A case report of carcinoma of the papilla of Vater associated with a hyperplasia-dysplasia-carcinoma sequence by pancreaticobiliary maljunction.

BACKGROUND: Pancreaticobiliary maljunction (PBM) is a known risk factor for biliary tract cancer. However, its association with carcinoma of the papilla of Vater (PVca) remains unknown. We report a case with PVca that was thought to be caused by the hyperplasia-dysplasia-carcinoma sequence, which is considered a mechanism underlying PBM-induced biliary tract cancer. CASE PRESENTATION: A 70-year-old woman presented with white stool and had a history of cholecystectomy for the diagnosis of a non-dilated biliary tract with PBM. Esophagogastroduodenoscopy revealed a tumor in the papilla of Vater, and PVca was histologically proven by biopsy. We finally diagnosed her with PVca concurrent with non-biliary dilated PBM (cT1aN0M0, cStage IA, according to the Union for International Cancer Control, 8th edition), and subsequently performed subtotal stomach-preserving pancreaticoduodenectomy. Pathological findings of the resected specimen revealed no adenomas and dysplastic and hyperplastic mucosae in the common channel slightly upstream of the main tumor, suggesting a PBM related carcinogenic pathway with hyperplasia-dysplasia-carcinoma sequence. Immunostaining revealed positivity for CEA. CK7 positivity, CK20 negativity, and MUC2 negativity indicated that this PVca was of the pancreatobiliary type. Genetic mutations were exclusively detected in tumors and not in normal tissues, and bile ducts from formalin-fixed paraffin-embedded samples included mutated-ERBB2 (Mutant allele frequency, 81.95%). Moreover, of the cell-free deoxyribonucleic acid (cfDNA) extracted from liquid biopsy mutated-ERBB2 was considered the circulating-tumor deoxyribonucleic acid (ctDNA) of this tumor. CONCLUSIONS: Herein, we report the first case of PVca with PBM potentially caused by a "hyperplasia-dysplasia-carcinoma sequence" detected using immunostaining and next-generation sequencing. Careful follow-up is required if pancreaticobiliary reflux persists, considering the possible development of PVca.

In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia.

Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy.

FRET probe for detecting two mutations in one EGFR mRNA.

Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

Ultrasound-dependent RNAi using TatU1A-rose bengal conjugate.

Tat-U1A-rose bengal conjugate (TatU1A-RB) was prepared as an ultrasound-sensitive RNA carrier molecule. This molecule consists of Tat cell-penetrating peptide, U1A RNA-binding protein, and rose bengal as a sonosensitizer. We demonstrated that TatU1A-RB delivered RNA via the endocytosis pathway, which was followed by ultrasound-dependent endosomal escape and cytosolic dispersion of the RNA. A short hairpin RNA (shRNA) delivered by TatU1A-RB mediated RNA interference (RNAi) ultrasound-dependently. Even by ultrasound irradiation through blood cells, RNAi could be induced with TatU1A-RB and the shRNA. This ultrasound-dependent cytosolic RNA delivery method will serve as the basis for a new approach to nucleic acid therapeutics.

Tumor-Informed Approach Improved ctDNA Detection Rate in Resected Pancreatic Cancer.

Pancreatic cancer is one of the cancers with very poor prognosis; there is an urgent need to identify novel biomarkers to improve its clinical outcomes. Circulating tumor DNA (ctDNA) from liquid biopsy has arisen as a promising biomarker for cancer detection and surveillance. However, it is known that the ctDNA detection rate in resected pancreatic cancer is low compared with other types of cancer. In this study, we collected paired tumor and plasma samples from 145 pancreatic cancer patients. Plasma samples were collected from 71 patients of treatment-naïve status and from 74 patients after neoadjuvant therapy (NAT). Genomic profiling of tumor DNA and plasma samples was conducted using targeted next-generation sequencing (NGS). Somatic mutations were detected in 85% (123/145) of tumors. ctDNA was detected in 39% (28/71) and 31% (23/74) of treatment-naïve and after-NAT groups, respectively, without referring to the information of tumor profiles. With a tumor-informed approach (TIA), ctDNA detection rate improved to 56% (40/71) and 36% (27/74) in treatment-naïve and after-NAT groups, respectively, with the detection rate significantly improved (p = 0.0165) among the treatment-naïve group compared to the after-NAT group. Cases who had detectable plasma ctDNA concordant to the corresponding tumor showed significantly shorter recurrence-free survival (RFS) (p = 0.0010). We demonstrated that TIA improves ctDNA detection rate in pancreatic cancer, and that ctDNA could be a potential prognostic biomarker for recurrence risk prediction.

Clinical implementation and current advancement of blood liquid biopsy in cancer.

Liquid biopsies have been receiving tremendous attentions as easy, rapid, and non-invasive tools for cancer diagnosis. Liquid biopsy can be performed repeatedly for disease monitoring and is expected to overcome the limitations of tissue biopsies. With the advancement of next generation sequencing technologies, it is now possible to detect minute amount of tumor-derived circulation tumor DNA (ctDNA) from blood samples. Importantly, ctDNA detection could be complementary to tissue biopsies or tumor biomarkers particularly in cases of which tumor biopsy is clinically difficult to obtain. Here, we introduce the up-to-date technologies used in cfDNA-based liquid biopsy and review the clinical utilities of ctDNA in cancer screening, detection of minimal residual diseases, selection of molecular-targeted drugs, as well as monitoring of treatment responsiveness. We also discuss the challenges and future perspectives of liquid biopsy implementation in clinical setting.

Fluorescence lifetime probes for detection of RNA degradation.

To investigate RNA degradation in live cells, detection methods that do not require RNA extraction from cells are necessary. In this study, we examined the utility of fluorescence lifetime measurements using a probe attached to the end of an RNA molecule for detecting RNA degradation. We optimized a short fluorescein-labeled RNA sequence whose fluorescence lifetime varied significantly before and after degradation. The selected HHG-fluorescein sequence (H = U, C, or A) is a promising RNA labeling unit (fluorescence lifetime probe) for live cell imaging of RNA degradation.

Minimization of apoptosis-inducing CPP-Bim peptide.

Pro-apoptotic peptides may be promising agents for cancer therapy owing to their ability to induce apoptosis in cancer cells. TatBim, a fusion peptide of Tat cell-penetrating peptide (CPP) and the BH3 domain derived from Bim apoptosis-inducing protein, is a pro-apoptotic peptide. In this study, based on the TatBim sequence, we attempted to minimize the CPP-Bim peptide while retaining apoptosis-inducing activity. The CPP and Bim parts were systematically shortened, and the pro-apoptotic activities of the shortened peptides were examined. We obtained TatBim-N1C2 and R8Bim-N1C2 as minimized peptides with efficient apoptotic activity. These peptides may have potential applications in future biomedical studies, such as cancer therapeutics.

Fluorophore-PNA-Quencher/Quencher-DNA probe for miRNA detection.

Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore-PNA-quencher (fPq) and a quencher-DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA-DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis.

Renal Dysfunction as a Predictor of Slow-Flow/No-Reflow Phenomenon and Impaired ST Segment Resolution After Percutaneous Coronary Intervention in ST-Elevation Myocardial Infarction With Initial Thrombolysis in Myocardial Infarction Grade 0.

BACKGROUND: The slow-flow/no-reflow phenomenon and impaired ST segment resolution (STR) following primary percutaneous coronary intervention (PCI) in ST-elevation myocardial infarction (STEMI) predict unfavorable prognosis and are characterized by obstruction of the coronary microvascular. Several predictors of slow-flow/no-reflow have been revealed, but few studies have investigated predictors of slow-flow/no-reflow and STR exclusively in acute myocardial infarction patients with initial Thrombolysis in Myocardial Infarction (TIMI) Grade 0.Methods and Results:In all, 279 STEMI patients with initial TIMI Grade 0 were enrolled in the study. Slow-flow/no-reflow was defined as TIMI Grade <3 by angiography after PCI, and impaired STR was defined as STR <50% on an electrocardiogram after PCI. Slow-flow/no-reflow was observed in 31 patients. In multivariate analysis, estimated glomerular filtration rate (eGFR; odds ratio [OR] 0.97; P=0.007), a history of cerebrovascular disease (OR 4.65, P=0.007), time to recanalization ≥4 h (OR 2.76, P=0.023), and systolic blood pressure ≤90 mmHg (OR 3.45, P=0.046) were independent predictors of slow-flow/no-reflow. Impaired STR was observed in 102 of 248 patients with TIMI Grade 3. In multivariate analysis, eGFR (OR 0.94, P<0.001) and occlusion of the left anterior descending artery (OR 4.48, P<0.001) were independent predictors of impaired STR; eGFR was the only independent predictor of both slow-flow/no-reflow and impaired STR. CONCLUSIONS: Renal dysfunction may be related to coronary microvascular dysfunction and obstruction.

Effects of empagliflozin in different phases of diabetes mellitus-related cardiomyopathy: a prospective observational study.

BACKGROUND: Diabetes mellitus-related cardiomyopathy (DMCMP), defined as left ventricular (LV) dysfunction caused by hyperglycemia in the absence of coronary artery disease, leads to heart failure (HF). Previous studies have shown that treatment with sodium-glucose co-transporter 2 inhibitor (SGLT2i) reduces the risk of exacerbation of HF. The beneficial effects of SGLT2i on HF depend not only on indirect actions such as osmotic diuresis but also on direct actions on the myocardium, leading to improvements in LV function. However, it remains unclear whether SGLT2i treatment is equally effective in any phase of DMCMP. The aim of this observational study was to compare the efficacy of SGLT2i treatment on LV dysfunction between early and advanced DMCMP. METHODS: Thirty-five symptomatic non-ischemic HF patients with LV ejection fraction > 40% and type 2 diabetes mellitus (T2DM) treated with empagliflozin (EMPA group) and 20 controls treated without SGLT2i were enrolled. According to the myocardial extracellular volume fraction (ECV), a reliable marker of cardiac fibrosis quantified by cardiac magnetic resonance, the EMPA group was further divided into early DMCMP (n = 16, ECV ≤ 30%) and advanced DMCMP (n = 19, ECV > 30%) groups and followed up prospectively. Echocardiography was performed at baseline and after 12 months. LV function assessed as LV global longitudinal strain (LVGLS) and the ratio of early diastolic mitral inflow velocity to early diastolic mitral annular velocity (E/e') were compared. RESULTS: ECV was strongly correlated with T2DM duration (r2 = 0.65, p < 0.001). At baseline, each group had a similar background. After 12 months, the EMPA group, especially the early DMCMP group, showed remarkable improvements in LVGLS (ΔLVGLS: 2.9 ± 3.0% (EMPA) vs. 0.6 ± 2.2% (controls), p = 0.005, and 4.6 ± 1.5% (early DMCMP) vs. 1.6 ± 3.3% (advanced DMCMP), p = 0.003) and E/e' (ΔE/e': - 1.5 ± 4.7 vs. - 0.3 ± 3.0, p = 0.253, and - 3.4 ± 5.5 vs. - 0.1 ± 3.5, p = 0.043). CONCLUSIONS: The positive effects of empagliflozin on LV dysfunction were more remarkable in early than in advanced DMCMP. Early intervention of SGLT2i for DMCMP may be preferable.

Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress.

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.

Rate Versus Rhythm Control in Tachycardia-Induced Cardiomyopathy Patients with Persistent Atrial Flutter.

Tachycardia-induced cardiomyopathy (TIC) is a potentially reversible cardiomyopathy caused by tachyarrhythmia. For atrial flutter (AFL) -induced TIC, a rhythm control strategy, such as catheter ablation, has been recommended. However, the efficacy of rate control has remained unclear due to the difficulty of achieving control using arrhythmic medications.We prospectively assessed 47 symptomatic heart failure (HF) patients with left ventricular ejection fraction (LVEF) < 50% and suspected persistent AFL-induced TIC. Patients were divided into the rhythm control strategy (n = 22; treatment with catheter ablation or electrical cardioversion) and rate control strategy (n = 25; treatment with bisoprolol) groups. The latter was further divided into the strict rate control strategy (average heart rate < 80 bpm) and lenient rate control strategy (average heart rate < 110 bpm) subgroups. The primary outcome was left ventricular (LV) function recovery, which was defined as an increase in LVEF ≥ 20% or to a value of ≥ 55% after 6 months.In the rhythm control strategy group, more patients achieved LV function recovery after 6 months (95.2% versus 60.9%, P = 0.010). The cumulative incidence of worsening HF events was significantly higher in the rate control strategy group than in the rhythm control strategy group (hazard ratio, 4.66; 95% confidence interval, 1.01-21.57). The subgroup study revealed the advantage of the strict rate control strategy for achieving LV function recovery (83.3% versus 36.4%, P = 0.036).The rate control strategy was significantly inferior to the rhythm control strategy for the LV function recovery in TIC patients with persistent AFL. Our findings suggest that the strict rate control strategy should be aimed if the rhythm control strategy cannot be performed.

Endosomal Escape of Peptide-Photosensitizer Conjugates Is Affected by Amino Acid Sequences near the Photosensitizer.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of peptides and proteins, but CPP fusion peptides and proteins are often transported by endocytosis and trapped in endosomes. Photochemical internalization (PCI) is a method for the endosomal escape of the trapped peptide or protein and release into the cytosol using light and photosensitizers. In PCI, endosomal membranes are thought to be destabilized by singlet oxygen (1O2) photogenerated from photosensitizers localized in endosomes. We previously developed CPP-cargo-photosensitizer (PS) conjugates able to photodependently enter the cytosol via the PCI mechanism. For example, TatU1A-PS (a covalent complex of Tat [CPP], U1A RNA-binding protein [cargo], and PS) can photodependently deliver RNAs into the cytosol, and TatBim-PS (a covalent complex of Tat, Bim [cargo], and PS) can photoinduce apoptosis in mammalian cells. However, for many newly created conjugates, the induction of PCI has been insufficient. We hypothesized that the amino acid linker sequence (XX) adjacent to the photosensitizer is an important determinant of PCI efficiency. In this study, using CPP-cargo-XX-PS platforms, we examined the relationship between PCI efficiency and the linker amino acid sequence near the photosensitizer. We found that hydrophobic FF and LL linkers enhanced the PCI efficiencies of both TatBim-XX-PS and TatU1A-XX-PS. The effectiveness of the linker depended, in part, on both the cargo moiety and the photosensitizer. These results may guide the design of CPP-cargo-PS conjugates conferring broad target functions for PCI and photodynamic therapy.

Photoinduced Endosomal Escape Mechanism: A View from Photochemical Internalization Mediated by CPP-Photosensitizer Conjugates.

Endosomal escape in cell-penetrating peptide (CPP)-based drug/macromolecule delivery systems is frequently insufficient. The CPP-fused molecules tend to remain trapped inside endosomes and end up being degraded rather than delivered into the cytosol. One of the methods for endosomal escape of CPP-fused molecules is photochemical internalization (PCI), which is based on the use of light and a photosensitizer and relies on photoinduced endosomal membrane destabilization to release the cargo molecule. Currently, it remains unclear how this delivery strategy behaves after photostimulation. Recent findings, including our studies using CPP-cargo-photosensitizer conjugates, have shed light on the photoinduced endosomal escape mechanism. In this review, we discuss the structural design of CPP-photosensitizer and CPP-cargo-photosensitizer conjugates, and the PCI mechanism underlying their application.

MicroRNA-664a-5p promotes neuronal differentiation of SH-SY5Y cells.

MicroRNAs (miRNAs) belong to a class of small noncoding RNAs that play important roles in the translational regulation of gene expression. A number of miRNAs are known to act as key regulators of diverse processes such as neuronal differentiation. In this study, we have attempted to identify novel miRNAs related to neuronal differentiation via microarray analysis in the human neuronal differentiation model neuroblastoma SH-SY5Y cells. We identified 15 up-regulated and eight down-regulated miRNAs in SH-SY5Y cells treated with all-trans retinoic acid to induce differentiation. We further showed that one of the up-regulated miRNAs, miR-664a-5p, promoted neuronal differentiation of SH-SY5Y cells. These findings enhance our understanding of the miRNAs involved in the process of neurogenesis and, in particular, highlight an important role of miR-664a-5p in SH-SY5Y cell neuronal differentiation. Further studies will be required to confirm the function of miR-664-5p in neuronal development and disease and to identify its relevant target genes.

Human soluble phospholipase A2 receptor is an inhibitor of the integrin-mediated cell migratory response to collagen-I.

Murine membrane-bound phospholipase A2 receptor 1 (PLA2R) is shed and released into plasma in a soluble form that retains all of the extracellular domains. Relatively little is known about human PLA2R. This study examined whether human soluble PLA2R has biological functions and whether soluble PLA2R exists in human plasma. Here, we showed that human recombinant soluble PLA2R (rsPLA2R) bound to collagen-I and inhibited interaction of collagen-I with the extracellular domain of integrin ß1 on the cell surface of human embryonic kidney 293 (HEK293) cells. As a result, rsPLA2R suppressed integrin ß1-mediated migratory responses of HEK293 cells to collagen-I in Boyden chamber experiments. Inhibition of phosphorylation of FAK Tyr397 was also observed. Similar results were obtained with experiments using soluble PLA2R released from HEK293 cells transfected with a construct encoding human soluble PLA2R. rsPLA2R lacking the fibronectin-like type II (FNII) domain had no inhibitory effects on cell responses to collagen-I, suggesting an important role of the FNII domain in the interaction of rsPLA2R with collagen-I. In addition, rsPLA2R suppressed the migratory response to collagen-IV and binding of collagen-IV to the cell surface of human podocytes that endogenously express membrane-bound, full-length PLA2R. Immunoprecipitation and Western blotting showed the existence of immunoreactive PLA2R in human plasma. In conclusion, human recombinant soluble PLA2R inhibits integrin ß1-mediated cell responses to collagens. Further studies are warranted to elucidate whether immunoreactive PLA2R in human plasma has the same properties as rsPLA2R.

Red and Near-Infrared Light-Directed Cytosolic Delivery of Two Different RNAs Using Photosensitive RNA Carriers.

Many cellular events are thought to be controlled by the temporal upregulation of multiple RNAs; the timing of the upregulation of these RNAs is not always the same. In this study, we first show that our light-directed intracellular RNA delivery method induced high concentrations of RNA in a short period. This effect was beneficial for the temporal control of cellular events by functional RNAs. Next, we stimulated the short-term upregulation of two different RNAs at different time points. Cytosolic delivery of a first RNA was induced by red light; thereafter, cytosolic delivery of a second RNA was induced by near-infrared light. The time difference between the introduction of the first and second RNA can be short (0.5-4 h) or long (>8 h). This strategy shows the potential for future applications of the deliberate control of time-dependent RNA concentration to guide various cellular functions by multiple RNAs.

Ultrasound-dependent cytoplasmic internalization of a peptide-sonosensitizer conjugate.

A method to induce cytoplasmic peptide delivery, using ultrasound, was demonstrated using a molecular conjugate of a cell-penetrating peptide (CPP), a functional peptide, and a sonosensitizer. As a model of such molecular conjugates, TatBim-RB, consisting of the Tat CPP, the Bim apoptosis inducing peptide, and the sonosensitizer rose bengal was synthesized. CPPs have been widely used for intracellular delivery of various cargos; however, CPP-fused molecules tend to become entrapped in endosomes, as was observed for TatBim-RB molecules applied to cells. To promote escape of the entrapped TatBim-RB molecules, cells were irradiated with ultrasound, which successfully induced endosomal escape and cytoplasmic dispersion of TatBim-RB, and subsequently apoptosis. Our results suggest that this peptide-sonosensitizer conjugate strategy may facilitate numerous kinds of medicinal chemistry studies, and furthermore, this specific conjugate may exhibit potential as a novel therapeutic agent for the promotion of apoptosis.

Photoinduced apoptosis using a peptide carrying a photosensitizer.

A novel molecule, TatBim-Alexa, consisting of the HIV1 Tat cell-penetrating peptide, the Bim apoptosis-inducing peptide, and Alexa Fluor 546 was synthesized for photoinducion of apoptosis. The Alexa Fluor 546 was used as a photosensitizer and covalently attached at the C-terminus of TatBim peptide by the thiol-maleimide reaction. Photo-dependent cytosolic internalization of TatBim-Alexa and photo-dependent apoptosis using TatBim-Alexa were demonstrated in several kinds of mammalian cells including human cancer cell lines.