ligands on the surface of target cells. Compared to Vδ2+ T cells in peripheral blood, cord blood Vδ1+ T cells showed varying cytotoxicity patterns depending on the target cells. Here, we determined the ideal conditions for culturing cord blood Vδ1+ T cells by observing that Vδ1+ T cells were more sensitive to CD3 signals than other subtypes of γδ T cells in cord blood. Cultured cord blood Vδ1+ T cells recognized target cells through activating receptors and secreted numerous cytotoxic cytokines. These results are useful for the development of tumor immunotherapy based on γδ T cells.

Neuronal mitochondrial morphology is significantly affected by both fixative and oxygen level during perfusion.

Neurons in the brain have a uniquely polarized structure consisting of multiple dendrites and a single axon generated from a cell body. Interestingly, intracellular mitochondria also show strikingly polarized morphologies along the dendrites and axons: in cortical pyramidal neurons (PNs), dendritic mitochondria have a long and tubular shape, while axonal mitochondria are small and circular. Mitochondria play important roles in each compartment of the neuron by generating adenosine triphosphate (ATP) and buffering calcium, thereby affecting synaptic transmission and neuronal development. In addition, mitochondrial shape, and thereby function, is dynamically altered by environmental stressors such as oxidative stress or in various neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. Although the importance of altered mitochondrial shape has been claimed by multiple studies, methods for studying this stress-sensitive organelle have not been standardized. Here we address pertinent steps that influence mitochondrial morphology during experimental processes. We demonstrate that fixative solutions containing only paraformaldehyde (PFA), or that introduce hypoxic conditions during the procedure, induce dramatic fragmentation of mitochondria both in vitro and in vivo. This disruption was not observed following the use of glutaraldehyde (GA) addition or oxygen supplementation, respectively. Finally, using pre-formed fibril α-synuclein treated neurons, we show fixative choice can alter experimental outcomes. Specifically, α-synuclein-induced mitochondrial remodeling could not be observed with PFA only fixation as fixation itself caused mitochondrial fragmentation. Our study provides optimized methods for examining mitochondrial morphology in neurons and demonstrates that fixation conditions are critical when investigating the underlying cellular mechanisms involving mitochondria in physiological and neurodegenerative disease models.

Dynamic changes in longitudinal circulating tumour DNA profile during metastatic colorectal cancer treatment.

BACKGROUND: Circulating tumour DNA (ctDNA) has been spotlighted as an attractive biomarker because of its easy accessibility and real-time representation of tumour genetic profile. However, the clinical utility of longitudinal ctDNA monitoring has not been clearly defined. METHODS: Serial blood samples were obtained from metastatic colorectal cancer patients undergoing first-line chemotherapy. ctDNA was sequenced using a targeted next-generation sequencing platform which included 106 genes. Changes in ctDNA profile and treatment outcome were comprehensively analysed. RESULTS: A total of 272 samples from 62 patients were analysed. In all, 90.3% of patients had detectable ctDNA mutation before treatment. ctDNA clearance after chemotherapy was associated with longer progression-free survival which was independent of radiological response (adjusted hazard ratio 0.22, 95% confidence interval 0.10-0.46). Longitudinal monitoring was able to detect ctDNA progression which preceded radiological progressive disease (PD) in 58.1% (median 3.3 months). Diverse resistant mutations (34.9%) and gene amplification (7.0%) at the time of PD were discovered. For 16.3% of the PD patients, the newly identified mutations could be potential candidates of targeted therapy or clinical trial. CONCLUSION: ctDNA profile provided a more accurate landscape of tumour and dynamic changes compared to radiological evaluation. Longitudinal ctDNA monitoring may improve personalised treatment decision-making.

USP37 Deubiquitinates CDC73 in HPT-JT Syndrome.

The CDC73/HRPT2 gene, a defect which causes hyperparathyroidism-jaw tumor (HPT-JT) syndrome, encodes CDC73/parafibromin. We aimed to investigate whether CDC73 would be a target for ubiquitin-proteasome degradation. We cloned full-length cDNAs encoding a family of 58 ubiquitin-specific deubiquitinating enzymes (DUBs), also known as ubiquitin-specific proteases (USPs). Use of the yeast two-hybrid system then enabled us to identify USP37 as interacting with CDC73. The biochemical interaction between the USP37 and CDC73 and their reciprocal binding domains were studied. Co-localization of CDC73 and USP37 was observed in cells. CDC73 was found to be polyubiquitinated, and polyubiquitination of CDC73 was prominent in mutants. CDC73 was deubiquitinated via K48-specific ubiquitin chains by USP37, but not by the catalytically inactive USP37C350S mutant. Observation of the binding between deletion mutants of CDC73 and USP37 revealed that the ß-catenin binding site of CDC73 and the ubiquitin-interacting motifs 2 and 3 (UIM2 and 3) of USP37 were responsible for the interaction between the two proteins. Moreover, these two enzymes co-existed within the nucleus of COS7 cells. We conclude that USP37 is a DUB for CDC73 and that the two proteins interact through specific domains, suggesting that USP37 is responsible for the stability of CDC73 in HPT-JT syndrome.

Mosloflavone-Resveratrol Hybrid TMS-HDMF-5z Exhibits Potent In Vitro and In Vivo Anti-Inflammatory Effects Through NF-κB, AP-1, and JAK/STAT Inactivation.

TMS-HDMF-5z is a hybrid of the natural products mosloflavone and resveratrol. It was discovered to show potent inhibitory effects against lipopolysaccharide (LPS)-induced production of inflammatory mediators in RAW 264.7 macrophages. However, its mechanism of action is unknown. Hence this study aimed to demonstrate and explore in vitro and in vivo anti-inflammatory effects of TMS-HDMF-5z and its mechanism of action employing RAW 264.7 macrophages and carrageenan-induced hind paw edema. This work revealed that TMS-HDMF-5z suppressed the LPS-induced inducible nitric-oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at the protein, mRNA, and promoter binding levels and tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6, and interferon-ß (IFN-ß) at the mRNA expression in RAW 264.7 macrophages. The results showed that TMS-HDMF-5z reduced the transcription and DNA binding activities of nuclear factor-κB (NF-κB) through inhibiting nuclear translocation of p65 and phosphorylation of κB inhibitor α (IκBα), IκB kinase (IKK), and TGF-ß activated kinase 1 (TAK1). Additionally, TMS-HDMF-5z attenuated the LPS-induced transcriptional and DNA binding activities of activator protein-1 (AP-1) by suppressing nuclear translocation of phosphorylated c-Fos, c-Jun, and activating transcription factor 2 (ATF2). TMS-HDMF-5z also reduced the LPS-induced phosphorylation of Janus kinase 1/2 (JAK1/2), signal transducers and activators of transcription 1/3 (STAT1/3), p38 mitogen-activated protein kinase (MAPK), and MAPK-activated protein kinase 2 (MK2). In rats, TMS-HDMF-5z alleviated carrageenan-induced hind paw edema through the suppressing iNOS and COX-2 via NF-κB, AP-1, and STAT1/3 inactivation. Collectively, the TMS-HDMF-5z-mediated inhibition of NF-κB, AP-1, and STAT1/3 offer an opportunity for the development of a potential treatment for inflammatory diseases.