Erratum: DNA Extraction and Comparison Between Old and Fresh Necrophilic Fly Samples.

This corrects the article 10.3791/66737.

Transperineal versus Transrectal MRI/TRUS fusion-guided prostate biopsy in a large, ethnically diverse, and multiracial cohort.

PURPOSE: To compare transperineal (TP) vs transrectal (TR) magnetic resonance imaging (MRI) and transrectal ultrasound (TRUS) fusion-guided prostate biopsy (PBx) in a large, ethnically diverse and multiracial cohort. MATERIALS AND METHODS: Consecutive patients who underwent multiparametric (mp) MRI followed by TP or TR TRUS-fusion guided PBx, were identified from a prospective database (IRB #HS-13-00663). All patients underwent mpMRI followed by 12-14 core systematic PBx. A minimum of two additional target-biopsy cores were taken per PIRADS≥3 lesion. The endpoint was the detection of clinically significant prostate cancer (CSPCa; Grade Group, GG≥2). Statistical significance was defined as p<0.05. RESULTS: A total of 1491 patients met inclusion criteria, with 480 undergoing TP and 1011 TR PBx. Overall, 11% of patients were Asians, 5% African Americans, 14% Hispanic, 14% Others, and 56% White, similar between TP and TR (p=0.4). For PIRADS 3-5, the TP PBx CSPCa detection was significantly higher (61% vs 54%, p=0.03) than TR PBx, but not for PIRADS 1-2 (13% vs 13%, p=1.0). After adjusting for confounders on multivariable analysis, Black race, but not the PBx approach (TP vs TR), was an independent predictor of CSPCa detection. The median maximum cancer core length (11 vs 8mm; p<0.001) and percent (80% vs 60%; p<0.001) were greater for TP PBx even after adjusting for confounders. CONCLUSIONS: In a large and diverse cohort, Black race, but not the biopsy approach, was an independent predictor for CSPCa detection. TP and TR PBx yielded similar CSPCa detection rates; however the TP PBx was histologically more informative.

Validation of the JAMR upper-arm pressure monitor B23 according to the AAMI/ESH/ISO Universal Standard (ISO 81060- 2:2018/AMD 1:2020).

The aim of this study was to evaluate the accuracy of the JAMR upper-arm blood pressure monitor B23 in the general population according to the AAMI/ESH/ISO Universal Standard (ISO 81060-2 : 2018/AMD 1 : 2020). The study recruited participants who met the criteria of the AAMI/ESH/ISO Universal Standard in terms of their number, sex, age, limb size, and blood pressure (BP) distribution. The study involved measuring BP, including both SBP and DBP, using both the test device and a standard mercury sphygmomanometer in sequential measurements. Of 90 participants, 85 qualified participants were analyzed. A total of 255 sets of comparison data (three sets for each subject) were obtained and analyzed. For the validation criterion 1, the mean ±â€…SD of the differences between the JAMR B23 and mercury sphygmomanometer BP readings was -0.24 ±â€…6.52/-2.67 ±â€…5.6 mmHg (SBP/DBP). For criterion 2, the SD of the averaged BP (SBP/DBP) differences between the JAMR B23 and reference BP (SBP/DBP) per participant was 5.61/5.13 mmHg (the requirement was ≤6.95/6.43 mmHg by calculation). The JAMR B23 passed all the requirements of the AAMI/ESH/ISO Universal Standard (ISO 81060-2 : 2018/AMD 1 : 2020) and can be recommended for clinical and self/home use in the general population.

Volumetric Stent Expansion Index to Assess Tapering Lesions Using Intravascular Ultrasound and Its Clinical Outcomes.

Background: This study aimed to assess the clinical significance of generating a volumetric stent expansion index for tapering lesions through intravascular ultrasound (IVUS). Previous IVUS studies have used minimal stent area (MSA) to predict adverse outcomes. Methods: A total of 251 tapering lesions were treated in this study via IVUS guidance in 232 patients. Eight stent expansion indices were evaluated to determine the association of these indices with device-oriented clinical endpoints (DoCEs) after two-year follow-ups. These were the ILUMIEN III and IV standards, the ULTIMATE (Intravascular Ultrasound Guided Drug Eluting Stents Implantation in "All-Comers" Coronary Lesions) standard, the IVUS-XPL (Impact of Intravascular Ultrasound Guidance on the Outcomes of Xience Prime Stents in Long Lesions) standard, the minimal volumetric expansion index (MVEI) using the Huo-Kassab or linear model, the MSA/vessel area at the MSA cross-section, the traditional stent expansion (MSA/mean proximal and distal reference lumen cross-sectional area), and MSA. Results: The MVEI was the only stent expansion index that correlated significantly with the two-year DoCEs (hazard ratio [HR], 1.91; 95% confidence interval [CI]: 1.16-3.96; p = 0.028). In the ROC analysis, the area under the curve for the MVEI was 0.71 (p = 0.002), with an optimal cut-off value of 62.2 for predicting the DoCEs. Conclusions: This is the first study to use IVUS for tapering lesions and demonstrate that the MVEI is an independent predictor of two-year DoCEs.

Clinical Implications of Acute Stent Mal-Apposition in the Left Main Coronary Artery.

Background: Intravascular ultrasound (IVUS) has been utilized to determine acute stent mal-apposition (ASM) after percutaneous coronary intervention (PCI) in the left main coronary artery (LMCA). However, the clinical consequences of this finding remain uncertain. This research aimed to evaluate the clinical implications of ASM in the LMCA using IVUS. Methods: In this study, 408 patients who underwent successful drug-eluting stent (DES) implantation in the LMCA were evaluated. We analyzed the prevalence and characteristics of ASM and its correlation with clinical outcomes. ASM is characterized by stent struts that are not in immediate proximity to the intimal surface of the vessel wall after initial stent deployment. Results: The observed incidence of LMCA-ASM post-successful PCI was 26.2%, both per patient and per lesion. Lesions with LMCA-ASM had a longer stent diameter, larger stent areas, and larger lumen areas compared to those without LMCA-ASM (4.0 ± 0.5 vs. 3.7 ± 0.4 mm, p < 0.001; 9.8 ± 2.0 vs. 9.0 ± 1.6 mm 2 , p < 0.001; 12.3 ± 1.9 vs. 10.1 ± 2.1 mm 2 , p < 0.001, respectively). The mean external elastic membrane (EEM) area (odds ratio (OR): 1.418 [95% confidence interval (CI): 1.295-1.556]; p < 0.001) emerged as an independent predictor of LMCA-ASM. During the observation period, LMCA-ASM did not display any association with device-oriented clinical endpoints (DoCE), which included cardiac death, target vessel-induced myocardial infarction (MI), stent thrombosis, and target lesion revascularization (TLR). Moreover, the DoCE incidence exhibited no significant disparity between patients with or without ASM (13.1 vs. 6.0%, p = 0.103). Conclusions: While LMCA-ASM was a not uncommon finding post-PCI, it did not correlate with adverse cardiac events in the present study.

Calcified Nodules in Non-Culprit Lesions with Acute Coronary Syndrome Patients.

Background: Calcified nodules (CN) have been linked to unfavorable clinical outcomes. However, there is a lack of systematic studies on non-culprit lesions with CN in patients with acute coronary syndromes (ACS). This study aims to investigate the frequency, distribution, predictors, and outcomes of CN in non-culprit lesions among ACS patients. Methods: We included 376 ACS patients who received successful stent placement in their culprit lesions. Intravascular ultrasound (IVUS) was performed to evaluate non-culprit lesions in left main arteries and all three coronary arteries (CA). CN was defined as accumulations of small nodular calcium deposits exhibiting a convex shape protruding into the lumen. Results: CNs was identified in 16.9% (121 of 712) per artery and 26.9% (101 of 376) per patient. They were predominantly located at the mid portion of the right coronary artery (26.3%) and the bifurcation site (59.9%). Patients with CN were older (63.57 ± 8.43 vs. 57.98 ± 7.15, p < 0.001) and had a higher prevalence of diabetes mellitus (55.4% vs. 42.2%, p = 0.022). However, there were no significant differences in baseline characteristics observed after propensity score matching (PSM). Multivariate analysis revealed that CN were independently associated with major adverse cardiovascular events (MACE) both before and after PSM (hazard ratio (HR): 0.341, 95% confidence interval (95% CI): 0.140-0.829, p = 0.018; HR: 0.275, 95% CI: 0.108-0.703, p = 0.007, respectively). During the observational period of 19.35 ± 10.59 months, the occurrence of MACE was significantly lower in patients with CN before and after PSM (5.9% vs. 16.7%, p = 0.046; 4.0% vs. 18.1%, p = 0.011; respectively). Conclusions: CN in non-culprit lesions with ACS patients was prevalent and caused fewer adverse clinical outcomes.

Targeting SOCS2 alleviates myocardial fibrosis by reducing nuclear translocation of ß-catenin.

BACKGROUND: Myocardial fibrosis is an important pathological feature of dilated cardiomyopathy (DCM). The roles of SOCS2 in fibrosis of different organs are controversial. Herein, we investigated the function and potential mechanism of SOCS2 in myocardial fibrosis. METHODS: Bioinformatics, immunohistochemistry (IHC), immunofluorescence (IF), western blot (WB), real-time fluorescence quantitative PCR (qPCR), rat primary myocardial fibroblasts (rCFs) culture, doxorubicin (DOX) induced mouse dilated cardiomyopathy (DCM) model, and in vivo adeno-associated virus (AAV) infection were used to explore the role of SOCS2 in DCM. RESULTS: Bioinformatics analysis showed that SOCS2 was positively correlated with fibrosis related factors. SOCS2 was significantly upregulated in patients and mice with DCM. In vivo experiments showed that targeted inhibition of cardiac SOCS2 could improve mouse cardiac function and alleviate myocardial fibrosis. Further research demonstrated that SOCS2 promoted the transformation of myofibroblasts. Knockdown of SOCS2 reduced the nuclear localization of ß-catenin, which inhibited the fibrogenic effect of Wnt/ß-catenin pathway. In addition, bioinformatics analysis suggested that lymphoid enhancer binding factor 1 (LEF1) was significantly positively correlated with SOCS2. Finally, dual luciferase assays demonstrated that LEF1 could bind to the promoter region of SOCS2, thereby mediating its transcriptional activation. CONCLUSION: SOCS2 could activate the Wnt/ß-catenin by regulating the nuclear translocation of ß-catenin, which induces the transcriptional activation of SOCS2. Overall, these results indicated a positive feedback activation phenomenon between SOCS2, ß-catenin and LEF1 in DCM. These results suggested that inhibition of SOCS2 could effectively alleviate the progression of myocardial fibrosis and improve cardiac function.

INT-1B3, an LNP formulated miR-193a-3p mimic, promotes anti-tumor immunity by enhancing T cell mediated immune responses via modulation of the tumor microenvironment and induction of immunogenic cell death.

microRNAs (miRNAs) are small, non-coding RNAs that regulate expression of multiple genes. MiR-193a-3p functions as a tumor suppressor in many cancer types, but its effect on inducing specific anti-tumor immune responses is unclear. Therefore, we examined the effect of our lipid nanoparticle (LNP) formulated, chemically modified, synthetic miR-193a-3p mimic (INT-1B3) on anti-tumor immunity. INT-1B3 inhibited distant tumor metastasis and significantly prolonged survival. INT-1B3-treated animals were fully protected against challenge with autologous tumor cells even in absence of treatment indicating long-term immunization. Protection against autologous tumor cell challenge was hampered upon T cell depletion and adoptive T cell transfer abrogated tumor growth. Transfection of tumor cells with our miR-193a-3p mimic (1B3) resulted in tumor cell death and apoptosis accompanied by increased expression of DAMPs. Co-culture of 1B3-transfected tumor cells and immature DC led to DC maturation and these mature DC were able to stimulate production of type 1 cytokines by CD4+ and CD8+ T cells. CD4-CD8- T cells also produced type 1 cytokines, even in response to 1B3-transfected tumor cells directly. Live cell imaging demonstrated PBMC-mediated cytotoxicity against 1B3-transfected tumor cells. These data demonstrate for the first time that miR-193a-3p induces long-term immunity against tumor development via modulation of the tumor microenvironment and induction of immunogenic cell death.

Fabrication of chitin-fibrin hydrogels to construct the 3D artificial extracellular matrix scaffold for vascular regeneration and cardiac tissue engineering.

As the cornerstone of tissue engineering and regeneration medicine research, developing a cost-effective and bionic extracellular matrix (ECM) that can precisely modulate cellular behavior and form functional tissue remains challenging. An artificial ECM combining polysaccharides and fibrillar proteins to mimic the structure and composition of natural ECM provides a promising solution for cardiac tissue regeneration. In this study, we developed a bionic hydrogel scaffold by combining a quaternized ß-chitin derivative (QC) and fibrin-matrigel (FM) in different ratios to mimic a natural ECM. We evaluated the stiffness of those composite hydrogels with different mixing ratios and their effects on the growth of human umbilical vein endothelial cells (HUVECs). The optimal hydrogels, QCFM1 hydrogels were further applied to load HUVECs into nude mice for in vivo angiogenesis. Besides, we encapsulated human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) into QCFM hydrogels and employed 3D bioprinting to achieve batch fabrication of human-engineered heart tissue (hEHT). Finally, the myocardial structure and electrophysiological function of hEHT were evaluated by immunofluorescence and optical mapping. Designed artificial ECM has a tunable modulus (220-1380 Pa), which determines the different cellular behavior of HUVECs when encapsulated in these. QCFM1 composite hydrogels with optimal stiffness (800 Pa) and porous architecture were finally identified, which could adapt for in vitro cell spreading and in vivo angiogenesis of HUVECs. Moreover, QCFM1 hydrogels were applied in 3D bioprinting successfully to achieve batch fabrication of both ring-shaped and patch-shaped hEHT. These QCFM1 hydrogels-based hEHTs possess organized sarcomeres and advanced function characteristics comparable to reported hEHTs. The chitin-derived hydrogels are first used for cardiac tissue engineering and achieve the batch fabrication of functionalized artificial myocardium. Specifically, these novel QCFM1 hydrogels provided a reliable and economical choice serving as ideal ECM for application in tissue engineering and regeneration medicine.

CISD2 regulates oxidative stress and mitophagy to maintain the balance of the follicular microenvironment in PCOS.

OBJECTIVES: To observe the CISD2 expression among PCOS patients and to explore its profound impact on the follicular microenvironment. Moreover, we want to elucidate the intricate mechanistic contribution of CISD2 to the onset and progression of PCOS. METHODS: Oxidase NOX2, mitophagy-related proteins, and CISD2 were detected by WB. The changes in mitochondrial structure and quantity were observed by transmission electron microscopy. Mitochondrial and lysosome colocalization was used to detect the changes of mitophagy. MDA kit, GSH and GSSG Assay kit and ROS probe were used to detect oxidative stress damage. RESULTS: We found that CISD2, mitophagy and oxidase in the GCs of PCOS patients were significantly increased. Testosterone stimulation leads to the increase of oxidase, mitophagy, and CISD2 in KGN cells. CISD2 inhibition promoted the increase of mitophagy, and the activation of mitochondria-lysosome binding, while alleviating the oxidative stress. CONCLUSIONS: Inhibition of CISD2 can improve the occurrence of oxidative stress by increasing the level of mitophagy, thus affecting the occurrence and development of PCOS diseases.

Flame spray pyrolyzed carbon-encapsulated Au/Fe3O4 nanoaggregates enabled efficient photothermal therapy and magnetic hyperthermia of esophageal cancer cells.

Multifunctional magneto-plasmonic nanoparticles with magnetic hyperthermia and photothermal therapy could kill cancer cells efficiently. Herein, carbon-encapsulated Au/Fe3O4 (Au/Fe3O4@C) was fabricated using an enclosed flame spray pyrolysis. The nanostructures, including an Fe3O4 core (51.9-55.2 nm) with a decreasing carbon shell thickness and an Au core (4.68-8.75 nm) coated with 2-4 graphite layers, were tailored by tuning the C2H4 content in the reacting gas mixture. Saturation magnetization (33.7-48.2 emu/g) and optical absorption were determined. The carbon shell facilitated the dispersion of Au/Fe3O4 and restrained their laser-induced and magnetic field-induced coalescence and growth. Au/Fe3O4@C exhibited excellent magnetic resonance imaging capability (91.4 mM-1 s-1) and photothermal performance (65.4°C for 0.8 mg/mL Au/Fe3O4@C at a power density of 1.0 W/cm2 after 300 s near-IR laser irradiation (808 nm)). Moreover, the combined application of photothermal and magnetic-heating properties reduced the required intensity of both laser and magnetic field compared to the intensity of separate situations. Our work provides a unique, intriguing approach to preparing multicomponent core/shell nanoaggregates that are promising candidates for esophageal cancer cell therapy.

Cardiomyocyte-specific Tbk1 deletion aggravated chronic doxorubicin cardiotoxicity via inhibition of mitophagy.

Doxorubicin (Dox) use is limited by Dox-induced cardiotoxicity. TANK-blinding kinase 1 (TBK1) is an important kinase involved in the regulation of mitophagy, but the role of TBK1 in cardiomyocytes in chronic Dox-induced cardiomyopathy remains unclear. Cardiomyocyte-specific Tbk1 knockout (Tbk1CKO) mice received Dox (6 mg/kg, injected intraperitoneally) once a week for 4 times, and cardiac assessment was performed 4 weeks after the final Dox injection. Adenoviruses encoding Tbk1 or containing shRNA targeting Tbk1, or a TBK1 phosphorylation inhibitor were used for overexpression or knockdown of Tbk1, or inhibit phosphorylation of TBK1 in isolated primary cardiomyocytes. Our results revealed that moderate Dox challenge decreased TBK1 phosphorylation (with no effect on TBK1 protein levels), resulting in compromised myocardial function, obvious mortality and overt interstitial fibrosis, and the effects were accentuated by Tbk1 deletion. Dox provoked mitochondrial membrane potential collapse and oxidative stress, the effects of which were exacerbated and mitigated by Tbk1 knockdown, specific inhibition of phosphorylation and overexpression, respectively. However, Tbk1 （Ser172A） overexpression did not alleviate these effects. Further scrutiny revealed that TBK1 exerted protective effects on mitochondria via SQSTM1/P62-mediated mitophagy. Tbk1 overexpression mediated cardioprotective effects on Dox-induced cardiotoxicity were cancelled off by Sqstm1/P62 knockdown. Moreover, TBK1-mitophagy-mitochondria cascade was confirmed in heart tissues from dilated cardiomyopathy patients. Taken together, our findings denoted a pivotal role of TBK1 in Dox-induced mitochondrial injury and cardiotoxicity possibly through its phosphorylation and SQSTM1/P62-mediated mitophagy.

Utilization of Pickering emulsion stabilized by chitin nanofibers for improving water and oxygen resistance of gelatin films.

The hydrophilicity of gelatin films obviously limits their application. In this work, novel protonated chitin nanofiber was prepared with green methods, surficial deacetylation combined with acidic and mechanical treatment. Composite films with excellent water and oxygen barrier properties were successfully prepared by blending O/W Pickering emulsions stabilized by chitin nanofibers with gelatin substrates. The films were characterized by Fourier-transform infrared, X-ray diffraction, mechanical properties, water and oxygen permeability, moisture content, water solubility, water contact angle and optical properties. The results demonstrated that the amino groups of chitin nanofibers bound to the carboxyl groups of gelatin via electrostatic interactions, which contributing to the excellent mechanical and barrier properties of composite films. The elongation at break of composite film (the concentration of chitin nanofiber was 0.2 wt%) was 2.66 times of gelatin film. And the water vapor and oxygen permeability of composite films lowered to 65.9 % and 52.9 %, respectively. The introduction of O/W Pickering emulsion significantly enhanced the hydrophobicity of gelatin-based film and the chitin nanofibers played a positive role in stabilizing the gelatin chains to a certain extent. This study expands the application fields of gelatin-based films and provides valuable technical route for the preparation of barrier films.

Comparison of the use of a spiral nasojejunal tube and transendoscopic enteral tubing in washed microbiota transplantation via the mid-gut route.

Background: Methods for washed microbiota transplantation (WMT) through the mid-gut include transendoscopic enteral tubing (TET) and manual spiral nasojejunal tube (SNT) placement have not been studied. Methods: This prospective interventional study was performed at a single centre. Patients were divided into the SNT and mid-gut TET groups based on their conditions and wishes. In the SNT group, an SNT was passively inserted into the stomach, and abdominal X-rays were taken within 24 h to confirm tube placement in the small intestine. In the mid-gut TET group, mid-gut TET was placed in the small intestine for gastroscopy. Data on the clinical efficacy of WMT, intubation time, cost, overall comfort score, adverse reactions, etc., were collected from the two groups. Results: Sixty-three patients were included in the study (SNT group (n = 40) and mid-gut TET group (n = 23)). The clinical efficacy of WMT in the SNT and mid-gut TET groups was 90 % and 95.7 %, respectively (P = 0.644). Compared with the mid-gut TET group, the SNT group showed a shorter operation time (120 s vs. 258 s, P = 0.001) and a lower average cost (641.7 yuan vs. 1702.1 yuan, P = 0.001). There was no significant difference in the overall comfort score or the incidence of common discomfort symptoms between the two groups. Conclusion: The different implantation methods have different advantages; compared with mid-gut TET placement, manual SNT placement provides some benefits.

The effect of childhood harshness and unpredictability on Internet addiction among college students: The mediating effect of self-control.

College students are inevitably online and at risk of becoming addicted. Life history theory provides an explanatory framework for individual differences in Internet addiction, and childhood harshness and unpredictability may be important antecedents. However, it is unclear whether and how childhood harshness and/or unpredictability affect Internet addiction during college. In this study, we recruited 483 Chinese college students and assessed their childhood harshness, unpredictability, self-control, and Internet addiction. The results of path analysis showed that childhood unpredictability was positively associated with Internet addiction among college students and was partially mediated by self-control. The effect of harshness on Internet addiction showed a suppression effect, i.e., the direct effect of harshness on Internet addiction was negative and the indirect effect through self-control was positive. This suggests that the high risk of Internet addiction stems from harshness and unpredictability in childhood, but that the effects of these factors are independent and distinct. Self-control plays an important role in this process, but many internal mechanisms remain to be tested in future research.

Injectable polyamide-amine dendrimer-crosslinked meloxicam-containing poly-γ-glutamic acid hydrogel for prevention of postoperative tissue adhesion through inhibiting inflammatory responses and balancing the fibrinolytic system.

Establishing a physical barrier between the peritoneum and the cecum is an effective method to reduce the risk of postoperative abdominal adhesions. Meloxicam (MX), a nonsteroidal anti-inflammatory drug has also been applied to prevent postoperative adhesions. However, its poor water solubility has led to low bioavailability. Herein, we developed an injectable hydrogel as a barrier and drug carrier for simultaneous postoperative adhesion prevention and treatment. A third-generation polyamide-amine dendrimer (G3) was exploited to dynamically combine with MX to increase the solubility and the bioavailability. The formed G3@MX was further used to crosslink with poly-γ-glutamic acid (γ-PGA) to prepare a hydrogel (GP@MX hydrogel) through the amide bonding. In vitro and in vivo experiments evidenced that the hydrogel had good biosafety and biodegradability. More importantly, the prepared hydrogel could control the release of MX, and the released MX is able to inhibit inflammatory responses and balance the fibrinolytic system in the injury tissues in vivo. The tunable rheological and mechanical properties (compressive moduli: from âˆ¼ 57.31 kPa to âˆ¼ 98.68 kPa;) and high anti-oxidant capacity (total free radical scavenging rate of âˆ¼ 94.56 %), in conjunction with their syringeability and biocompatibility, indicate possible opportunities for the development of advanced hydrogels for postoperative tissue adhesions management.

DNA Extraction and Comparison between Old and Fresh Necrophilic Fly Samples.

A total of five samples of Chrysomya megacephala samples - three fresh samples, one sample stored in alcohol for 2 years, and one sample stored in dry sealed storage for 2 years protected from light only - were selected to investigate whether a blood DNA extraction kit could extract DNA from necrophilous flies and to determine whether alcohol could prolong the preservation of necrophilous flies' DNA. First, the blood DNA extraction kit was used to extract DNA from their thorax tissues. Then, the DNA purity and concentration were examined using a microplate reader and a fluorometer. Finally, PCR amplification and electrophoresis of the extracted DNA were done with necrophilic fly-specific primers located in the mitochondrial CO I gene sequence. The results showed that the DNA purity of all samples was greater than 2.0. The DNA concentration was observed to be of the following order: fresh samples > alcohol-preserved old samples > untreated, old samples. All samples had specific electrophoretic bands after PCR amplification. In conclusion, a blood DNA extraction kit can be used to extract DNA from necrophilic flies successfully, and the DNA concentration of fresh fly samples is greater than that of old fly samples. The flies can be stored in alcohol for a long time.