Prognostic utility of circulating tumor DNA methylation analysis in stage IV colorectal cancer.

BACKGROUND AND OBJECTIVES: Our aim in this study was to investigate the usefulness of circulating tumor (ct) DNA methylation analysis for predicting long-term outcomes after resection in Stage IV colorectal cancer (CRC). METHODS: Methylation analyses were performed on 95 plasma samples from patients with CRC who underwent surgery. The methylation status (relative methylation value: RMV) of CpG within the promoter region of three genes (CHFR, SOX11, and CDO1) was assessed to quantitative methylation-specific PCR (qMSP) analysis. RESULTS: In the patients who had undergone resection of the primary tumor and metastatic organs with curative intent, the CHFR-RMV high group had significantly worse recurrence-free survival (RFS) compared with the CHFR-RMV low group (p = 0.001). Multivariate analysis revealed that CHFR-RMV was a significant independent prognostic factor (hazard ratio = 2.63 (1.29-5.36); p = 0.008). In the patients who had undergone resection of the primary tumor with metastatic organs with curative intent after neoadjuvant systemic chemotherapy, the SOX11-RMV high group had significantly worse RFS compared with the SOX11-RMV low group (p = 0.004). CONCLUSIONS: The current study showed the usefulness of ctDNA methylation analysis for predicting the possibility of curative resection and long-term outcomes after resection in Stage IV CRC. A future prospective study is needed to obtain more conclusive results.

Dopa-responsive Rest Tremor Preceding Tachyphemia in Progressive Supranuclear Palsy: A Case Report.

Progressive supranuclear palsy (PSP) is characterized by progressive postural instability, falls, and supranuclear vertical gaze abnormalities. In this report, we present the case of a 71-year-old woman with dopa-responsive rest tremor followed by tachyphemia and postural instability. She initially presented with dopa-responsive slowness and tremor in the right hand. Two years later, she developed speech difficulties (tachyphemia) and a propensity for falls. Based on the diagnostic criteria for PSP, the patient was diagnosed with probable PSP-RS. The clinical manifestations observed in our patient are unique and are considered important for illustrating a broad spectrum of PSP syndrome.

Use of 3D-CT-derived psoas major muscle volume in defining sarcopenia in colorectal cancer.

BACKGROUND: Sarcopenia is characterized by reduced skeletal muscle volume and is a condition that is prevalent among elderly patients and associated with poor prognosis as a comorbidity in malignancies. Given the aging population over 80 years old in Japan, an understanding of malignancies, including colorectal cancer (CRC), complicated by sarcopenia is increasingly important. Therefore, the focus of this study is on a novel and practical diagnostic approach of assessment of psoas major muscle volume (PV) using 3-dimensional computed tomography (3D-CT) in diagnosis of sarcopenia in patients with CRC. METHODS: The subjects were 150 patients aged ≥ 80 years with CRC who underwent primary tumor resection at Juntendo University Hospital between 2004 and 2017. 3D-CT measurement of PV and conventional CT measurement of the psoas major muscle cross-sectional area (PA) were used to identify sarcopenia (group S) and non-sarcopenia (group nS) cases. Clinicopathological characteristics, operative results, postoperative complications, and prognosis were compared between these groups. RESULTS: The S:nS ratios were 15:135 for the PV method and 52:98 for the PA method. There was a strong positive correlation (r = 0.66, p < 0.01) between PVI (psoas major muscle volume index) and PAI (psoas major muscle cross-sectional area index), which were calculated by dividing PV or PA by the square of height. Surgical results and postoperative complications did not differ significantly in the S and nS groups defined using each method. Overall survival was worse in group S compared to group nS identified by PV (p < 0.01), but not significantly different in groups S and nS identified by PA (p = 0.77). A Cox proportional hazards model for OS identified group S by PV as an independent predictor of a poor prognosis (p < 0.05), whereas group S by PA was not a predictor of prognosis (p = 0.60). CONCLUSIONS: The PV method for identifying sarcopenia in elderly patients with CRC is more practical and sensitive for prediction of a poor prognosis compared to the conventional method.

Dysregulation of SNX1-retromer axis in pharmacogenetic models of Parkinson's disease.

Since the identification of vacuolar protein sorting (VPS) 35, as a causative molecule for familial Parkinson's disease (PD), retromer-mediated endosomal machinery has been a rising factor in the pathogenesis of the disease. The retromer complex cooperates with sorting nexin (SNX) dimer and DNAJC13, another causal molecule in PD, to transport cargoes from endosomes to the trans-Golgi network, and is also involved in mitochondrial dynamics and autophagy. Retromer dysfunction may induce neuronal death leading to PD via several biological cascades, including misfolded, insoluble α-synuclein (aS) accumulation and mitochondrial dysfunction; however, the detailed mechanisms remain poorly understood. In this study, we showed that the stagnation of retromer-mediated retrograde transport consistently occurs in different PD-mimetic conditions, i.e., overexpression of PD-linked mutant DNAJC13, excess aS induction, or toxin-induced mitochondrial dysfunction. Mechanistically, DNAJC13 was found to be involved in clathrin-dependent retromer transport as a functional modulator of SNX1 together with heat shock cognate 70 kDa protein (Hsc70), which was controlled by the binding and dissociation of DNAJC13 and SNX1 in an Hsc70 activity-dependent manner. In addition, excess amount of aS decreased the interaction between SNX1 and VPS35, the core component of retromer. Furthermore, R33, a pharmacological retromer chaperone, reduced insoluble aS and mitigated rotenone-induced neuronal apoptosis. These findings suggest that retrograde transport regulated by SNX1-retromer may be profoundly involved in the pathogenesis of PD and is a potential target for disease-modifying therapy for the disease.

Reduced histone H3K4 trimethylation in oral mucosa of patients with DYT-KMT2B.

BACKGROUND: DYT-KMT2B, also known as DYT28, is a childhood-onset hereditary dystonia caused by KMT2B mutation. The pathogenesis of DYT-KMT2B involves haploinsufficiency of KMT2B, an enzyme that catalyzes specific histone methylation (H3K4me3). Dysmorphic features in patients with DYT-KMT2B suggest that KMT2B dysfunction may extend beyond the neuronal system. Therefore, valuable diagnostic insights may be obtained from readily available tissue samples. OBJECTIVES: To explore the altered H3K4me3 levels in non-neural tissue of DYT-KMT2B patients. METHODS: A database analysis was performed to determine in which parts of the body and in which cells KMT2B is highly expressed. Twelve clinically and genetically diagnosed patients with DYT-KMT2B and 12 control subjects participated in this study. Oral mucosa-derived purified histone proteins were analyzed using Western blotting with anti-H3K4me3 and anti-H4 antibodies. RESULTS: Higher expression of KMT2B was observed in oral keratinocytes and gingival fibroblasts, constituting the oral mucosa. In oral mucosa analyses, DYT-KMT2B cases exhibited markedly reduced H3K4me3 levels compared with the controls. Using a cutoff window of 0.90-0.98, the H3K4me3/H4 expression ratio was able to distinguish patient groups. CONCLUSIONS: Oral mucosa H3K4me3 analysis is currently not sufficient as a diagnostic tool for DYT-KMT2B, but has the advantage for screening test since it is a non-invasive means.

Recurrent Ipsilateral C5 Nerve Palsy Associated With Hereditary Neuropathy With Liability to Pressure Palsy.

Hereditary neuropathy with liability to pressure palsy (HNPP) is an autosomal dominant disorder caused by heteroplasmic deletion of the peripheral myelin protein 22 (PMP22) gene. HNPP typically presents with clinical features such as peroneal nerve palsy or cubital tunnel syndrome, which are caused by mechanical compression. Diagnosing cases where neuropathy is absent at the pressure site can be challenging. This is a case study of an 18-year-old man who underwent surgery on the left side of his neck over 10 years ago to remove lymphadenopathy. Following the surgery, he experienced recurrent weakness but only sought medical attention when muscle weakness persisted for longer than a week postoperatively. Upon admission, the patient exhibited neurological symptoms consistent with C5 neuropathy, mainly affecting the deltoid muscles. No serological abnormalities were found to be associated with neuropathy. Neither magnetic resonance imaging nor computed tomography scans detected any lesions around the C5 nerve root. The posture during sleep was believed to cause excessive extension of the C5 nerve root, leading to the assumption that there was some vulnerability in the nerve. A transient sensory loss in the area innervated by the ulnar nerve prompted us to examine the fluorescence in situ hybridization study on the blood sample, which revealed a deletion of the PMP22 gene. The patient was diagnosed with HNPP and was advised to avoid risky postures. Following the implementation of these lifestyle changes, he did not experience any further weakness in his shoulders.

Alpha-synuclein promotes PRMT5-mediated H4R3me2s histone methylation by interacting with the BAF complex.

α-Synuclein (αS) is a key molecule in the pathomechanism of Parkinson's disease. Most studies on αS to date have focused on its function in the neuronal cytosol, but its action in the nucleus has also been postulated. Indeed, several lines of evidence indicate that overexpressed αS leads to epigenomic alterations. To clarify the functional role of αS in the nucleus and its pathological significance, HEK293 cells constitutively expressing αS were used to screen for nuclear proteins that interact with αS by nanoscale liquid chromatography/tandem mass spectrometry. Interactome analysis of the 229 identified nuclear proteins revealed that αS interacts with the BRG1-associated factor (BAF) complex, a family of multi-subunit chromatin remodelers important for neurodevelopment, and protein arginine methyltransferase 5 (PRMT5). Subsequent transcriptomic analysis also suggested a functional link between αS and the BAF complex. Based on these results, we analyzed the effect of αS overexpression on the BAF complex in neuronally differentiated SH-SY5Y cells and found that induction of αS disturbed the BAF maturation process, leading to a global increase in symmetric demethylation of histone H4 on arginine 3 (H4R3me2s) via enhanced BAF-PRMT5 interaction. Chromatin immunoprecipitation sequencing confirmed accumulated H4R3me2s methylation near the transcription start site of the neuronal cell adhesion molecule (NRCAM) gene, which has roles during neuronal differentiation. Transcriptional analyses confirmed the negative regulation of NRCAM by αS and PRMT5, which was reconfirmed by multiple datasets in the Gene Expression Omnibus (GEO) database. Taken together, these findings suggest that the enhanced binding of αS to the BAF complex and PRMT5 may cooperatively affect the neuronal differentiation process.

Detection of Modified Histones from Oral Mucosa of a Patient with DYT-KMT2B Dystonia.

Introduction: DYT-KMT2B is a rare childhood-onset, hereditary movement disorder typically characterized by lower-limb dystonia and subsequently spreads into the craniocervical and laryngeal muscles. Recently, KMT2B-encoding lysine (K)-specific histone methyltransferase 2B was identified as the causative gene for DYT-KMT2B, also known as DYT28. In addition to the fact that many physicians do not have sufficient experience or knowledge of hereditary dystonia, the clinical features of DYT-KMT2B overlap with those of other hereditary dystonia, and limited clinical biomarkers make the diagnosis difficult. Methods: Histone proteins were purified from the oral mucosa of patients with de novo KMT2B mutation causing premature stop codon, and then trimethylated fourth lysine residue of histone H3 (H3K4me3) which was catalyzed by KMT2B was analyzed by immunoblotting with specific antibody. We further analyzed the significance of H3K4me3 in patients with DYT-KMT2B using publicly available datasets. Results: H3K4me3 histone mark was markedly lower in the patient than in the control group. Additionally, a reanalysis of publicly available datasets concerning DNA methylation also demonstrated that KMT2B remained inactive in DYT-KMT2B. Discussion: Although only one case was studied due to the rarity of the disease, the reduction of H3K4me3 in the patient's biological sample supports the dysfunction of KMT2B in DYT-KMT2B. Together with informatics approaches, our results suggest that KMT2B haploinsufficiency contributes to the DYT-KMT2B pathogenic process.

ME3BP-7 is a targeted cytotoxic agent that rapidly kills pancreatic cancer cells expressing high levels of monocarboxylate transporter MCT1.

Nearly 30% of Pancreatic ductal adenocarcinoma (PDAC)s exhibit a marked overexpression of Monocarboxylate Transporter 1 (MCT1) offering a unique opportunity for therapy. However, biochemical inhibitors of MCT1 have proven unsuccessful in clinical trials. In this study we present an alternative approach using 3-Bromopyruvate (3BP) to target MCT1 overexpressing PDACs. 3BP is a cytotoxic agent that is known to be transported into cells via MCT1, but its clinical usefulness has been hampered by difficulties in delivering the drug systemically. We describe here a novel microencapsulated formulation of 3BP (ME3BP-7), that is effective against a variety of PDAC cells in vitro and remains stable in serum. Furthermore, systemically administered ME3BP-7 significantly reduces pancreatic cancer growth and metastatic spread in multiple orthotopic models of pancreatic cancer with manageable toxicity. ME3BP-7 is, therefore, a prototype of a promising new drug, in which the targeting moiety and the cytotoxic moiety are both contained within the same single small molecule. One Sentence Summary: ME3BP-7 is a novel formulation of 3BP that resists serum degradation and rapidly kills pancreatic cancer cells expressing high levels of MCT1 with tolerable toxicity in mice.

Positive Circumferential Resection Margin in Rectal Cancer Is a Robust Predictor of Poor Long-term Prognosis With Clinicopathological Bias Between Groups Compensated by Propensity-score Matching Analysis.

BACKGROUND/AIM: Circumferential resection margin (CRM) is the most reliable predictor of local and distant recurrence in locally-advanced rectal cancer (LARC). The present study was conducted to compare the long-term outcomes between CRM (+) and (-) groups using propensity-score (PS) matching analysis to compensate for bias between groups. PATIENTS AND METHODS: Of 563 consecutive patients with Stage II/III rectal cancer who were treated surgically with curative-intent at Juntendo University Hospital between Jan 1989 and Mar 2018, 412 patients were enrolled retrospectively in the study. The patients were divided into a CRM (+) group (n=21; 5.1%) and a CRM (-) group (n=391; 94.9%). RESULTS: In the entire cohort, recurrence-free survival (RFS), local recurrence-free survival (LRFS), non-local recurrence-free survival (NLRFS), and cancer-specific survival (CSS) were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. Univariate analysis demonstrated patients in the CRM (+) group had significantly larger primary tumors (p=0.02), more frequently had open surgery (p=0.009), had an abdominoperineal resection (APR) procedure (p=0.01) and a T4 primary tumor (p<0.0001). After PS matching analysis, in the propensity-matched cohort, RFS, LRFS, NLRFS and CSS were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. CONCLUSION: PS matching analysis demonstrated that RFS, LRFS, NLRFS, and CSS were significantly worse among patients in the CRM (+) group compared with those in the CRM (-) group. The present results indicate that CRM (+) is a robust predictor of long-term outcome of LARC, independent of tumor size.

Sortilin acts as an endocytic receptor for α-synuclein fibril.

Cell-to-cell spreading of misfolded α-synuclein (αSYN) is supposed to play a key role in the pathological progression of Parkinson's disease (PD) and other synucleinopathies. Receptor-mediated endocytosis has been shown to contributes to the uptake of αSYN in both neuronal and glial cells. To determine the receptor involved in αSYN endocytosis on the cell surface, we performed unbiased, and comprehensive screening using a membrane protein library of the mouse whole brain combined with affinity chromatography and mass spectrometry. The candidate molecules hit in the initial screening were validated by co-immunoprecipitation using cultured cells; sortilin, a vacuolar protein sorting 10 protein family sorting receptor, exhibited the strongest binding to αSYN fibrils. Notably, the intracellular uptake of fibrillar αSYN was slightly but significantly altered, depending on the expression level of sortilin on the cell surface, and time-lapse image analyses revealed the concomitant internalization and endosomal sorting of αSYN fibrils and sortilin. Domain deletion in the extracellular portion of sortilin revealed that the ten conserved cysteines (10CC) segment of sortilin was involved in the binding and endocytosis of fibrillar αSYN; importantly, pretreatment with a 10CC domain-specific antibody significantly hindered αSYN fibril uptake. The presence of sortilin in the core structure of Lewy bodies and glial cytoplasmic inclusions in the brain of synucleinopathy patients was confirmed via immunohistochemistry, and the expression level of sortilin in mesencephalic dopaminergic neurons may be altered with disease progression. These results provide compelling evidence that sortilin acts as an endocytic receptor for pathogenic form of αSYN, and yields important insight for the development of disease-modifying targets for synucleinopathies.

HMGA1 induces FGF19 to drive pancreatic carcinogenesis and stroma formation.

High mobility group A1 (HMGA1) chromatin regulators are upregulated in diverse tumors where they portend adverse outcomes, although how they function in cancer remains unclear. Pancreatic ductal adenocarcinomas (PDACs) are highly lethal tumors characterized by dense desmoplastic stroma composed predominantly of cancer-associated fibroblasts and fibrotic tissue. Here, we uncover an epigenetic program whereby HMGA1 upregulates FGF19 during tumor progression and stroma formation. HMGA1 deficiency disrupts oncogenic properties in vitro while impairing tumor inception and progression in KPC mice and subcutaneous or orthotopic models of PDAC. RNA sequencing revealed HMGA1 transcriptional networks governing proliferation and tumor-stroma interactions, including the FGF19 gene. HMGA1 directly induces FGF19 expression and increases its protein secretion by recruiting active histone marks (H3K4me3, H3K27Ac). Surprisingly, disrupting FGF19 via gene silencing or the FGFR4 inhibitor BLU9931 recapitulates most phenotypes observed with HMGA1 deficiency, decreasing tumor growth and formation of a desmoplastic stroma in mouse models of PDAC. In human PDAC, overexpression of HMGA1 and FGF19 defines a subset of tumors with extremely poor outcomes. Our results reveal what we believe is a new paradigm whereby HMGA1 and FGF19 drive tumor progression and stroma formation, thus illuminating FGF19 as a rational therapeutic target for a molecularly defined PDAC subtype.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice.

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Local recurrence of submucosal invasive colorectal cancer after endoscopic submucosal dissection revealed by copy number variation.

We report a case in which analysis of copy number variation revealed local recurrence of submucosal invasive colorectal cancer after curative endoscopic submucosal dissection (ESD). An 86-year-old man with a history of abdominoperineal resection of the rectum for rectal cancer underwent resection with ESD for early-stage sigmoid cancer 5 cm away from the stoma opening. At the same time, ileocecal resection was performed for advanced cecal cancer. Twelve months after ESD, advanced cancer occurred in the area of the ESD lesion. It was unclear if the cancer was a local recurrence after ESD, implantation of cecal cancer, or a new lesion. Copy number variation analysis performed for the three lesions revealed that the new lesion originated from residual tumor cells from ESD and was unlikely to be cecal cancer.

Examination of colorectal cancer cases with metal allergy.

PURPOSE: To report our initiatives and treatment results for patients with colorectal cancer with metal allergy. METHODS: A total of 27 patients (2.6%) with a history of metal contact dermatitis were identified among 1027 patients who underwent curative resection of colorectal cancer from 2014 to 2020. The results of the patch test, perioperative results, and postoperative colonoscopy findings were also investigated. RESULTS: The patch test for metal allergens and staples was performed in 21 patients (77.8%), and 13 of them (61.9%) tested positive for at least one metal allergen. Ni (38.1%), Co (28.6%), and Pd (19.0%) showed higher positive rates than other metals, and 1 patient (4.8%) tested positive for staples. Stapled anastomosis/suturing was performed as planned in 15 of 27 patients. In 10 patients, the anastomosis method was changed from stapled to hand-sewn according to the no-patch test results (60%), positivity for multiple metals (20%), positivity for staples (10%), and surgeon's judgment (10%). No complications and abnormal colonoscopy findings were found to be associated with stapled anastomosis/suturing. CONCLUSION: The patch test is useful for selecting an optimal anastomosis method for patients with suspected metal allergy.

pfeRNAs-A Novel Class of Small Non-coding RNAs With Real Translational Potential.

Small non-coding RNAs (sncRNAs) are defined by being less than 200 nucleotides (nt) in length, and consequently, have been divided into many different subclasses including mature microRNA (miRNA), small interfering RNA (siRNA), piwi-interacting RNA (piRNA), protein functional effector sncRNA (pfeRNA), precursor miRNA (pre-miRNA), small nucleolar RNA (snoRNA), 5S ribosome RNA (5SrRNA), 5.8SrRNA, and small nuclear RNA (snRNA). Except for the class of pfeRNAs, the discovery, identification, biogenesis, characterization, and function of other sncRNAs have been well documented. Herein, we provide a review, written especially for clinicians, of the least understood class of functional sncRNAs, the pfeRNAs, focusing on their initial discovery, identification, unique features, function, as well as their exciting clinical translational potential.

Benefits and risks of diverting stoma creation during rectal cancer surgery.

Purpose: A consensus has been reached regarding diverting stoma (DS) construction in rectal cancer surgery to avoid reoperation related to anastomotic leakage. However, the incidence of stoma-related complications (SRCs) remains high. In this study, we examined the perioperative outcomes of DS construction in patients who underwent sphincter-preserving surgery for rectal cancer. Methods: We included 400 participants who underwent radical sphincter-preserving surgery for rectal cancer between 2005 and 2017. These participants were divided into the DS (+) and DS (-) groups, and the outcomes, including postoperative complications, were compared. Results: The incidence of ileus was higher in the DS (+) group than in the DS (-) group (P<0.01); however, no patients in the DS (+) group showed grade 3 anastomotic leakage. Furthermore, early SRCs were observed in 33 patients (21.6%) and bowel obstruction-related stoma outlet syndrome occurred in 19 patients (12.4%). There was no significant intergroup difference in the incidence of grade 3b postoperative complications. However, the most common reason for reoperation was different in the 2 groups: anastomotic leakage in 91.7% of patients with grade 3b postoperative complications in the DS (-) group, and SRCs in 85.7% of patients with grade 3b postoperative complications in the DS (+) group. Conclusion: Patients with DS showed higher incidence rates of overall postoperative complications, severe postoperative complications (grade 3), and bowel obstruction, including stoma outlet syndrome, than patients without DS. Therefore, it is important to construct an appropriate DS to avoid SRCs and to be more selective in assigning patients for DS construction.

Efficacy and safety of regorafenib dose-escalation therapy for Japanese patients with refractory metastatic colorectal cancer (RECC study).

BACKGROUND: Regorafenib significantly improves overall survival in previously treated metastatic colorectal cancer patients. However, various toxicities, such as hand-foot skin reaction (HFSR), fatigue, and liver dysfunction have limited the use of regorafenib. These toxicities appear soon after treatment initiation. The ReDOS study demonstrated the effectiveness of a weekly dose-escalation therapy of regorafenib starting with a lower daily dose; however, its usefulness in Asian subjects is unknown. We conducted a phase II study to evaluate the safety and survival benefit of regorafenib dose-escalation therapy for Japanese patients. METHODS: Patients with sufficient organ function, who had previously received more than two lines of chemotherapy were included. Regorafenib was started at 80 mg/day and escalated to 120 mg/day in Week 2 and 160 mg/day in Week 3, if no severe drug-related toxicities were observed. The primary endpoint was cancer progression-free survival (PFS). Tumor response and progression were assessed radiologically every 8 weeks. This study was registered in the University Hospital Medical Information Network (UMIN#UMIN000028933). RESULTS: 57 patients were enrolled and all started regorafenib at 80 mg/day. 32 patients (56.1%) were subsequently escalated to 120 mg/day and 19 (33.3%) to 160 mg/day. Only 8 patients (14.0%) discontinued treatment because of adverse events. Median PFS was 1.9 months. Median overall survival was 8.9 months, the response rate was 0%, and the disease control rate was 31.6%. The most frequent adverse event greater than grade 3 was hypertension (19.3%), followed by HFSR (14.0%). CONCLUSIONS: Regorafenib dose-escalation therapy is well tolerated with PFS-like regorafenib standard therapy.

A TRIzol-based method for high recovery of plasma sncRNAs approximately 30 to 60 nucleotides.

Protein functional effector sncRNAs (pfeRNAs) are approximately 30-60 nucleotides (nt), of which the extraction method from plasma has not yet been reported. Silver staining in a high-resolution polyacrylamide gel suggested that the majority of plasma sncRNAs extracted by some broadly used commercial kits were sncRNAs from 100 nt upwards. Additionally, TRIzol's protocol is for long RNA but not sncRNA recovery. Here, we report a TRIzol-based frozen precipitation method (TFP method), which shows rigor and reproducibility in high yield and quality for plasma sncRNAs approximately 30-60 nt. In contrast to the yields by the commercial kit, plasma sncRNAs extracted by the TFP method enriched more sncRNAs. We used four different pfeRNAs of 34 nt, 45 nt, 53 nt, and 58 nt to represent typical sizes of sncRNAs from 30 to 60 nt and compared their levels in the recovered sncRNAs by the TFP method and by the commercial kit. The TFP method showed lower cycle threshold (CT) values by 2.01-9.17 cycles in 38 plasma samples from 38 patients, including Caucasian, Asian, African American, Latin, Mexican, and those who were a mix of more than one race. In addition, pfeRNAs extracted by two organic-based extraction methods and four commercial kits were undetermined in 22 of 38 samples. Thus, the quick and unbiased TFP method enriches plasma sncRNA ranging from 30 to 60 nt.

Prenatal stress enhances atherosclerosis and telomere shortening in ApoE knockout mouse offspring.

Children born to women who experience stress during pregnancy have an increased risk of atherosclerosis in later life, but few animal models have explored mechanisms. To study this phenomenon, timed-bred ApoE knockout mice were determined pregnant with ultrasound and randomly assigned on gestation day 8.5 to either a control (no stress) or prenatal stress (PS) group using 2 h of restraint for five consecutive days. PS significantly increased plasma corticosterone levels in pregnant mice. The litters from PS mice showed increased neonatal mortality within the first week of life. Body weights (at euthanasia) of adult offspring at 25 wk from the PS group were significantly increased compared with weights of controls. Adult offspring from these pregnancies were serially imaged with ultrasound to measure plaque thickness and were compared with plaque macroscopic and microscopic pathology. PS groups had increased plaque thickness determined by ultrasound, gross, histological evaluation and increased aortic root and valve macrophage infiltration at 25 wk. Five-week-old mice from PS group had significant decrease in mean arterial pressure, yet blood pressure normalized by 10 wk. As prenatal stress induced increased atherosclerosis, and telomeres are susceptible to stress, aortas from 10-wk-old mice were compared for telomere lengths and were found to be significantly shorter in PS mice compared with control mice. These studies support future investigation of how stress impacts telomere shortening in animal models and human aortas. This model could be further used to investigate the role of prenatal stress, telomere biology, and atherosclerosis pathogenesis in adults.