Harnessing enhanced CRISPR/Cas12a trans-cleavage activity with extended reporters and reductants for early diagnosis of Helicobacter pylori, the causative agent of peptic ulcers and stomach cancer.

Developing rapid and non-invasive diagnostics for Helicobacter pylori (HP) is imperative to prevent associated diseases such as stomach gastritis, ulcers, and cancers. Owing to HP strain heterogeneity, not all HP-infected individuals incur side effects. Cytotoxin-associated gene A (CagA), and vacuolating cytotoxin A (VacA) genes predominantly drive HP pathogenicity. Therefore, diagnosing CagA and VacA genotypes could alert active infection and decide suitable therapeutics. We report an enhanced LbCas12a trans-cleavage activity with extended reporters and reductants (CEXTRAR) for early detection of HP. We demonstrate that extended ssDNA reporter acts as an excellent signal amplifier, making it a potential alternative substrate for LbCas12a collateral activity. Through a systematic investigation of various buffer components, we demonstrate that reductants improve LbCas12a trans-cleavage activity. Overall, our novel reporter and optimal buffer increased the trans-cleavage activity to an order of 16-fold, achieving picomolar sensitivity (171 pM) without target pre-amplification. Integrated with loop-mediated isothermal amplification (LAMP), CEXTRAR successfully attained attomolar sensitivity for HP detection using real-time fluorescence (43 and 96 aM), in-tube fluorescence readouts (430 and 960 aM), and lateral flow (4.3 and 9.6 aM) for CagA and VacA, respectively. We also demonstrate a rapid 2-min Triton X-100 lysis for clinical sample analysis, which could provide clinicians with actionable information for rapid diagnosis. CEXTRAR could potentially spot the 13C urea breath test false-negatives. For the first time, our study unveils an experimental outlook to manipulate reporters and reconsider precise cysteine substitution via protein engineering for Cas variants with enhanced catalytic activities for use in diagnostics and genetic engineering.

Transcription factor EB-mediated mesenchymal stem cell therapy induces autophagy and alleviates spinocerebellar ataxia type 3 defects in neuronal cells model.

Defects in ataxin-3 proteins and CAG repeat expansions in its coding gene ATXN3 cause Spinocerebellar Ataxia Type 3 (SCA3) or Machado-Joseph disease (MJD) polyglutamine neurodegenerative disease. The mutant proteins aggregate as inclusion bodies in cells and compete with wild-type ataxin-3, which leads to neuronal dysfunction or death and impairs Beclin1-mediated autophagy. It has been reported that Mesenchymal stem cells (MSCs) can reliably treat several neurodegenerative diseases. Herein, we used a Transcription Factor EB (TFEB) nuclear translocation-mediated MSCs co-culture approach to reconstitute autophagy and lysosomal biogenesis, and reduce SCA3-like behaviors in induced pluripotent stem cells (iPSCs)-derived neuron cells models. Our iPSCs model showed enhanced expression of autophagy proteins, attenuated the expression and toxic effects of mutant ataxin-3 on neurons, and alleviated the effects of ataxin-3 on autophagy. Therefore, MSCs are associated with autophagy-inducing therapy and compared to animal models, our MSCs co-culture could be used as a novel and potential therapeutic approach to study SCA3 disease and other neurodegenerative diseases.

Structure elucidation, biogenesis, and bioactivities of acylphloroglucinol-derived meroterpenoid enantiomers from Dryopteris crassirhizoma.

Twenty-four racemic acylphloroglucinol meroterpenoids including eighteen unusual stuctures (3 âˆ¼ 10, 13, 14, and 17 âˆ¼ 24), and a major component filixic acid ABA (25), were isolated from Dryopteris crassirhizoma. Structurally, the dimeric acylphloroglucinol derivatives possess unprecedented skeletons of mixed acylphloroglucinol and sesquiterpene biosynthetic origin. The stereochemistries of six reported meroterpenoids with undefined chiral centers were reassigned. Two intriguing methods by analyzing a) the regularity of chemical shift variation of protons and carbons around the stereogenic centers, and b) pyridine-induced deshielding effect of hydroxy groups, to discriminate relative configurations of flexible long-chain alcohol with chiral centers separated by three or seven covalent bonds, were successfully applied. A non-enzymatic biosynthesis of 1 âˆ¼ 24 was assumed based on a rare single-crystal cluster formed with two diastereomeric enantiomer pairs (±1/±2) and chiral HPLC analyses. Meroterpenoids 13 and 14 showed obvious inhibitory effects on NO production in LPS-induced RAW264.7, and suppressed the expression of iNOS, COX-2, IL-1ß, and IL-18. Their anti-inflammatory activity was closely related to the inhibition of the formation and function of inflammasomes. Additionally, the known 25 showed antiviral efficacy against the influenza viruse A/Puerto Rico/8/1934 (H1N1).

Hair follicle-derived mesenchymal stem cells decrease alopecia areata mouse hair loss and reduce inflammation around the hair follicle.

BACKGROUND: Alopecia areata (AA) is a common autoimmune hair loss disease with increasing incidence. Corticosteroids are the most widely used for hair loss treatment; however, long-term usage of hormonal drugs is associated with various side effects. Mesenchymal stem cells (MSCs) therapy has been studied extensively to curb autoimmune diseases without affecting immunity against diseases. METHODS: Hair follicle-derived MSCs (HF-MSCs) were harvested from the waste material of hair transplants, isolated and expanded. The therapeutic effect of HF-MSCs for AA treatment was investigated in vitro AA-like hair follicle organ model and in vivo C3H/HeJ AA mice model. RESULTS: AA-like hair follicle organ in vitro model was successfully established by pre-treatment of mouse vibrissa follicles by interferon-γ (IFN-γ). The AA-like symptoms were relieved when IFN-γ induced AA in vitro model was co-cultured with HF-MSC for 2 days. In addition, when skin grafted C3H/HeJ AA mice models were injected with 106 HF-MSCs once a week for 3 weeks, the transcription profiling and immunofluorescence analysis depicted that HF-MSCs treatment significantly decreased mouse hair loss and reduced inflammation around HF both in vitro and in vivo. CONCLUSIONS: This study provides a new therapeutic approach for alopecia areata based on HF-MSCs toward its future clinical application.

S100A4 plays a key role in TRPV3 ion channel expression and its electrophysiological function.

Transient receptor potential vanilloid 3 (TRPV3), a non-selective cation ion channel, is regulated by small molecules such as Ca2+ and calmodulin (CaM). Together with S100A4 (S100 calcium-binding protein family), is critical in cell proliferation and progression. Although TRPV3 has been proved to play a role in Ca2+ regulation and participate in Ca2+-related cellular processes, its molecular mechanism remains unclear. In this study, we found that TRPV3 and S100A4 were co-expressed in the same region of the cell, and surprisingly, the protein expression level of TRPV3 significantly increased with the overexpression of S100A4. Moreover, co-immunoprecipitation results showed that these two proteins could bind with each other. Functionally, we found that when S100A4 was simultaneously expressed in cells, more Ca2+ would be transferred into the cells through the TRPV3 ion channel. Consistent with Ca2+ regulation results, electrophysiological recordings demonstrated that S100A4 improved the function of TRPV3 in ions' flux, suggesting that the S100A4 could bind with TRPV3 and simultaneously promoted its expression, thus affecting its functions on related ions' flux. Our findings identified the link between S100A4 and TRPV3 and provided a novel molecular mechanism for TRPV3 regulation.

Generation of a tdTomato-GAD67 reporter human epilepsia mutation induced pluripotent stem cell line, USTCi001-A-2, using CRISPR/Cas9 editing.

Dravet syndrome is an epileptic encephalopathy largely due to haploinsufficiency of the voltage-gated sodium channel Nav1.1 that is expressed primarily in GABAergic neurons. In order to distinguish the different subtypes, we used gene editing to introduce tdTomato gene into the genome of iPSCs to label the GABAergic neurons in the differentiated neuronal networks. The gene-edited cell line demonstrates normal karyotype, expresses the main pluripotency markers, and shows the presence of differentiation into the three embryonic germ layers in teratomas.

Transient receptor potential ankyrin 1 contributes to the ATP-elicited oxidative stress and inflammation in THP-1-derived macrophage.

P2X7 receptor (P2X7R) is an ATP-gated non-selective cation channel which mediates ATP-induced inflammation in macrophages. Transient receptor potential (TRP) receptors are nociceptors in cellular membrane which can perceive the stimuli of environmental irritant. The interaction between TRP channels and P2X7R has been found while the details about inflammation are still unclear. In this study, we suggested that transient receptor potential ankyrin 1 (TRPA1), a member of TRP superfamily, participates in ATP-induced oxidative stress and inflammation in human acute monocytic leukemia cell line (THP-1)-derived macrophage. The co-localization between TRPA1 and P2X7R was detected using immunofluorescence in THP-1-derived macrophage and transfected human embryonic kidney cell line (HEK293T). The mechanism by which ATP or 3'-O-(4-Benzoylbenzoyl)-ATP (BzATP) induces the activation of macrophages was verified by calcium imaging, mitochondrial reactive oxygen species (mtROS) detection, mitochondrial membrane potential (∆Ψm) measurement, flow cytometry, enzyme-linked immunosorbent assay (ELISA), western blotting, CCK-8 assay, and the lactate dehydrogenase (LDH) release cytotoxic assay. The BzATP and ATP induced calcium overload, mitochondria injury, interleukin-1ß (IL-1ß) secretion, and cytotoxicity can be inhibited by TRPA1 antagonists. These results indicated that TRPA1 can co-localize with P2X7R and mediate ATP-induced oxidative stress and inflammation. Therefore, the inhibition of TRPA1 may provide a potential therapy for ATP-elicited inflammatory diseases, including atherosclerosis.

Transient Receptor Potential Ankyrin 1 Contributes to Lysophosphatidylcholine-Induced Intracellular Calcium Regulation and THP-1-Derived Macrophage Activation.

Lysophosphatidylcholine (LPC) is a major atherogenic lipid that stimulates an increase in mitochondrial reactive oxygen species (mtROS) and the release of cytokines under inflammasome activation. However, the potential receptors of LPC in macrophages are poorly understood. Members of the transient receptor potential (TRP) channel superfamily, which is crucially involved in transducing environmental irritant stimuli into nociceptor activity, are potential receptors of LPC. In this study, we investigated whether LPC can induce the activation of transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily. The functional expression of TRPA1 was first detected by quantitative real-time polymerase chain reaction (qRT-PCR), western blotting and calcium imaging in human acute monocytic leukemia cell line (THP-1)-derived macrophages. The mechanism by which LPC induces the activation of macrophages through TRPA1 was verified by cytoplasmic and mitochondrial calcium imaging, mtROS detection, a JC-1 assay, enzyme-linked immunosorbent assay, the CCK-8 assay and the lactate dehydrogenase (LDH) cytotoxic assay. LPC induced the activation of THP-1-derived macrophages via calcium influx, and this activation was suppressed by potent and selective inhibitors of TRPA1. These results indicated that TRPA1 can mediate mtROS generation, mitochondrial membrane depolarization, the secretion of IL-1ß and cytotoxicity through cellular and mitochondrial Ca2+ influx in LPC-treated THP-1-derived macrophages. Therefore, the inhibition of TRPA1 may protect THP-1-derived macrophages against LPC-induced injury.

Recurrent activations of transient receptor potential vanilloid-1 and vanilloid-4 promote cellular proliferation and migration in esophageal squamous cell carcinoma cells.

Some members of the transient receptor potential vanilloid (TRPV) subfamily of cation channels are thermosensitive. Earlier studies have revealed the distribution and functions of these thermo-TRPVs (TRPV1-4) in various organs, but their expression and function in the human esophagus are not fully understood. Here, we probed for the expression of the thermo-TRPVs in one nontumor human esophageal squamous cell line and two esophageal squamous cell carcinoma (ESCC) cell lines. TRPV1, TRPV2, and TRPV4 proteins were found to be upregulated in ESCC cells, while TRPV3 was not detectable in any of these cell lines. Subsequently, channel function was evaluated via monitoring of Ca2+ transients by Ca2+ imaging and nonselective cation channel currents were recorded by whole-cell patch clamp. We found that TRPV4 was activated by heat at 28 °C-35 °C, whereas TRPV1 and TRPV2 were activated by higher, noxious temperatures (44 °C and 53 °C, respectively). Furthermore, TRPV1 was activated by capsaicin (EC 50 = 20.32 µm), and this effect was antagonized by AMG9810; TRPV2 was activated by a newly developed cannabinoid compound, O1821, and inhibited by tranilast. In addition, TRPV4 was activated by hypotonic solutions (220 m Osm), and this effect was abolished by ruthenium red. The effects of TRPV1 and TRPV4 on ESCC were also explored. Our data, for the first time, showed that the overactivation of TRPV1 and TRPV4 promoted the proliferation and/or migration of ESCC cells. In summary, TRPV1, TRPV2, and TRPV4 were functionally expressed in human esophageal squamous cells, and thermo-TRPVs might play an important role in the development of ESCC.

γ-secretase binding sites in aged and Alzheimer's disease human cerebrum: the choroid plexus as a putative origin of CSF Aß.

Deposition of ß -amyloid (Aß) peptides, cleavage products of ß-amyloid precursor protein (APP) by ß-secretase-1 (BACE1) and γ-secretase, is a neuropathological hallmark of Alzheimer's disease (AD). γ-Secretase inhibition is a therapeutical anti-Aß approach, although changes in the enzyme's activity in AD brain are unclear. Cerebrospinal fluid (CSF) Aß peptides are thought to derive from brain parenchyma and thus may serve as biomarkers for assessing cerebral amyloidosis and anti-Aß efficacy. The present study compared active γ-secretase binding sites with Aß deposition in aged and AD human cerebrum, and explored the possibility of Aß production and secretion by the choroid plexus (CP). The specific binding density of [(3) H]-L-685,458, a radiolabeled high-affinity γ-secretase inhibitor, in the temporal neocortex and hippocampal formation was similar for AD and control cases with similar ages and post-mortem delays. The CP in post-mortem samples exhibited exceptionally high [(3) H]-L-685,458 binding density, with the estimated maximal binding sites (Bmax) reduced in the AD relative to control groups. Surgically resected human CP exhibited APP, BACE1 and presenilin-1 immunoreactivity, and ß-site APP cleavage enzymatic activity. In primary culture, human CP cells also expressed these amyloidogenic proteins and released Aß40 and Aß42 into the medium. Overall, our results suggest that γ-secretase activity appears unaltered in the cerebrum in AD and is not correlated with regional amyloid plaque pathology. The CP appears to be a previously unrecognised non-neuronal contributor to CSF Aß, probably at reduced levels in AD.