Development of a Rapid Epstein-Barr Virus Detection System Based on Recombinase Polymerase Amplification and a Lateral Flow Assay.

The quality of cellular products used in biological research can directly impact the ability to obtain accurate results. Epstein-Barr virus (EBV) is a latent virus that spreads extensively worldwide, and cell lines used in experiments may carry EBV and pose an infection risk. The presence of EBV in a single cell line can contaminate other cell lines used in the same laboratory, affecting experimental results. We developed three EBV detection systems: (1) a polymerase chain reaction (PCR)-based detection system, (2) a recombinase polymerase amplification (RPA)-based detection system, and (3) a combined RPA-lateral flow assay (LFA) detection system. The minimum EBV detection limits were 1 × 103 copy numbers for the RPA-based and RPA-LFA systems and 1 × 104 copy numbers for the PCR-based system. Both the PCR and RPA detection systems were applied to 192 cell lines, and the results were consistent with those obtained by the EBV assay methods specified in the pharmaceutical industry standards of the People's Republic of China. A total of 10 EBV-positive cell lines were identified. The combined RPA-LFA system is simple to operate, allowing for rapid result visualization. This system can be implemented in laboratories and cell banks as part of a daily quality control strategy to ensure cell quality and experimental safety and may represent a potential new technique for the rapid detection of EBV in clinical samples.

Investigation of the mixed origins of the MGC-803 cell line reveals that it is a hybrid cell line derived from HeLa.

Human cancer cell lines have an essential role in cancer research, but only authentic cell lines should be used as biological models. Authentication testing using short tandem repeat (STR) loci has shown that MGC-803 cells, which were reported to come from gastric adenocarcinoma, are similar to HeLa. In this study, we confirmed that the MGC-803 cell line contains genetic material from HeLa, including genetic sequence from human papilloma virus 18 (HPV18). Additional alleles were present on STR analysis that remained stable after extensive passaging and generation of mono-clones. This behavior is consistent with a hybrid cell line arising from cell-cell fusion. Further genetic analysis revealed that MGC-803 originated from donors with different genetic ancestries, one African (HeLa) and the other Asian. Transcriptomic analysis demonstrated that MGC-803 closely resembles HeLa and another nasopharyngeal-HeLa hybrid cell line CNE-2. Based on these findings, we conclude that MGC-803 is a hybrid cell line derived from HeLa and other cells, the latter derived from a different patient with Asian genetic ancestry.

A novel protocol to derive cervical motor neurons from induced pluripotent stem cells for amyotrophic lateral sclerosis.

Sporadic amyotrophic lateral sclerosis (sALS) is the majority of ALS, and the lack of appropriate disease models has hindered its research. Induced pluripotent stem cell (iPSC) technology now permits derivation of iPSCs from somatic cells of sALS patients to investigate disease phenotypes and mechanisms. Most existing differentiation protocols are time-consuming or low efficient in generating motor neurons (MNs). Here we report a rapid and simple protocol to differentiate MNs in monolayer culture using small molecules, which led to nearly pure neural stem cells in 6 days, robust OLIG2+ pMNs (73%-91%) in 12 days, enriched CHAT+ cervical spinal MNs (sMNs) (88%-97%) in 18 days, and functionally mature sMNs in 28 days. This simple and reproducible protocol permitted the identification of hyperexcitability phenotypes in our sALS iPSC-derived sMNs, and its application in neurodegenerative diseases should facilitate in vitro disease modeling, drug screening, and the development of cell therapy.

BACH1 encourages ferroptosis by activating KDM4C-mediated COX2 demethylation after cerebral ischemia-reperfusion injury.

It has been confirmed that BTB domain and CNC homologue 1 (BACH1) are involved in ferroptosis-related diseases. However, the function of BACH1 in cerebral ischemia-reperfusion injury (CIRI)-induced ferroptosis remains to be largely unrevealed. First, analysis of differentially expressed genes in CIRI based on the GEO dataset GSE119121 revealed that BACH1 was upregulated in CIRI. BACH1 level was prominently increased in middle cerebral artery occlusion (MCAO)/reperfusion model and oxygen-glucose deprivation/reoxygenation cell model. Further, knock-down of BACH1 markedly reduced iron ion concentration, ROS production, 4-HNE and lipid peroxidation levels and facilitated GSH content, cell viability and protein levels of GPX4 and SLC7A11, while an pcDNA-KDM4C or pcDNA-COX2 combined with BACH1 siRNA could not enhance this effect. Mechanistically, BACH1 bound on the KDM4C promoter to transcriptionally activate its expression. Besides, KDM4C could occupy the promoter locus of the COX2 gene, promoting the COX2 expression by eliminating H3K9me3. Overexpression of KDM4C or COX2 overturned the effects of BACH1 inhibition. In vivo findings displayed that brain infraction, pathological damage and neuronal loss rate in MCAO mice were conspicuously decreased after BACH1 knock-down. This study reveals that BACH1 encourages ferroptosis in neuroblastoma cells and CIRI mouse brain tissues by activating KDM4C-mediated COX2 demethylation.

Nasal mucosal care before and after nasal endoscopic surgery based on computational information system resource sharing technology.

This study presents an in-depth analysis on a machine-designed computational web-based information system, which was used to conduct nasal mucosal care before and after nasal endoscopic surgery for chronic sinusitis. The system was developed and implemented using the mainstream B/S structure model with a Java development framework and MySQL database. Sinus irrigation solution has been shown to be effective for postoperative flushing after nasal endoscopy, by eliminating mucosal edema and promoting mucosal epithelialization at the operative cavity, and it is currently a desirable method that deserves promotion. By comparing the time required for surgical cavity cleaning, the rinsing solution was shown to be key of the physical flushing effect in the initial period after nasal endoscopy. It could remove blood cemented and surgical cavity surface cemented skin and secretions. In addition, the sinus irrigation solution can accelerate the mucosal epithelialization of the operative cavity more effectively than compounded saline. It could effectively eliminate mucosal edema, restore its protective and defensive functions, and help local blood circulation, secretion absorption, mucosal growth, mucosal regeneration and repair, and mucus cilia removal.

A silver lining in cell line authentication: Short tandem repeat analysis of 1373 cases in China from 2010 to 2019.

Continuous cell lines are practical models that are widely used in the study of disease mechanisms and particularly cancers. However, the issue of cell line cross-contamination has existed since the 1960s, despite repeated advocation for cell line authentication by many experts. Furthermore, cell line abuse has been underestimated and underreported. The China Center for Type Culture Collection (CCTCC) received 1373 cell samples for authentication from 2010 to 2019, and has found that the quality of cell lines has improved during this time, offering a positive outlook for the future.

Generation and characterization of three induced pluripotent stem cell lines (NUIGi046-A, NUIGi046-B, NUIGi046-C) from a 51-year-old healthy individual.

Skin punch biopsy was donated by a healthy 51-year-old Caucasian male and the dermal fibroblasts were reprogrammed into human induced pluripotent stem cell (hiPSC) lines by using non-integrative Sendai viruses expressing OCT4, SOX2, KLF4 and c-MYC. Three iPSC lines (NUIGi046-A, NUIGi046-B, NUIGi046-C) highly expressed the pluripotent markers and were capable of differentiating into cells of endodermal, mesodermal, and ectodermal origin. These iPSCs can be offered as controls and in combination with genome-editing and three-dimensional (3D) system. They may be used for human disease modelling and drug screening.

Advantages of a 21-loci short tandem repeat method for detection of cross-contamination in human cell lines.

Cross-contamination of cell lines is a highly relevant and pervasive problem. The analysis of short tandem repeats (STR) is a simple and commercially available technique to authenticate cell lines for more than two decades. At present, STR multiple amplification kits have been developed up to 21 loci while the current STR databases only provide 9-loci STR profiles. Here, we compared the advantages of 21-loci STR methodology using the same algorithm as 9-loci method. The 21-loci method reduced the uncertainty ratio for authentications by 97.5% relative to the 9-loci method and exclude effectively false positive. We show that the additional 12 loci helped to greatly reduce sample-site marker specificity arising from genetic isolation and the occurrence of null alleles, suggesting that inclusion of additional loci in these databases will ultimately improve the efficiency and accuracy of authentication of cell lines. Taken together, we demonstrate the utility of a 21-loci method in human cells, providing a novel marker panel for use as a valuable alternative to 9-loci analyses to minimize cell line authentication errors and reduce costs due to erroneous experiments.

Agonist-specific voltage-dependent gating of lysosomal two-pore Na+ channels.

Mammalian two-pore-channels (TPC1, 2; TPCN1, TPCN2) are ubiquitously- expressed, PI(3,5)P2-activated, Na+-selective channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and Ebola viral infection. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of the presumed 'S4 voltage-sensing' domains. By performing high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tricyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, referred to as Lysosomal Na+ channel Voltage-dependent Activators (LyNa-VAs). We also identified another compound which, like PI(3,5)P2, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal functions.

Rapamycin directly activates lysosomal mucolipin TRP channels independent of mTOR.

Rapamycin (Rap) and its derivatives, called rapalogs, are being explored in clinical trials targeting cancer and neurodegeneration. The underlying mechanisms of Rap actions, however, are not well understood. Mechanistic target of rapamycin (mTOR), a lysosome-localized protein kinase that acts as a critical regulator of cellular growth, is believed to mediate most Rap actions. Here, we identified mucolipin 1 (transient receptor potential channel mucolipin 1 [TRPML1], also known as MCOLN1), the principle Ca2+ release channel in the lysosome, as another direct target of Rap. Patch-clamping of isolated lysosomal membranes showed that micromolar concentrations of Rap and some rapalogs activated lysosomal TRPML1 directly and specifically. Pharmacological inhibition or genetic inactivation of mTOR failed to mimic the Rap effect. In vitro binding assays revealed that Rap bound directly to purified TRPML1 proteins with a micromolar affinity. In both healthy and disease human fibroblasts, Rap and rapalogs induced autophagic flux via nuclear translocation of transcription factor EB (TFEB). However, such effects were abolished in TRPML1-deficient cells or by TRPML1 inhibitors. Hence, Rap and rapalogs promote autophagy via a TRPML1-dependent mechanism. Given the demonstrated roles of TRPML1 and TFEB in cellular clearance, we propose that lysosomal TRPML1 may contribute a significant portion to the in vivo neuroprotective and anti-aging effects of Rap via an augmentation of autophagy and lysosomal biogenesis.

Prognosis of Symptoms and Complications After Microvascular Decompression for Hemifacial Spasm: A Single-Center Experience.

OBJECTIVE: The aim of this study was to identify potential prognostic factors of hemifacial spasm (HFS) after microvascular decompression (MVD), to establish the appropriate way to tackle postprocedure symptoms and complications (PPSCs), and to find the incidence and duration of PPSCs. METHODS: Two hundred and forty-eight patients with HFS were monitored between December 2009 and December 2014. The mean follow-up duration was 24 months (range, 6-67 months). We divided patients based on their PPSC status and investigated the following factors: age, sex, spasm side, facial nerve block before MVD (botulinum toxin treatment), acupuncture before MVD, duration of HFS, hypertension, diabetes, hepatitis B virus (HBV) infection status, herpes simplex virus infection status, smoking status and alcohol use, offending vessels, Chiari malformation, electrophysiologic monitoring results, and postoperative HFS. Univariable analysis and multivariate logistic regression were used to find potential risk factors. Kaplan-Meier analysis was used to show the duration of postprocedure facial palsy. RESULTS: Age (odds ratio [OR], 1.037; 95% confidence interval [CI], 1.004-1.072; P = 0.03) and HBV status (OR, 18.256; 95% CI, 2.723-122.415; P = 0.03) were positive predictors of PPSCs. Postoperative HFS (OR, 0.249; 95% CI, 0.084-0.0739; P = 0.012) may be a protective factor for postprocedure facial palsy. Most PPSCs related to cranial nerves recovered spontaneously in 3 months. Infections and cerebrospinal fluid leakages were controlled by medical intervention in 1-2 weeks. The permanent complication rate was only 4.8%. CONCLUSIONS: Although the incidence of PPSCs after MVD is very high, most PPSCs related to cranial nerves recovered spontaneously in several days. Permanent complications after MVD for HFS are rare. Age may relate to the occurrence of PPSCs, and postoperative HFS may be a protective factor for patients with facial palsy after MVD.

Streptozotocin inhibits synaptic transmission and edaravone attenuates streptozotocin-induced electrophysiological changes in CA1 pyramidal neurons of rat hippocampal slices.

The purpose of this study was to investigate the acute and chronic effects of streptozotocin (STZ) upon synaptic transmission and the effects of edaravone (EDA, a free radical scavenger) on STZ-induced electrophysiological changes in CA1 pyramidal neurons of rat hippocampal slices. To accomplish this goal, spontaneous excitatory postsynaptic current (sEPSC), miniature excitatory postsynaptic current (mEPSC), spontaneous inhibitory postsynaptic current (sIPSC) and miniature inhibitory postsynaptic current (mIPSC) were recorded within hippocampal slices using whole-cell patch clamp techniques. The results showed that the amplitudes and frequencies of sEPSC, mEPSC, sIPSC and mIPSC were inhibited by 1000µM STZ, while treatment of EDA (1000µM) attenuated these STZ-induced changes. The degree of these neurotoxic effects of STZ and effects of EDA increased as a function of drug duration as assessed at 2, 4 or 8h of exposure. Taken together, our results demonstrate that STZ induces neurotoxicity within these hippocampal slices through its capacity to alter synaptic transmission and these STZ-induced alterations in electrophysiological responses are attenuated by EDA.

Up-regulation of lysosomal TRPML1 channels is essential for lysosomal adaptation to nutrient starvation.

Upon nutrient starvation, autophagy digests unwanted cellular components to generate catabolites that are required for housekeeping biosynthesis processes. A complete execution of autophagy demands an enhancement in lysosome function and biogenesis to match the increase in autophagosome formation. Here, we report that mucolipin-1 (also known as TRPML1 or ML1), a Ca(2+) channel in the lysosome that regulates many aspects of lysosomal trafficking, plays a central role in this quality-control process. By using Ca(2+) imaging and whole-lysosome patch clamping, lysosomal Ca(2+) release and ML1 currents were detected within hours of nutrient starvation and were potently up-regulated. In contrast, lysosomal Na(+)-selective currents were not up-regulated. Inhibition of mammalian target of rapamycin (mTOR) or activation of transcription factor EB (TFEB) mimicked a starvation effect in fed cells. The starvation effect also included an increase in lysosomal proteostasis and enhanced clearance of lysosomal storage, including cholesterol accumulation in Niemann-Pick disease type C (NPC) cells. However, this effect was not observed when ML1 was pharmacologically inhibited or genetically deleted. Furthermore, overexpression of ML1 mimicked the starvation effect. Hence, lysosomal adaptation to environmental cues such as nutrient levels requires mTOR/TFEB-dependent, lysosome-to-nucleus regulation of lysosomal ML1 channels and Ca(2+) signaling.

Galantamine inhibits calpain-calcineurin signaling activated by beta-amyloid in human neuroblastoma SH-SY5Y cells.

Galantamine, which is currently used in the treatment of patients with Alzheimer's disease (AD), has been shown to have a neuroprotective effect against beta-amyloid (Abeta) peptide-induced toxicity, which is involved in the pathogenesis of AD. In this study, we investigated the mechanism underlying the protective effect of galantamine on Abeta-induced toxicity in human neuroblastoma cells (SH-SY5Y). Using MTT and LDH leakage assays, we observed that galantamine pretreatment significantly prevented Abeta1-40-induced cell death. Abeta1-40-induced overexpression and increased cleavage of both calpain and calcineurin were observed by Western blotting and double immunofluorescent staining. Increased calcineurin phosphatase activity and decreased level of pSer112 BAD were also observed in Abeta1-40-damaged cells. However, all these alterations were found to be reversed by galantamine pretreatment. We also found that the neuroprotection of galantamine can be blocked by an alpha7 nAChR antagonist. Overall, our results suggest that galantamine may prevent the neuronal damage induced by Abeta1-40 through a mechanism related to the regulation of calpain-calcineurin activation and BAD phosphorylation, which may involve the participation of alpha7 nAChR.

Eosinophilia in very low birth weight infants.

BACKGROUND: Eosinophilia is common in premature infants, though its clinical significance remains unknown. This study investigated the pattern of eosinophilia and related factors in very low birth-weight (VLBW) infants. METHODS: The medical records of VLBW infants (birth body weight < 1500 g) admitted to the neonatal intensive care unit of a tertiary care center of Cheng Kung University Hospital between January 2005 and June 2007 were analyzed. Complete blood counts (CBC) with differential leukocyte counts were performed weekly. Eosinophilia was defined as an eosinophil count of more than 0.700 x 10(9)/L. The possible related factors were analyzed. RESULTS: A total of 142 infants were recruited into the study. Those who did not survive after the first 28 days and those with less than four available CBCs were excluded, leaving 107 infants and 828 CBC measurements. Overall, 19.0% of CBCs (157/828) indicated eosinophilia and 69.0% of all infants had at least one instance of eosinophilia during their hospital stay. Eosinophilia mainly occurred in the third week of life (27.1%), with an average peak eosinophil count of 0.520 x 10(9)/L. There were 37.3% of patients with mild eosinophilia (0.700-0.999 x 10(9)/L), 50.7% with moderate eosinophilia (1.000-2.999 x 10(9)/L), and 12% with severe eosinophilia (> or =3.000 x 10(9)/L). The demographic data and perinatal characteristics of infants with and without eosinophilia were comparable. Medical treatments including mechanical ventilation, antibiotic administration, total parenteral nutrition, intravenous catheterization, transfusion, and body weight gains were similar between the two groups. The eosinophil counts in the first week of life were significantly higher in infants with bronchopulmonary dysplasia (p < 0.05). They were also greater in VLBW infants with sepsis at the first, the third, the fourth, the fifth and the seventh weeks (p < 0.05). CONCLUSION: Eosinophilia is common in VLBW infants and occurs mainly in the third week of life. Higher eosinophil counts were associated with sepsis and family history of atopic eczema. Bronchopulmonary dysplasia was associated with higher eosinophil counts in the first week of life.