Protein-Labeling Reagents Selectively Activated by Copper(I).

Copper is an essential trace element that participates in many biological processes through its unique redox cycling between cuprous (Cu+) and cupric (Cu2+) oxidation states. To elucidate the biological functions of copper, chemical biology tools that enable selective visualization and detection of copper ions and proteins in copper-rich environments are required. Herein, we describe the design of Cu+-responsive reagents based on a conditional protein labeling strategy. Upon binding Cu+, the probes generated quinone methide via oxidative bond cleavage, which allowed covalent labeling of surrounding proteins with high Cu+ selectivity. Using gel- and imaging-based analyses, the best-performing probe successfully detected changes in the concentration of labile Cu+ in living cells. Moreover, conditional proteomics analysis suggested intramitochondrial Cu+ accumulation in cells undergoing cuproptosis. Our results highlight the power of Cu+-responsive protein labeling in providing insights into the molecular mechanisms of Cu+ metabolism and homeostasis.

Feasibility of self-measurement telemonitoring using a handheld heart sound recorder in patients with heart failure - SELPH multicenter pilot study.

BACKGROUND: Multi-parametric assessment, including heart sounds in addition to conventional parameters, may enhance the efficacy of noninvasive telemonitoring for heart failure (HF). We sought to assess the feasibility of self-telemonitoring with multiple devices including a handheld heart sound recorder and its association with clinical events in patients with HF. METHODS: Ambulatory HF patients recorded their own heart sounds, mono­lead electrocardiograms, oxygen saturation, body weight, and vital signs using multiple devices every morning for six months. RESULTS: In the 77 patients enrolled (63 ±â€¯13 years old, 84 % male), daily measurements were feasible with a self-measurement rate of >70 % of days in 75 % of patients. Younger age and higher Minnesota Living with Heart Failure Questionnaire scores were independently associated with lower adherence (p = 0.002 and 0.027, respectively). A usability questionnaire showed that 87 % of patients felt self-telemonitoring was helpful, and 96 % could use the devices without routine cohabitant support. Six patients experienced ten HF events of re-hospitalization and/or unplanned hospital visits due to HF. In patients who experienced HF events, a significant increase in heart rate and diastolic blood pressure and a decrease in the time interval from Q wave onset to the second heart sound were observed 7 days before the events compared with those without HF events. CONCLUSIONS: Self-telemonitoring with multiple devices including a handheld heart sound recorder was feasible even in elderly patients with HF. This intervention may confer a sense of relief to patients and enable monitoring of physiological parameters that could be valuable in detecting the deterioration of HF.

Role of proteoglycan synthesis genes in osteosarcoma stem cells.

Osteosarcoma stem cells (OSCs) contribute to the pathogenesis of osteosarcoma (OS), which is the most common malignant primary bone tumor. The significance and underlying mechanisms of action of proteoglycans (PGs) and glycosaminoglycans (GAGs) in OSC phenotypes and OS malignancy are largely unknown. This study aimed to investigate the role of PG/GAG biosynthesis and the corresponding candidate genes in OSCs and poor clinical outcomes in OS using scRNA-seq and bulk RNA-seq datasets of clinical OS specimens, accompanied by biological validation by in vitro genetic and pharmacological analyses. The expression of ß-1,3-glucuronyltransferase 3 (B3GAT3), one of the genes responsible for the biosynthesis of the common core tetrasaccharide linker region of PGs, was significantly upregulated in both OSC populations and OS tissues and was associated with poor survival in patients with OS with high stem cell properties. Moreover, the genetic inactivation of B3GAT3 by RNA interference and pharmacological inhibition of PG biosynthesis abrogated the self-renewal potential of OSCs. Collectively, these findings suggest a pivotal role for B3GAT3 and PG/GAG biosynthesis in the regulation of OSC phenotypes and OS malignancy, thereby providing a potential target for OSC-directed therapy.

Evaluation of the efficacy of combined device strategies for long femoropopliteal artery disease.

One of the major problems associated with bare nitinol stent implantation is stent fracture, particularly in the popliteal artery. The purpose of this study was to determine whether drug coated balloons (DCB), interwoven stents (IWS), or plain old balloon angioplasty (POBA) would be suitable for use in distal femoropopliteal (FP) long lesions when an Eluvia stent was implanted proximal to a lesion. This was a multi-center retrospective study enrolling patients undergoing concomitant use of Eluvia with DCB, IWS or POBA for symptomatic atherosclerotic femoropopliteal disease (lesion length > 15 cm) [Rutherford category 2-6] between January 2018 and September 2021. 79 patients with 89 femoropopliteal lesions were enrolled in this study. The mean lesion length and the percentage of the popliteal artery involvement was 24.3 ± 6.4 cm vs 24.0 ± 9.0 cm vs 26.6 ± 6.2 cm and 65.8% vs 89.4% vs 67.8% for the Eluvia + DCB, Eluvia + IWS, and Eluvia + POBA groups, respectively. The 1-year Kaplan-Meier estimates of primary patency and freedom from major adverse limb events (MALEs) were 53.3% vs 44.1% vs 24.2% and 62.4% vs 51.0% vs 28.1%, respectively. Eluvia + POBA was associated with a lower rate for 1-year primary patency (HR 2.49; 95% confidence interval (CI): 1.28-4.87; p = 0.007 and HR 2.38; 95% CI: 1.13-5.77; p = 0.04). In SFA long lesions with proximal Eluvia implantation, distal implantations of either a DCB or IWS were comparable, as opposed to POBA alone which generated worse results.

Magnetic resonance elastography for the prediction of hepatocellular carcinoma in chronic hepatitis B.

Background and Aim: Magnetic resonance elastography (MRE) is used for the evaluation of liver fibrosis; however, it remains unclear whether MRE-based liver stiffness is associated with hepatocellular carcinoma (HCC) development, particularly in patients with chronic hepatitis B. Methods: A total of 504 patients with chronic hepatitis B receiving MRE were enrolled. The primary endpoint was the association between MRE-based liver stiffness and HCC. Results: In a cross-sectional analysis at the time of MRE measurement, the median (interquartile range) liver stiffness values in patients with presence or history of HCC and those without HCC were 3.68 (2.89-4.96) and 2.60 (2.22-3.45) kPa, respectively, and liver stiffness was significantly higher in patients with presence or history of HCC than in those without HCC (P < 0.001). In a longitudinal analysis of patients without HCC, the 1-, 3-, and 5-year cumulative incidence of HCC in patients with liver stiffness ≥3.6 kPa and those with liver stiffness <3.6 kPa were 3.8%, 7.0%, and 22.9%, and 0%, 0.9%, and 1.5%, respectively (P < 0.001). In the multivariable analysis, MRE-based liver stiffness (per 1 kPa) or liver stiffness ≥3.6 kPa was an independent factor for HCC development with an adjusted hazard ratio (aHR) of 1.61 (95% confidence interval [CI], 1.3-2.0) or aHR of 8.22 (95% CI, 2.1-31). Conclusion: MRE-based liver stiffness is associated with HCC risk in patients with chronic hepatitis B and may be used for the early prediction of HCC development and determination of indications for treatment.

Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids.

BACKGROUND: The clinical application of human induced pluripotent stem cell-derived cardiomyocytes (CMs) for cardiac repair commenced with the epicardial delivery of engineered cardiac tissue; however, the feasibility of the direct delivery of human induced pluripotent stem cell-derived CMs into the cardiac muscle layer, which has reportedly induced electrical integration, is unclear because of concerns about poor engraftment of CMs and posttransplant arrhythmias. Thus, in this study, we prepared purified human induced pluripotent stem cell-derived cardiac spheroids (hiPSC-CSs) and investigated whether their direct injection could regenerate infarcted nonhuman primate hearts. METHODS: We performed 2 separate experiments to explore the appropriate number of human induced pluripotent stem cell-derived CMs. In the first experiment, 10 cynomolgus monkeys were subjected to myocardial infarction 2 weeks before transplantation and were designated as recipients of hiPSC-CSs containing 2×107 CMs or the vehicle. The animals were euthanized 12 weeks after transplantation for histological analysis, and cardiac function and arrhythmia were monitored during the observational period. In the second study, we repeated the equivalent transplantation study using more CMs (6×107 CMs). RESULTS: Recipients of hiPSC-CSs containing 2×107 CMs showed limited CM grafts and transient increases in fractional shortening compared with those of the vehicle (fractional shortening at 4 weeks after transplantation: 26.2±2.1%; 19.3±1.8%; P<0.05), with a low incidence of posttransplant arrhythmia. Transplantation of increased dose of CMs resulted in significantly greater engraftment and long-term contractile benefits (fractional shortening at 12 weeks after transplantation: 22.5±1.0%; 16.6±1.1%; P<0.01, left ventricular ejection fraction at 12 weeks after transplantation: 49.0±1.4%; 36.3±2.9%; P<0.01). The incidence of posttransplant arrhythmia slightly increased in recipients of hiPSC-CSs containing 6×107 CMs. CONCLUSIONS: We demonstrated that direct injection of hiPSC-CSs restores the contractile functions of injured primate hearts with an acceptable risk of posttransplant arrhythmia. Although the mechanism for the functional benefits is not fully elucidated, these findings provide a strong rationale for conducting clinical trials using the equivalent CM products.

Subchronic toxicity study of indium-tin oxide nanoparticles following an intratracheal administration into the lungs of rats.

OBJECTIVES: We aimed to analyze the subchronic toxicity and tissue distribution of indium after the intratracheal administration of indium-tin oxide nanoparticles (ITO NPs) to the lungs of rats. METHODS: Male Wistar rats were administered a single intratracheal dose of 10 or 20 mg In/kg body weight (BW) of ITO NPs. The control rats received only an intratracheal dose of distilled water. A subset of rats was periodically euthanized throughout the study from 1 to 20 weeks after administration. Indium concentrations in the serum, lungs, mediastinal lymph nodes, kidneys, liver, and spleen as well as pathological changes in the lungs and kidneys were determined. Additionally, the distribution of ionic indium and indium NPs in the kidneys was analyzed using laser ablation-inductively coupled plasma mass spectrometry. RESULTS: Indium concentrations in the lungs of the two ITO NP groups gradually decreased over the 20-week observation period. Conversely, the indium concentrations in the mediastinal lymph nodes of the two ITO groups increased and were several hundred times higher than those in the kidneys, spleen, and liver. Pulmonary and renal toxicities were observed histopathologically in both the ITO groups. Both indium NPs and ionic indium were detected in the kidneys and their distributions were similar to the strong indium signals detected at the sites of inflammatory cell infiltration and tubular epithelial cells. CONCLUSIONS: Our results demonstrate that intratracheal administration of 10 or 20 mg In/kg body weight of ITO NPs in male rats produces pulmonary and renal toxicities.

TAS3681, an androgen receptor antagonist, prevents drug resistance driven by aberrant androgen receptor signaling in prostate cancer.

Second-generation androgen receptor (AR) signaling inhibitors (ARSIs), such as abiraterone and enzalutamide, prolong the life of patients with castration-resistant prostate cancer (CRPC). However, patients receiving ARSIs ultimately develop resistance through various complex mechanisms, including AR mutations, constitutively active AR-splice variants (AR-Vs), and AR overexpression. Here, we characterized a novel AR pure antagonist, TAS3681, which inhibits AR transcriptional activity and downregulates AR-full length (AR-FL) and AR-Vs. TAS3681 reduced the protein levels of AR-FL and AR-Vs including AR-V7 in enzalutamide-resistant cells (SAS MDV No. 3-14), in vitro and in vivo, showing strong antitumor efficacy in an AR-V7-positive xenograft model. In AR-overexpressing VCaP (prostate cancer) cells, conversely to enzalutamide, TAS3681 effectively suppressed cell proliferation and downregulated AR expression. Importantly, TAS3681 blocked the transcriptional activity of various mutant ARs, including mutations F877L/T878A and H875Y/T878A, which confer resistance to enzalutamide, and V716M and H875Y mutations, which confer resistance to darolutamide. Our results demonstrate that TAS3681 suppresses the reactivation of AR signaling, which causes resistance to ARSIs, via a newly identified mechanism of action. Therefore, TAS3681 could be a new therapeutic option for CRPC treatment.

Aging-associated changes of optical coherence tomography-measured ganglion cell-related retinal layer thickness and visual sensitivity in normal Japanese.

PURPOSE: To report aging-associated change rates in circumpapillary retinal nerve fiber layer thickness (cpRNFLT) and macular ganglion cell-inner plexiform layer and complex thickness (MGCIPLT, MGCCT) in normal Japanese eyes and to compare the data in linear scaled visual field (VF) sensitivity of central 4 points of Humphrey Field Analyzer (HFA) 24-2 test (VF4TestPoints) to that in MGCIPLT in four 0.6-mm-diameter circles corresponding to the four central points of HFA 24-2 adjusted for retinal ganglion cell displacement (GCIPLT4TestPoints). STUDY DESIGN: Prospective observational study METHODS: HFA 24-2 tests and spectral-domain optical coherence tomography (SD-OCT) measurements of cpRNFLT, MGCIPLT, MGCCT and GCIPLT4TestPoints were performed every 3 months for 3 years in 73 eyes of 37 healthy Japanese with mean age of 50.4 years. The time changes of SD-OCT-measured parameters and VF4TestPoints were analyzed using a linear mixed model. RESULTS: The aging-associated change rates were -0.064 µm/year for MGCIPLT and and -0.095 for MGCCT (P=0.020 and 0.017), but could not be detected for cpRNFLT. They accelerated with aging at -0.009µm/year/year of age for MGCIPLT (P<0.001), at 0.011 for MGCCT (P<0.001) and at 0.013 for cpRNFLT(0.031). The aging-associated decline of -82.1 [1/Lambert]/year of VF4TestPoints corresponded to -0.095 µm/year of GCIPLT4TestPoints. CONCLUSION: We report that aging-associated change rates of cpRNFLT, MGCIPLT and MGCCT in normal Japanese eyes were found to be significantly accelerated along with aging. Relationship between VF sensitivity decline rates and SD-OCT measured GCIPLT decline rates during physiological aging in the corresponding parafoveal retinal areas are also documented.

Predictive role of regional thigh tissue oxygen saturation monitoring during cardiopulmonary bypass in lung injury after cardiac surgery.

Acute respiratory distress syndrome (ARDS) is a serious complication following cardiac surgery mainly associated with the use of cardiopulmonary bypass (CPB), which could increase the risk of mortality and morbidity. This study investigated the association of regional oxygen saturation (rSO2) during CPB with postoperative outcomes, including respiratory function. Patients who underwent cardiac surgery with CPB from 2015 to 2019 were included. Near-infrared spectroscopy was used to monitor rSO2 at the forehead, abdomen, and thighs throughout the surgery. Postoperative markers associated with CPB were assessed for correlations with PaO2/FiO2 (P/F) ratios at intensive care unit (ICU) admission. Postoperative lung injury (LI) was defined as moderate or severe ARDS based on the Berlin criteria, and its incidence was 29.9% (20/67). On multiple regression analysis, the following were associated with P/F ratios at ICU admission: vasoactive-inotropic scores at CPB induction (P = 0.03), thigh rSO2 values during CPB (P = 0.04), and body surface area (P < 0.001). A thigh rSO2 of 71% during CPB was significantly predictive of postoperative LI with an area under the curve of 0.71 (P = 0.03), sensitivity of 0.70, and specificity of 0.68. Patients with postoperative LI had longer ventilation time and ICU stays. Thigh rSO2 values during CPB were a potential predictor of postoperative pulmonary outcomes.

Impact of immune-related adverse events in patients with hepatocellular carcinoma treated with atezolizumab plus bevacizumab.

BACKGROUND AND AIM: Immune checkpoint inhibitors pose the risk of immune-related adverse events (irAEs). Recent data suggest that irAEs may be associated with a favorable prognosis. This study aimed to investigate and analyze the association between these adverse events and the clinical benefits in patients with unresectable hepatocellular carcinoma. METHODS: The study enrolled 130 patients with advanced hepatocellular carcinoma treated with atezolizumab plus bevacizumab between November 2020 and January 2023 at a single center. The relationship between irAEs and both response rate and post-treatment outcomes was investigated. RESULTS: Out of the 130 patients, irAEs developed in 36 (27.7%) patients. The irAE group exhibited a significantly longer progression-free survival (PFS) than the non-irAE group, with a median PFS of 8.9 compared with 4.6 months (P < 0.01). No difference was found in the overall survival between the irAE and non-irAE groups. The irAE group demonstrated significantly higher disease control rate (DCR) than the non-irAE group (97.0% vs 65.5%, P < 0.01). The analysis by irAE severity revealed that the grade 1/2 group exhibited significantly longer PFS (7.9 vs 4.6 months, P = 0.007) and higher DCR (100% vs 65.5%, P < 0.01) than the non-irAE group. Furthermore, hypothyroidism correlated with a favorable PFS (8.9 vs 5.4 months, P = 0.02), DCR (100% vs 71.3%, P = 0.03), and overall response rate (58.3% vs 18.5%, P = 0.005). CONCLUSION: The presence of irAEs is associated with prolonged PFS and higher DCR. Specifically, mild irAEs (grade 1/2) and hypothyroidism displayed prolonged PFS and higher DCR.

The gateway reflex regulates tissue-specific autoimmune diseases.

The dynamic interaction and movement of substances and cells between the central nervous system (CNS) and peripheral organs are meticulously controlled by a specialized vascular structure, the blood-brain barrier (BBB). Experimental and clinical research has shown that disruptions in the BBB are characteristic of various neuroinflammatory disorders, including multiple sclerosis. We have been elucidating a mechanism termed the "gateway reflex" that details the entry of immune cells, notably autoreactive T cells, into the CNS at the onset of such diseases. This process is initiated through local neural responses to a range of environmental stimuli, such as gravity, electricity, pain, stress, light, and joint inflammation. These stimuli specifically activate neural pathways to open gateways at targeted blood vessels for blood immune cell entry. The gateway reflex is pivotal in managing tissue-specific inflammatory diseases, and its improper activation is linked to disease progression. In this review, we present a comprehensive examination of the gateway reflex mechanism.

Electron Localization Induced by Disordered Anions in an Organic Conductor.

We report on a new organic conductor κ″-(ET)2Cu[N(CN)2]Br (κ″-Br), which is the first polymorph of an organic superconductor κ-(ET)2Cu[N(CN)2]Br (κ-Br), where ET denotes bis(ethylenedithio)tetrathiafulvalene. κ″-Br has a similar κ-type arrangement of ET molecules to κ-Br, but, in contrast to the orthorhombic κ-Br, which has ordered polyanion chains, presents a monoclinic crystal structure with disordered polymeric anion chains. To elucidate the electronic state of κ″-Br, we performed band calculations as well as transport, magnetic, and optical measurements. The calculated band dispersion, magnitude of electron correlation, and room-temperature optical conductivity spectra of κ″-Br were comparable to those of κ-Br. Despite these similarities, the κ″-Br salt exhibited a semiconducting behavior. The electron spin resonance and Raman spectroscopies indicated that there is neither magnetic nor charge order in κ″-Br, suggesting the occurrence of Anderson localization due to disordered anion layers.

The stiffness and collagen control differentiation of osteoclasts with an altered expression of c-Src in podosome.

Osteoclasts are hematopoietic cells attached to the bones containing type I collagen-deposited hydroxyapatite during bone resorption. Two major elements determine the stiffness of bones: regular calcified bone (bone that is resorbable by osteoclasts) and un-calcified osteoid bone (bone that is un-resorbable by osteoclasts). The osteolytic cytokine RANKL promotes osteoclast differentiation; however, the roles of the physical interactions of osteoclasts with calcified and un-calcified bone at the sealing zones and the subsequent cellular signaling remain unclear. In this study, we investigated podosomes, actin-rich adhesion structures (actin-ring) in the sealing zone that participates in sensing hard stiffness with collagen in the physical environment during osteoclast differentiation. RANKL-induced osteoclast differentiation induction was promoted when Raw264.7 cells were cultured on collagen-coated plastic dishes but not on non-coated plastic dishes, which was associated with the increased expression of podosome-related genes and Src. In contrast, when cells were cultured on collagen gel, expression of podosome-related genes and Src were not upregulated. The induction of podosome-related genes and Src requires hard stiffness with RGD-containing substratum and integrin-mediated F-actin polymerization. These results indicate that osteoclasts sense both the RGD sequence and stiffness of calcified collagen through their podosome components regulating osteoclast differentiation via the c-Src pathway.

DDX6 is involved in the pathogenesis of inflammatory diseases via NF-κB activation.

The IL-6 amplifier was originally discovered as a mechanism for the enhanced activation of NF-κB in non-immune cells. In the IL-6 amplifier, IL-6-STAT3 and NF-κB stimulation is followed by an excessive production of IL-6, chemokines, and growth factors to develop chronic inflammation preceding the development of inflammatory diseases. Previously, using a shRNA-mediated genome-wide screening, we found that DEAD-Box Helicase 6 (DDX6) is a candidate positive regulator of the amplifier. Here, we investigate whether DDX6 is involved in the pathogenesis of inflammatory diseases via the IL-6 amplifier. We found that DDX6-silencing in non-immune cells suppressed the NF-κB pathway and inhibited activation of the IL-6 amplifier, while the forced expression of DDX6 enhanced NF-κB promoter activity independent of the RNA helicase activity of DDX6. The imiquimod-mediated dermatitis model was suppressed by the siRNA-mediated gene downregulation of DDX6. Furthermore, silencing DDX6 significantly reduced the TNF-α-induced phosphorylation of p65/RelA and IκBα, nuclear localization of p65, and the protein levels of IκBα. Mechanistically, DDX6 is strongly associated with p65 and IκBα, but not TRADD, RIP, or TRAF2, suggesting a novel function of DDX6 as an adaptor protein in the NF-κB pathway. Thus, our findings demonstrate a possible role of DDX6 beyond RNA metabolism and suggest DDX6 is a therapeutic target for inflammatory diseases.

Quantitative determination of the intracellular uptake of silica nanoparticles using asymmetric flow field flow fractionation coupled with ICP mass spectrometry and their cytotoxicity in HepG2 cells.

We established a size separation method for silica nanoparticles (SiNPs) measuring 10, 30, 50, 70, and 100 nm in diameter using asymmetric flow field flow fractionation hyphenated with inductively coupled plasma mass spectrometry (AF4-ICP-MS), and evaluated the cytotoxicity of SiNPs in human hepatoma HepG2 cells. Analysis of the mixture sample revealed that nanoparticles of different sizes were eluted at approximately 2-min intervals, with no effect on each elution time or percentage recovery. Compared with larger SiNPs, smaller SiNPs exhibited high cytotoxicity when the volume of SiNPs exposed to the cells was the same. We measured SiNPs in culture medium and inside cells by AF4-ICP-MS and found that approximately 17% of SiNPs in the mixture of five differently sized particles were absorbed by the cells. Transmission electron microscopy revealed that 10 nm SiNPs formed aggregates and accumulated in the cells. Based on AF4-ICP-MS analysis, there is no clear difference in the particle volume absorbed by the cells among different sizes. Therefore, the high toxicity of small SiNPs can be explained by the fact that their large surface area relative to particle volume efficiently induces toxicological influences. Indeed, the large surface area of 10 nm SiNPs significantly contributed to the production of reactive oxygen species.

Concordance between metabolic dysfunction-associated steatotic liver disease and nonalcoholic fatty liver disease.

AIM: A multisociety consensus group proposed a new nomenclature for metabolic dysfunction-associated steatotic liver disease (MASLD). Although patients with nonalcoholic fatty liver disease (NAFLD) are expected to be reclassified as patients with MASLD under the new nomenclature, the concordance between MASLD and NAFLD remains unclear. Moreover, waist circumference could be adjusted by ethnicity for diagnosing MASLD; however, there are limited data on the optimal waist circumference in the Japanese population. METHODS: This cross-sectional study was conducted on 3709 Japanese patients with NAFLD. The primary endpoint was the prevalence of MASLD in patients with NAFLD. The difference between the original waist circumference criteria (>94 cm for men and >80 cm for women) and the Japanese metabolic syndrome criteria (≥85 cm for men and ≥90 cm for women) for concordance between NAFLD and MASLD was also investigated. RESULTS: According to the original criteria, the prevalence of MASLD in patients with NAFLD was 96.7%. Similarly, according to the Japanese waist circumference criteria, 96.2% of patients with NAFLD could be reclassified as those with MASLD. The concordance rate was significantly higher in the original criteria than in the Japanese criteria (p = 0.02). CONCLUSIONS: NAFLD could be considered MASLD using the original MASLD criteria in the Japanese population, and insights from NAFLD research could be applied to MASLD.

Quality assessment of enzymatic methyl-seq library constructed using crude cell lysate.

Enzymatic methyl-seq (EM-seq), an enzyme-based method, identifies genome-wide DNA methylation, which enables us to obtain reliable methylome data from purified genomic DNA by avoiding bisulfite-induced DNA damage. However, the loss of DNA during purification hinders the methylome analysis of limited samples. The crude DNA extraction method is the quickest and minimal sample loss approach for obtaining useable DNA without requiring additional dissolution and purification. However, it remains unclear whether crude DNA can be used directly for EM-seq library construction. In this study, we aimed to assess the quality of EM-seq libraries prepared directly using crude DNA. The crude DNA-derived libraries provided appropriate fragment sizes and concentrations for sequencing similar to those of the purified DNA-derived libraries. However, the sequencing results of crude samples exhibited lower reference sequence mapping efficiencies than those of the purified samples. Additionally, the lower-input crude DNA-derived sample exhibited a marginally lower cytosine-to-thymine conversion efficiency and hypermethylated pattern around gene regulatory elements than the higher-input crude DNA- or purified DNA-derived samples. In contrast, the methylation profiles of the crude and purified samples exhibited a significant correlation. Our findings indicate that crude DNA can be used as a raw material for EM-seq library construction.

Zinc Deficiency as a General Feature of Cancer: a Review of the Literature.

Trace elements are minerals that are present in very low concentrations in the human body and yet are crucial for a wide range of physiological functions. Zinc, the second most abundant trace element, is obtained primarily from the diet. After being taken up in the intestine, zinc is distributed to various target organs, where it plays key roles in processes such as immunity, protein folding, apoptosis, and antioxidant activity. Given the important role of zinc in a wide range of enzymatic reactions and physiological processes, zinc deficiency has been identified in a variety of diseases, notably cancer. In recent years, multiple meta-analyses and reviews looking at zinc levels in individual cancer types have been published, as have a plethora of primary studies demonstrating a link between low zinc levels and specific types of cancer. In this review, we summarize recent evidence implicating low zinc concentrations in serum or tissues as a characteristic in a wide range of cancers. We also discuss preliminary findings indicating that zinc level measurement could ultimately become a useful clinical tool for cancer diagnosis and predicting outcomes in patients with cancer. Finally, we suggest future directions for further elucidating the role of zinc deficiency in cancer development and progression.