Fluorescent Spots Research Articles

α-Lipoic acid improves mitochondrial biogenesis and dynamics by enhancing antioxidant and inhibiting Wnt/Ca2+ pathway to relieve fluoride-induced hepatotoxic injury

Fluoride (F), widely present in water and food, poses a serious threat to liver health, and oxidative damage and mitochondrial damage are its main causes. As a natural mitochondrial protector and antioxidant, α-lipoic acid (ALA)'s alleviating effect on fluorosis liver injury and its underlying mechanism are still unclear. Therefore, this study established a fluorosis ALA intervention mice model to explore the mechanism of mitochondrial biogenesis, mitochondrial dynamics, and Wnt/Ca2+ pathway in ALA attenuating fluorosis liver injury. The results showed that ALA mitigated F-induced weight loss, hepatic structural and functional damage, hepatocytes mitochondrial damage, and decreased antioxidant levels. However, ALA did not reduce F accumulation in the femur. Further mRNA and protein detection results showed that F increased the expression levels of key genes in the mitochondrial fission (Drp1, Mff, and Fis1), mitophagy (Parkin, Pink1, and Prdx3), Wnt/Ca2+ pathway (Wnt5a and CaMK2), and rised the number and intensity of fluorescent spots of Drp1, but decreased the expression levels of key genes in the mitochondrial biogenesis (Sirt1, Sirt3, and PGC-1α) and fusion (OPA1, Mfn2, and Mfn1), and reduced the number and intensity of fluorescent spots of PGC-1α in the liver. However, the intervention of ALA relieved the F-induced changes in the expressions of the above genes. In conclusion, ALA mitigated F-induced hepatic injury through enhancing antioxidant capacity and inhibiting Wnt/Ca2+ pathway to improve mitochondrial biogenesis and dynamics disturbance. This study further reveals the hepatotoxic mechanism of F and the protective mechanism of ALA, and provides a theoretical basis for ALA as a potential preventive and palliative agent for F-induced hepatotoxic injury.

Characterization of metal components in white-based polyester single fibers by X-ray fluorescence spectrometry and X-ray absorption fine structure analysis utilizing synchrotron radiation for forensic discrimination

Discrimination of single fibers is important in forensic investigation. Conventional microscopic observation and analytical methods such as micro-Fourier transform-infrared spectroscopy, a suitable technique for polymer component identification, can discriminate single white fibers made from different polymers. Polyesters contain metal compounds derived from polymerization catalysts, transesterification catalysts, and matting agents. Here we propose a forensic method for discriminating white-based polyester single fibers. The X-ray intensity ratios, distribution of metal elements, and chemical states are identified by semi-microbeam X-ray fluorescence (XRF) spot analysis, nanobeam XRF imaging, and X-ray absorption fine structure analysis (XAFS) utilizing synchrotron radiation, respectively. The analytical specimens were white-based polyester single fibers from the standard forensic fiber collection (Microtrace LLC, USA) and commercially available polyester products. Semi-microbeam XRF spot analysis detected metallic elements such as Ti, Mn, Co, Ge, and Sb derived from polymerization catalysts, transesterification catalysts, and matting agents. The X-ray intensity ratios of Ti/Mn, Ti/Ge, Ti/Sb, and Mn/Sb were obtained with good reproducibility but those of Ti/Co, Mn/Co, and Co/Sb, which are related to cobalt, were lowly reproducible in some samples. The poor reproducibility of the cobalt-related X-ray intensity ratios was attributed to uneven distributions of the metallic elements in the polyester single fibers, as visualized by nanobeam XRF imaging. The images showing elemental heterogeneity have proven to be good markers for identification. The XAFS results showed that three types of Ti compounds and two types of cobalt compounds are utilized in different applications of polyester single fibers; moreover, the chemical states are useful indices of single-fiber discrimination.

EGFR-targeted fluorescence molecular imaging for intraoperative margin assessment in oral cancer patients: a phase II trial

Inadequate surgical margins occur frequently in oral squamous cell carcinoma surgery. Fluorescence molecular imaging (FMI) has been explored for intraoperative margin assessment, but data are limited to phase-I studies. In this single-arm phase-II study (NCT03134846), our primary endpoints were to determine the sensitivity, specificity and positive predictive value of cetuximab-800CW for tumor-positive margins detection. Secondary endpoints were safety, close margin detection rate and intrinsic cetuximab-800CW fluorescence. In 65 patients with 66 tumors, cetuximab-800CW was well-tolerated. Fluorescent spots identified in the surgical margin with signal-to-background ratios (SBR) of ≥2 identify tumor-positive margins with 100% sensitivity, 85.9% specificity, 58.3% positive predictive value, and 100% negative predictive value. An SBR of ≥1.5 identifies close margins with 70.3% sensitivity, 76.1% specificity, 60.5% positive predictive value, and 83.1% negative predictive value. Performing frozen section analysis aimed at the fluorescent spots with an SBR of ≥1.5 enables safe, intraoperative adjustment of surgical margins.

Performance Comparison Between Conventional Fluorescent Spot Test and Quantitative Assay in Detecting G6PD Deficiency in Neonates.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy worldwide. The fluorescent spot test (FST) is the conventional method for screening neonates for G6PD. However, it has limitations and quantitative assays such as the CareStart Biosensor 1 are being increasingly recommended. This study aimed to compare FST and CareStart Bioensor 1 in their ability to detect G6PD levels in neonates. This cross-sectional study involved 455 neonates between June and December 2020. Two milliliters of cord blood were analyzed with CareStart Biosensor 1 and dried cord blood spots with FST. Data was recorded and statistically analyzed. Sensitivity, specificity, positive predictive value, and negative predictive value were calculated to determine the performance of FST at specific G6PD cut-off values; Cohen's kappa analysis assessed the agreement between the two methods. The sensitivity of FST at 30% cut-off G6PD activity level was 91.0%, (95% CI: 57.0-100) and specificity of 97.0% (95% CI: 95.0-98.0). At 60% cut-off, the FST sensitivity sharply declined to 29.0% (95% CI: 19.0-40.0) with a specificity of 100% (95% CI: 98.0-100). The overall prevalence of G6PD deficiency was 5.1% as measured by FST and 17.8% by Biosensor 1 (p < 0.001). In this study, FST missed a significant proportion of cases of intermediate G6PD levels. FST also misclassified several G6PD intermediate individuals as normal, rendering them susceptible to oxidative stress. Biosensor 1 reported a significantly higher prevalence of G6PD deficiency.

Difference in Intestine Content of Caenorhabditis elegans When Fed on Non-Pathogenic or Pathogenic Bacteria.

We investigated the bacterial food digestion and accumulation in wild-type adult Caenorhabditis elegans (C. elegans) worms that have fed on either non-pathogenic RFP-expressing Escherichia coli (E. coli) OP50 or pathogenic-RFP-expressing Pseudomonas aeruginosa (P. aeruginosa) PAO1 during the first 4 days of adulthood. Once the worms had completed their planned feeding cycles, they were loaded on microfluidic chips, where they were fixed to allow high-resolution z-stack fluorescence imaging of their intestines utilizing a Spinning Disk Confocal Microscope (SDCM) equipped with a high-resolution oil-immersion objective (60×). IMARIS software was used to visualize and analyze the obtained images, resulting in the production of three-dimensional constructs of the intestinal bacterial load. We discovered two distinct patterns for the bacteria-derived fluorescence signal in the intestine: (i) individual fluorescent spots, originating from intact bacteria, were present in the fluorescent E. coli-OP50-fed worms, and (ii) individual fluorescent spots (originating from intact bacteria) were dispersed in large regions of diffuse fluorescence (RDF), originating from disrupted bacteria, in fluorescent P. aeruginosa-PAO1-fed worms. We performed a semi-automated single-worm-resolution quantitative analysis of the intestinal bacterial load, which showed that the intestinal bacterial load generally increases with age of the worms, but more rapidly for the fluorescent P. aeruginosa-PAO1-fed worms.

Investigating the Prevalence of Glucose-6-Phosphate Dehydrogenase (G6PD) Deficiency among Patients with Favism Symptoms in Kerman City, Southern Iran

Background and Aims: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is humans’ most common erythrocyte enzyme defect. About 400 million people are estimated to be affected by this disorder worldwide. Antimalarial drugs, especially primaquine, and other oxidative stress, can cause hemolytic complications in G6PD deficient individuals. This study aimed to evaluate the prevalence of G6PD deficiency in Kerman City in southern Iran.&#x0D; Materials and Methods: This descriptive cross-sectional study was conducted from 2016 to 2021. Blood samples were taken from all patients with symptoms of G6PD deficiency who were referred to a general hospital in Kerman City in southern Iran. The G6PD enzyme activity was measured qualitatively by fluorescent spot test.&#x0D; Results: A total of 6369 patients were included in this study. G6PD deficiency was seen in 424 (6.7%) subjects. Of 424 patients, 359 (84.7%) were severely G6PD deficient, and 65 (15.3%) patients exhibited partial deficiency. G6PD deficiency was seen in 324 (9.3%) males and 100 (3.4%) females (p &lt; 0.001).&#x0D; Conclusion: The results of our study confirmed the existence of G6PD deficiency in a significant percentage of patients in Kerman City. Therefore, many people in this city are exposed to hemolytic complications if they use antimalarial drugs and other oxidative substances. According to our results, testing G6PD deficiency and monitoring the potential primaquine toxicity in patients who receive primaquine are highly recommended.

Whole-mount smFISH allows combining RNA and protein quantification at cellular and subcellular resolution

Multicellular organisms result from complex developmental processes largely orchestrated through the quantitative spatiotemporal regulation of gene expression. Yet, obtaining absolute counts of messenger RNAs at a three-dimensional resolution remains challenging, especially in plants, owing to high levels of tissue autofluorescence that prevent the detection of diffraction-limited fluorescent spots. In situ hybridization methods based on amplification cycles have recently emerged, but they are laborious and often lead to quantification biases. In this article, we present a simple method based on single-molecule RNA fluorescence in situ hybridization to visualize and count the number of mRNA molecules in several intact plant tissues. In addition, with the use of fluorescent protein reporters, our method also enables simultaneous detection of mRNA and protein quantity, as well as subcellular distribution, in single cells. With this method, research in plants can now fully explore the benefits of the quantitative analysis of transcription and protein levels at cellular and subcellular resolution in plant tissues.

Infection tracing and organ tropism of Siniperca chuatsi rhabdovirus expressing enhanced green fluorescent protein

Siniperca chuatsi Rhabdovirus (SCRV) is one of the important pathogens that infect mandarin fish. Recovery of recombinant viruses expressing fluorescent protein is an important method for virology research. In this study, the enhanced green fluorescent protein (EGFP) gene was inserted between the leader and the N gene of the SCRV genome. The recombinant SCRV of rS3-LeNEGFP expressing the EGFP was rescued, and the virus titer was as high as 2 × 106 PFU/mL in EPC cells. The insertion of the EGFP gene in the genome of SCRV was stable. EGFP was expressed efficiently after 10 passages in EPC cells. In vitro growth characterization, pathogenicity and organ tropism in vivo were studied. Compared to the parental rS3, rS3-LeNEGFP was defective in vitro, but not attenuated in vivo. The mortality of red zebrafish caused by rS3-LeNEGFP was the same as the parental SCRV rS3. The earliest expression of EGFP was detectable at 2 h, and clear fluorescence spots appeared at 8 h post-infection in EPC cells. The liver, heart, and brain were the major organ targets of rS3-LeNEGFP in vivo, and the kidney, gill, and spermatocyte were also invaded. The organ infection in vitro was different from that in vivo. This study is of great significance for revealing the pathogenesis of SCRV and developing vaccines and antiviral drugs for SCRV in the future.

131 Alveolar Macrophage Degranulation Initiates the Spatially Restricted Innate Immune Response During Ventilator-Assisted Nanoparticle Inhalation

Abstract To visualize and measure in real-time the cellular pulmonary innate immune response elicited by inhaled NPs, we apply state-of-the-art intravital microscopy on the alveolar region of the murine lung. Fluorescent carboxyl-quantum-dot-NPs (deposited dose of 16 cm2/g) applied via ventilator-assisted inhalation accumulated within seconds as distinct fluorescent spots at the alveolar walls. Already at 30min, cQD-NPs elicited an increase in microvascular neutrophil numbers as compared to controls, which subsequently transmigrated into the alveolar space. This early immune response was not specific to cQD-NPs, since a comparable increase in neutrophil numbers was also observed upon inhalation of a bioequivalent dose of carbon black NPs, a typical component of urban air pollution. AMs actively accumulated cQD-NPs over time, starting few minutes after inhalation. Significantly more neutrophils were attracted in proximity to cQD-NP-laden AMs, as compared to NP-free AMs or control areas with epithelial associated cQD-NP. Prior airway application of a cellular degranulation inhibitor prevented cQD-NP and CNP induced neutrophil recruitment at 2h and 24 h after inhalation. Inhibition of Fcγ-receptor mediated AM cQD-NP uptake by prior application of anti-CD64 mAbs or neutralizing of TNFα by anti-TNFα mAbs in the airspace, abolished this early immune response. Importantly, initial ImageStream flow cytometry analysis of BAL samples indicated an increase of macrophage-derived microvesicles upon NP inhalation. Taken together, our data links NP-induced AM degranulation and cytokine release to the rapid and site-specific recruitment of neutrophils, suggesting this process to be a key event in mounting the immune response upon NP inhalation.

Rapid selection of potyviral cross-protection effective mutants from the local lesion host after nitrous acid mutagenesis.

Zucchini yellow mosaic virus (ZYMV) seriously damages cucurbits worldwide. Control of ZYMV by cross-protection has been practised for decades, but selecting useful mild viruses is time-consuming and laborious. Most attenuated potyviruses used for cross-protection do not induce hypersensitive reaction (HR) in Chenopodium quinoa, alocal lesion host Chenopodium quinoa. Here, severe ZYMV TW-TN3 tagged with green fluorescent protein (GFP), designated ZG, was used for nitrous acid mutagenesis. From three trials, 11 mutants were identified from fluorescent spots without HR in inoculated C. quinoa leaves. Five mutants caused attenuated symptoms in squash plants. The genomic sequences of these five mutants revealed that most of the nonsynonymous changes were located in the HC-Pro gene. The replacement of individual mutated HC-Pros in the ZG backbone and an RNA silencing suppression (RSS) assay indicated that each mutated HC-Pro is defective in RSS function and responsible for reduced virulence. Four mutants provided high degrees of protection (84%-100%) against severe virus TW-TN3 in zucchini squash plants, with ZG 4-10 being selected for removal of the GFP tag. After removal of the GFP gene, Z 4-10 induced symptoms similar to ZG 4-10 and still provided 100% protection against TW-TN3 in squash, thus is considered not a genetically engineered mutant. Therefore, using a GFP reporter to select non-HR mutants of ZYMV from C. quinoa leaves is an efficient way to obtain beneficial mild viruses for cross-protection. This novel approach is being applied to other potyviruses.

Stellera chamaejasme extract against multidrug resistance of breast cancer cell line MCF-7

This study explored the effect and underlying mechanism of Stellera chamaejasme extract(SCE) on multidrug resistance of breast cancer. The chemotherapy-sensitive breast cancer cell line MCF-7 and adriamycin(ADR)-resistant cell line MCF-7/ADR were used as experimental subjects. MTT assay was used to detect cell proliferation activity. Pi staining was used to detect the cell cycle. 4',6-Diamidino-2-phenylindole, dihydrochloride(DAPI) staining and flow cytometry were used to detect apoptosis. Dansylcadaverine(MDC) staining and GFP-LC3B-Mcherry adenovirus transfection were used to detect autophagy. The protein expression of Bcl-2, Bax, caspase-9, caspase-3, LC3B, p62, and Beclin-1 was detected by Western blot. The results showed that SCE could significantly inhibit the proliferation of both sensitive and resistant breast cancer cell lines. The drug resistance factor was 0.53, which was significantly lower than 59 of ADR. Meanwhile, the proportion of sensitive/resistant cells in the G_0/G_1 phase increased significantly after SCE treatment. In addition, DAPI staining showed that a series of apoptosis phenomena such as nuclear pyknosis, staining deepening, and nuclear fragmentation appeared in sensitive/resistant cell lines after SCE administration. Moreover, the results of flow cytometry double staining showed that the proportion of apoptotic cells in sensitive/resistant cell lines increased significantly after SCE administration. Besides, Western blot showed that the protein expression levels of caspase-3, caspase-9, and Bcl-2 significantly decreased and the expression level of Bax protein significantly increased in both breast cancer cell lines after SCE administration. Furthermore, SCE could also increase the positive fluorescent spots after MDC staining and yellow fluorescent spots after GFP-LC3B-mcherry transfection, and up-regulate the expression levels of autophagy-related proteins LC3B-Ⅱ, p62, and Beclin-1 in breast cancer cells. In summary, SCE may play the role of anti-multidrug resistance by blocking the cell cycle of breast cancer multidrug-resistant cells, blocking autophagy flow, and ultimately interfering with the apoptosis resistance of drug-resistant cells.

Fluorescent Labeling of Peroxisome and Nuclear in Colletotrichum aenigma.

Anthracnose is one of the most widespread and destructive diseases in grapes. Grape anthracnose can be caused by various Colletotrichum species, such as Colletotrichum gloeosporioides and Colletotrichum cuspidosporium. In recent years, Colletotrichum aenigma was reported as a causal agent of Grape anthracnose in China and South Korea. Peroxisome is an important organelle in eukaryotes, which plays a very important role in the growth, development, and pathogenicity of several plant-pathogenic fungal species i, but it has not been reported in C. aenigma. In this work, the peroxisome of C. aenigma was labeled with a fluorescent protein, using green fluorescent protein (GFP) and red fluorescent protein (DsRED and mCherry) as reporter genes. Via Agrobacterium tumefaciens-mediated transformation (AtMT), two fluorescent fusion vectors to mark the peroxisomes, with GFP and DsRED, respectively, were introduced into a wild-type strain of C. aenigma. In the transformants, bright dots of green or red fluorescence in hyphae and spores could be seen in the strains labeled peroxisome. The nuclei labeled by the same method showed bright round fluorescent spots. In addition, we also combined fluorescent protein labeling with chemical staining to show the localization more clearly. The ideal peroxisome and nuclear fluorescence-labeled C. aenigma strain was obtained, which provided a reference for the study of its growth, development, and pathogenicity.

Prevalence of Glucose-6-Phosphate Dehydrogenase Deficiency in Male Newborn Infants and Its Relationship with Neonatal Jaundice in Thailand.

The study aimed to explore the prevalence of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency among male newborn infants in northeastern Thailand and its relationship with neonatal jaundice (NJ). This prospective cohort study included male newborn infants with gestational age (GA) ≥35 weeks born between July 1, 2019, and March 1, 2021. Cord blood was sent for G-6-PD fluorescent spot test (FST) and results were reported as normal, partial, or complete deficiency. Infants with NJ would have blood tested for total serum bilirubin (TSB) level and other possible causes of NJ. Duration of phototherapy, length of hospital stays, and complications were documented. There were 922 male infants included in this study with 854 (93.1%) term and 63 (6.9%) preterm infants. FST showed 132 infants (14.4%) had G-6-PD deficiency. Incidence of NJ was significantly higher among infants with G-6-PD deficiency compared with infants with normal G-6-PD level (47.7 vs. 25.8%; relative risk [RR]: 2.62, 95% confidence interval [CI]: 1.79-3.82; p < 0.001). Regardless of G-6-PD level, preterm infants had significantly higher incidence of NJ than term infants (52.4 vs. 27.3%; RR: 2.93, 95% CI: 1.75-4.92; p < 0.001). Duration of phototherapy was significantly longer in infants with G-6-PD deficiency with NJ but hospital stays were similar. Infants with combined G-6-PD deficiency and other causes of hemolysis did not have higher TSB level than infants with isolated G-6-PD deficiency. Risk factors associated with NJ were G-6-PD deficiency and preterm infants, whereas more advance GA was associated with reduced risk for NJ. G-6-PD deficiency and preterm infants were important risk factors for NJ. Routine G-6-PD screening, close monitoring for signs of NJ in infant with risks, and appropriate parental counseling should be implemented. · G-6-PD deficiency increases risk of neonatal jaundice.. · Preterm infants have higher risk for neonatal jaundice.. · G-6-PD deficiency does not link with severe jaundice..

Toward High‐Throughput Single‐Spin Optically Detected Magnetic Resonance Spectroscopy with Spatially Programmable Laser Illumination

AbstractThe single nitrogen‐vacancy (NV) centers in diamond are shown as a quantum sensor with broad applications in nanoscale sensing of magnetic field, electric field, temperature, and strain. However, the basic technique, namely, optically detected magnetic resonance to manipulate and measure single NV centers needs to be highly sped up for broad and practical applications. Here, this work shows a parallel optically detected magnetic resonance (ODMR) platform of single NV centers including the programmable excitation laser spots generated by a digital micromirror device, a four‐channel uniform microwave delivery, and the fluorescence detector based on scientific complementary metal–oxide–semiconductor camera. In the viewing field of 26 µm, hundreds of distinguishable fluorescent spots are addressed and 413 NV center spots are recognized. The magnetic resonance spectrum, Rabi oscillation, and spin echo of each NV center spot are measured in parallel. As a result, a preliminary speedup of ≈20‐fold is achieved compared to the confocal‐based ODMR and can be further extended to thousands of folds after updating the digital micromirror device and camera.

Development of a chemical tracer and a chromatographic fingerprint for the quality control of finished products based on hydroethanol extract of fresh leaves of Cassia alata (L.) Roxb (Fabaceae.)

SUMMARY&#x0D; Introduction: Cassia alata (L) Roxb belonging to the Fabaceae family is widely used in the treatment of dermatoses. The quality control of herbal products remains very difficult due to their complex phytochemical composition. This study aims to develop a chromatographic fingerprint by thin layer chromatography (TLC) and a chemical tracer characterized by UV spectrophotometry that will be used for phytochemical analysis of a hydroethanol extract of fresh leaves of Cassia alata (L) Roxb (EHCA) and a finished product based on this extract (mitraca soap).&#x0D; Methodology: Phytochemical screening was performed on EHCA. A spectrophotometric analysis was then performed. The tracer of the selected extract was searched in a soap following the guidelines of the European Medicines Agency (EMA).&#x0D; Results: Phytochemical screening revealed the presence of anthracenosides, saponosides, steroidal and triterpene glycosides, leucoanthocyanosides, tannins and flavonoids. A more visible fluorescent orange spot with an Rf of 0.52 ± 0.01 observed at 365 nm was established as tracer. It is detected at a threshold of 0.69 ± 0.01 mg/mL. TLC performed on the extract provided a good separation of this tracer characterized as an anthracenoside. The resolution factor to evaluate the separation with the other peaks was estimated to be 1.83 ± 0.01. This anthracenoside compound showed four absorption maxima at 223, 274, 284, 292 nm. It was also found in the C. alata soap. The orange compound (anthracenoside) is located at Rf 0.52 ± 0.01 at 365 nm similar to that of the initial pure extract.&#x0D; Conclusion: The selected tracer could be used to control the quality of the finished products based on this extract and in particular the soap.&#x0D; Key words: Cassia alata, quality control, tracer, anthracenoside, hydroethanol extract.

The dynamic change in the degradation and in vitro digestive properties of porcine myofibrillar protein during freezing storage

The myofibrillar protein (MP) degradation and in vitro digestive properties of porcine longissimus during freezing at −8, −18, −25 and − 40 °C for 1, 3, 6, 9 and 12 months were investigated. As the freezing temperature and duration of frozen storage increased, the amino nitrogen and TCA (trichloroacetic acid)-soluble peptides of the samples were significantly increased, while the total sulfhydryl content and band intensity of myosin heavy chain, actin, troponin T, tropomyosin were significantly decreased (P < 0.05). At higher freezing storage temperatures and durations, the particle size of MP samples and the green fluorescent spots detected using a laser particle size analyzer and confocal laser scanning microscopy became large. After 12 months of freezing, the digestibility and the degree of hydrolysis of the trypsin digestion solution of the samples frozen at −8 °C were significantly decreased by 15.02 % and 14.28 %, respectively, when compared to fresh samples, whereas, the mean surface diameter (d3,2) and mean volume diameter (d4,3) were significantly increased by 14.97 % and 21.53 %, respectively. Therefore, frozen storage induced protein degradation and impaired the ability of digestion in the pork proteins. This phenomenon was more evident as the samples were frozen at high temperatures over a long storage period.

Single-molecule assay for highly sensitive SARS-CoV-2 gene detection based on CRISPR-Cas12a.

A trans-cleavage activity of some CRISPR-associated (Cas) proteins including Cas12a and Cas 13a that non-specifically cleaves single-stranded DNA or RNA, has been wildly used to develop sensitive gene detection methods because it is a multiple-turnover enzymatic process, only activated via guide RNA-target gene pairing. Combined with a pre-amplification step, CRISPR-Cas12a-based gene detection methods achieved even attomolar detection limits. However, the pre-amplification step often accompanies the tedious design of primers and inevitable base substitution errors by DNA polymerases, which is not suitable for quantitative analysis. Here, we report a single-molecule assay for ultrasensitive and quantitative gene detection utilizing the trans-cleavage activity of Cas12a without pre-amplification. To monitor the trans-cleavage of Cas12a at the single-molecule level, we designed a hairpin DNA probe with a stem-loop structure where the single-stranded DNA loop can be cleaved via the trans-cleavage of Cas12a, labeled fluorophore and quencher pairs at the end of the stem and immobilized it at the surface. Only in the existence of target gene, the trans-cleavage of Cas12a is activated, cleaving the loop of the hairpin DNA probe, making the quencher leave from the surface, and resulting in the appearance of a fluorescent spot. In other words, the number of appearing fluorescent spots reflects the amount of target gene quantitatively. After optimization of various experimental conditions such as different Cas12a orthologs, monovalent and divalent ion concentration in the reaction buffer, reaction temperature and the mixing ratio of Cas12a to guide RNA, we successfully achieved to detect the E gene of SARS-CoV-2 down to sub picomolar range without any pre-amplification step.

Cover Feature: A Tool, an App and a Field: Fluorescent PET Sensors, Blood Electrolyte Analysis and Molecular Logic as Products of Supramolecular Photoscience from Northern Ireland and Sri Lanka (ChemPlusChem 4/2023)

The cover feature image shows chemical species (in gold) rushing in to bind with receptors (in green) so that light signals of associated fluorophores (in blue) are switched from ‘off’ to ‘on’. Plastic chips containing such fluorescent PET sensor spots help medical personnel to save lives everyday worldwide. These ‘fluorophore-receptor’ constructs also emulate logic gates (in orange) of the semiconductor IT revolution and they can operate in tiny living spaces where semiconductors fear to tread. Molecular information technology underpins all living systems. Cover design by Chao-Yi Yao. More information can be found in the Perspective article by Chao-Yi Yao and A. Prasanna de Silva.

Sex Determination in Dioscorea dumetorum: Evidence of Heteromorphic Sex Chromosomes and Sex-Linked NORs

Yams (Dioscorea spp.) are a pantropical genus located worldwide that constitute an important source of nutrients and pharmaceutical substances. Some Dioscorea crop species are widely grown in West Africa. One species that is mainly cultivated in Cameroon is Dioscorea dumetorum. This is a dioecious root crop whose sex-determining system was unknown until now. To address the possible presence of sex chromosomes in D. dumetorum, we performed a karyotype characterization of male and female individuals using classical and molecular cytogenetic approaches. It was determined that 2n = 40 was the most common number of chromosomes in all of the investigated samples. One chromosome pair was longer than the others in the chromosome set and was a heteromorph in male and homomorph in female individuals. This pair corresponded to sex chromosomes, and we also confirmed this with molecular cytogenetic experiments. The results of chromomycin banding revealed the presence of strong positive signals on this chromosome pair. The signals, corresponding to GC-rich DNA regions, were similar in size on the chromosomes of the female individuals, whereas they were different in size in the male individuals. This size difference in the GC-rich heterochromatin regions was also apparent in the interphase nuclei as one small and one large fluorescent spot. The results of the in situ hybridization experiment showed that these chromomycin positive signals on the sex chromosomes also corresponded to the 35S rDNA cluster. The mean 2C DNA value (genome size) obtained for D. dumentorum was 0.71 pg (±0.012), which represents a small genome size. We found no difference in the genome size between the male and female individuals. The results of this study contribute to increasing our knowledge of sex determination in D. dumetorum (standard sex-determining XX/XY system) and may have some agronomic applications.

Alpha-lipoic acid inhibits sodium arsenite-mediated autophagic death of rat insulinoma cells.

To investigate the protective effect of α-lipoic acid on sodium arsenite (NaAsO2) induced INS-1 cells injury and its mechanism. The cell viability was measured by CCK-8 assay. The autophagosomes was observed under transmission electron microscopy. The autophagosomes in cells transfected with green fluorescent protein microtubule-associated protein light chain 3 (GFP-LC3) plasmids were observed under a laser scanning con-focal microscope. The expression of LC3-II, P62, PI3K, and mTOR proteins in INS-1 cells treated with a combination of chloroquine (CQ, autophagy inhibitor) and NaAsO2 were detected by Western blot assay. The expression of LC3-II, P62, PI3K, and mTOR proteins were detected in INS-1 cells treated with a combination of rapamycin (autophagy inducer, mTOR inhibitor) and α-LA. The cytotoxicity induced by NaAsO2 was reversed by α-LA, and the viability of NaAsO2-treated INS-1 cells increased. α-LA pretreatment decreased the autophagosome accumulation induced by NaAsO2. α-LA also reduced the fluorescence spot aggregation of GFP-LC3 in INS-1 cells exposed to NaAsO2 as observed under a laser scanning con-focal microscope. α-LA inhibited NaAsO2 induced autophagy by up-regulating PI3K and mTOR and down-regulating LC3-II and P62. CQ inhibited NaAsO2 induced autophagy by up-regulating PI3K, mTOR, P62 and down-regulating LC3-II. α-LA inhibited rapamycin-induced autophagy by up-regulating PI3K, mTOR and P62 and down-regulating LC3-II. The results showed that NaAsO2 could induce autophagy activation in INS-1 cells. The α-LA may inhibit autophagy activation by regulating the PI3K/mTOR pathway. The data indicated that α-LA might inhibit the NaAsO2-induced autophagic death of INS-1 cells by regulating the PI3K/mTOR pathway.