Downregulation of a CT10 regulator of kinase (Crk) promotes the formation of diapause embryos in the brine shrimp Artemia.

To survive under harsh environments, embryonic development of Artemia was arrested at the gastrula stage and released as the diapause embryo. Cell cycle and metabolism were highly suppressed in this state of quiescence. However, cellular mechanisms underlying diapause remain largely unclear. In this study, we found that the expression level of a CT10 regulator of kinase-encoding gene (Ar-Crk) in diapause embryos was significantly lower than non-diapause embryos at the early embryogenetic stage of Artemia. Knockdown of Ar-Crk by RNA interference induced formation of diapause embryos, while the control group produced nauplii. Western blot analysis and metabolic assays revealed that the diapause embryos produced by Ar-Crk-knocked-down Artemia had similar characteristics of diapause markers, arrested cell cycle, and suppressed metabolism with those diapause embryos produced by natural oviparous Artemia. Transcriptomic analysis of Artemia embryos revealed knockdown of Ar-Crk induced downregulation of the aurora kinase A (AURKA) signaling pathway, as well as energetic and biomolecular metabolisms. Taken together, we proposed that Ar-Crk is a crucial factor in determining the process of diapause in Artemia. Our results provide insight into the functions of Crk in fundamental regulations such as cellular quiescence.

Tryptophan metabolism as bridge between gut microbiota and brain in chronic social defeat stress-induced depression mice.

Backgrounds: Gut microbiota plays a critical role in the onset and development of depression, but the underlying molecular mechanisms are unclear. This study was conducted to explore the relationships between gut microbiota and host's metabolism in depression. Methods: Chronic social defeat stress (CSDS) model of depression was established using C57BL/6 male mice. Fecal samples were collected from CSDS group and control group to measure gut microbiota and microbial metabolites. Meanwhile, tryptophan metabolism-related metabolites in hippocampus were also analyzed. Results: CSDS successfully induced depressive-like behaviors in CSDS group. The 24 differential bacterial taxa between the two groups were identified, and 14 (60.87%) differential bacterial taxa belonged to phylum Firmicutes. Functional analysis showed that tryptophan metabolism was significantly affected in CSDS mice. Meanwhile, 120 differential microbial metabolites were identified, and two key tryptophan metabolism-related metabolites (tryptophan and 5-hydroxytryptophan (5-HTP)) were significantly decreased in feces of CSDS mice. The correlation analysis found the significant relationships between tryptophan and differential bacterial taxa under Firmicutes, especially genus Lactobacillus (r=0.801, p=0.0002). In addition, the significantly decreased 5-hydroxytryptamine (5-HT) in hippocampus of depressed mice was also observed. Conclusions: Our results showed that tryptophan metabolism might have an important role in the crosstalk between gut microbioa and brain in depression, and phylum Firmicutes, especially genus Lactobacillus, might be involved in the onset of depression through regulating tryptophan metabolism.

From ecology to oncology: To understand cancer stem cell dormancy, ask a Brine shrimp (Artemia).

The brine shrimp (Artemia), releases embryos that can remain dormant for up to a decade. Molecular and cellular level controlling factors of dormancy in Artemia are now being recognized or applied as active controllers of dormancy (quiescence) in cancers. Most notably, the epigenetic regulation by SET domain-containing protein 4 (SETD4), is revealed as highly conserved and the primary control factor governing the maintenance of cellular dormancy from Artemia embryonic cells to cancer stem cells (CSCs). Conversely, DEK, has recently emerged as the primary factor in the control of dormancy exit/reactivation, in both cases. The latter has been now successfully applied to the reactivation of quiescent CSCs, negating their resistance to therapy and leading to their subsequent destruction in mouse models of breast cancer, without recurrence or metastasis potential. In this review, we introduce the many mechanisms of dormancy from Artemia ecology that have been translated into cancer biology, and herald Artemia's arrival on the model organism stage. We show how Artemia studies have unlocked the mechanisms of the maintenance and termination of cellular dormancy. We then discuss how the antagonistic balance of SETD4 and DEK fundamentally controls chromatin structure and consequently governs CSCs function, chemo/radiotherapy resistance, and dormancy in cancers. Many key stages from transcription factors to small RNAs, tRNA trafficking, molecular chaperones, ion channels, and links with various pathways and aspects of signaling are also noted, all of which link studies in Artemia to those of cancer on a molecular and/or cellular level. We particularly emphasize that the application of such emerging factors as SETD4 and DEK may open new and clear avenues for the treatment for various human cancers.

A cytological revisit on parthenogenetic Artemia and the deficiency of a meiosis-specific recombinase DMC1 in the possible transition from bisexuality to parthenogenesis.

Although parthenogenesis is widespread in nature and known to have close relationships with bisexuality, the transitional mechanism is poorly understood. Artemia is an ideal model to address this issue because bisexuality and "contagious" obligate parthenogenesis independently exist in its congeneric members. In the present study, we first performed chromosome spreading and immunofluorescence to compare meiotic processes of Artemia adopting two distinct reproductive ways. The results showed that, unlike conventional meiosis in bisexual Artemia, meiosis II in parthenogenic Artemia is entirely absent and anaphase I is followed by a single mitosis-like equational division. Interspecific comparative transcriptomics showed that two central molecules in homologous recombination (HR), Dmc1 and Rad51, exhibited significantly higher expression in bisexual versus parthenogenetic Artemia. qRT-PCR indicated that the expression of both genes peaked at the early oogenesis and gradually decreased afterward. Knocking-down by RNAi of Dmc1 in unfertilized females of bisexual Artemia resulted in a severe deficiency of homologous chromosome pairing and produced univalents at the middle oogenesis stage, which was similar to that of parthenogenic Artemia, while in contrast, silencing Rad51 led to no significant chromosome morphological change. Our results indicated that Dmc1 is vital for HR in bisexual Artemia, and the deficiency of Dmc1 may be correlated with or even possibly one of core factors in the transition from bisexuality to parthenogenesis.

Multi-omics data reveals the important role of glycerophospholipid metabolism in the crosstalk between gut and brain in depression.

BACKGROUND: Gut microbiota plays a critical role in the onset and development of depression, but the underlying molecular mechanisms are unclear. This study was conducted to observe the characteristics of gut microbiota, lipid metabolism and neurotransmitters in Gut-Liver-Brain axis in depressed mice (DM), and identify some novel perceptions on relationships between gut microbiota and depression. METHODS: A mouse model of depression was built used chronic unpredictable mild stress (CUMS). Fecal samples (measuring gut microbiota compositions, microbial genes and lipid metabolites), liver samples (measuring lipid metabolites), and hippocampus (measuring neurotransmitters) were collected. Both univariate and multivariate statistical analyses were used to identify the differential gut microbiota, metabolic signatures and neurotransmitters in DM. RESULTS: There were significant differences on both microbial and metabolic signatures between DM and control mice (CM): 71 significantly changed operational taxonomic units (OTUs) (60.56% belonged to phylum Firmicutes) and 405 differential lipid metabolites (51.11% belonged to Glycerophospholipid (GP) metabolism) were identified. Functional analysis showed that depressive-like behaviors (DLB)-related differential microbial genes were mainly enriched in GP metabolism. Weighted correlation network analysis (WGCNA) showed that DLB-related differential metabolites mainly belonged to GPs. Meanwhile, seven differential neurotransmitters were identified. Comprehensive analysis found that Lachnospiraceae and gamma-aminobutyric acid (GABA) were significantly correlated with 94.20% and 53.14% differential GPs, respectively, and GABA was significantly correlated with three main DLB phenotypes. CONCLUSION: Our results provided novel perceptions on the role of Gut-Liver-Brain axis in the onset of depression, and showed that GP metabolism might be the bridge between gut microbiota and depression. "Lachnospiraceae-GP metabolism-GABA" held the promise as a potential way between gut microbiota and brain functions in DM.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

An anti-poisoning nanosensor for in situ monitoring of intracellular endogenous hydrogen sulfide.

Intracellular H2S plays an important regulatory role in cell metabolism. The limited sensing materials and severe sensor passivation hinder its quantification. We functionalized conductive nanowires with MoS2 and quercetin in a large-scale manner, developed single nanowire sensors with excellent electrocatalytic and anti-poisoning performance, and achieved the accurate quantification of H2S within single cells.

Dual-channel nanoelectrochemical sensor for monitoring intracellular ROS and NADH kinetic variations of their concentrations.

Reactive oxygen species (ROS) and nicotinamide adenine dinucleotide (NADH) are important intracellular redox-active molecules involved in various pathological processes including inflammation, neurodegenerative diseases, and cancer. However, the fast dynamic changes and mutual regulatory kinetic relationship between intracellular ROS and NADH in these biological processes are still hard to simultaneously investigate. A dual-channel nanowire electrode (DC-NWE) integrating two conductive nanowires, one functionalized with platinum nanoparticles and the other with conductive polymer, was nanofabricated for the selective and simultaneous real-time monitoring of intracellular ROS and NADH release by mitochondria in single living MCF-7 tumoral cells stimulated by resveratrol. The production of ROS was observed to occur tenths of a second before the release of NADH, a significant new piece of information suggesting a mechanism of action of resveratrol. Beyond the importance of the specific data gathered in this study, this work established the feasibility of simultaneously monitoring multiple species and analyzing their kinetics relationships over sub-second time scales thanks to dual-channel nanowire electrodes. It is believed that this concept and its associated nanoelectrochemical tools might benefit to a deeper understanding of mutual regulatory relationship between intracellular crucial molecular markers during physiological and pathological processes as well as for evaluating medical treatments.

Investigation of the Distribution of Thalassemia in Children in Jiangxi Province, the People's Republic of China.

Thalassemia is one of southern China's most common inherited disorders. Little is known about the genotypes of thalassemia in children in Jiangxi Province, the People's Republic of China (PRC). Two thousand, nine hundred and fifty-two children with suspected thalassemia were recruited from August 2016 to December 2020 at the Jiangxi Provincial Children's Hospital, Nanchang, PRC. Reverse dot-blot hybridization was used to detect α- and ß-thalassemia (α- and ß-thal) genotypes. A rare mutation was detected using gap-polymerase chain reaction (gap-PCR) and gene sequencing. The overall distribution of thalassemia (1534 cases) was 51.96%, and the detection rate of α-thal (616 cases), ß-thal (888 cases) and concurrent α- and ß-thalassemias (30 cases) was 20.86, 30.08, and 1.02%, respectively. A rare α-thal genotype, -α27.6/- -SEA (Southeast Asian), was identified. Seventy-eight cases of severe ß-thal were detected, accounting for 8.78% of the cases, including 56 double heterozygous cases and 22 cases that were homozygous. Both α- and ß-thalassemias are widely distributed in the children of Jiangxi Province. Thalassemia genetic testing is essential to establish a comprehensive thalassemia prevention program and improve public education.

Homeostasis inside Single Activated Phagolysosomes: Quantitative and Selective Measurements of Submillisecond Dynamics of Reactive Oxygen and Nitrogen Species Production with a Nanoelectrochemical Sensor.

Reactive oxygen and nitrogen species (ROS/RNS) are generated by macrophages inside their phagolysosomes. This production is essential for phagocytosis of damaged cells and pathogens, i.e., protecting the organism and maintaining immune homeostasis. The ability to quantitatively and individually monitor the four primary ROS/RNS (ONOO-, H2O2, NO, and NO2-) with submillisecond resolution is clearly warranted to elucidate the still unclear mechanisms of their rapid generation and to track their concentration variations over time inside phagolysosomes, in particular, to document the origin of ROS/RNS homeostasis during phagocytosis. A novel nanowire electrode has been specifically developed for this purpose. It consisted of wrapping a SiC nanowire with a mat of 3 nm platinum nanoparticles whose high electrocatalytic performances allow the characterization and individual measurements of each of the four primary ROS/RNS. This allowed, for the first time, a quantitative, selective, and statistically robust determination of the individual amounts of ROS/RNS present in single dormant phagolysosomes. Additionally, the submillisecond resolution of the nanosensor allowed confirmation and measurement of the rapid ability of phagolysosomes to differentially mobilize their enzyme pools of NADPH oxidases and inducible nitric oxide synthases to finely regulate their homeostasis. This reveals an essential key to immune responses and immunotherapies and rationalizes its biomolecular origin.

Redox Homeostasis Alteration in Endothelial Mechanotransduction Monitored by Dual Stretchable Electrochemical Sensors.

In vivo, endothelial cells are permanently subjected to dynamic cyclic stretch and adapt to it through the release of vasoactive substances. Among them, reactive oxygen species (ROS) and nitric oxide (NO) are indispensable redox molecules, the contents of which and their ratio are closely implicated with endothelial redox homeostasis. However, simultaneous and quantitative monitoring of ROS and NO release in endothelial mechanotransduction remains a great challenge. Herein, a stretchable electrochemical device is developed with a dual electrode based on gold nanotubes decorated with uniform and tiny platinum nanoparticles. This hybrid nanostructure endows the sensor with high sensitivity toward both hydrogen peroxide (H2O2) (as the most stable ROS) and NO electrooxidation. Importantly, the two species can be well discriminated by applying different potentials, which allows simultaneous monitoring of H2O2 and NO release in stretch-induced endothelial mechanotransduction by the same device. The results of quantitative analysis suggest that endothelial redox homeostasis and its alteration are strongly related to vascular biomechanical and biochemical milieus. Further investigation reveals that the interplay of ROS and NO signaling has an important role in the regulation of endothelial redox state. This work will greatly facilitate the deep understanding of the molecular mechanism of endothelial dysfunction and vascular disorder.

Versatile Construction of Biomimetic Nanosensors for Electrochemical Monitoring of Intracellular Glutathione.

The current strategies for nanoelectrode functionalization usually involve sophisticated modification procedures, uncontrollable and unstable modifier assembly, as well as a limited variety of modifiers. To address this issue, we propose a versatile strategy for large-scale synthesis of biomimetic molecular catalysts (BMCs) modified nanowires (NWs) to construct functionalized electrochemical nanosensors. This design protocol employs an easy, controllable and stable assembly of diverse BMCs-poly(3,4-ethylenedioxythiophene) (PEDOT) composites on conductive NWs. The intrinsic catalytic activity of BMCs combined with outstanding electron transfer ability of conductive polymer enables the nanosensors to sensitively and selectively detect various biomolecules. Further application of sulfonated cobalt phthalocyanine functionalized nanosensors achieves real-time electrochemical monitoring of intracellular glutathione levels and its redox homeostasis in single living cells for the first time.

Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells.

Recently, stretchable electrochemical sensors have stood out as a powerful tool for the detection of soft cells and tissues, since they could perfectly comply with the deformation of living organisms and synchronously monitor mechanically evoked biomolecule release. However, existing strategies for the fabrication of stretchable electrochemical sensors still face with huge challenges due to scarce electrode materials, demanding processing techniques and great complexity in further functionalization. Herein, we report a novel and facile strategy for one-step preparation of stretchable electrochemical biosensors by doping ionic liquid and catalyst into a conductive polymer (poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate), PEDOT:PSS). Bis(trifluoromethane) sulfonimide lithium salt as a small-molecule plasticizer can significantly improve the stretchability and conductivity of the PEDOT:PSS film, and cobalt phthalocyanine as an electrocatalyst endows the film with excellent electrochemical sensing performance. Moreover, the functionalized PEDOT:PSS retained good cell biocompatibility with two extra dopants. These satisfactory properties allowed the real-time monitoring of stretch-induced transient hydrogen peroxide release from cells. This work presents a versatile strategy to fabricate conductive polymer-based stretchable electrodes with easy processing and excellent performance, which benefits the in-depth exploration of sophisticated life activities by electrochemical sensing.

Expansion of targetable sites for the ribonucleoprotein-based CRISPR/Cas9 system in the silkworm Bombyx mori.

BACKGROUND: With the emergence of CRISPR/Cas9 technology, multiple gene editing procedures became available for the silkworm. Although binary transgene-based methods have been widely used to generate mutants, delivery of the CRISPR/Cas9 system via DNA-free ribonucleoproteins offers several advantages. However, the T7 promoter that is widely used in the ribonucleoprotein-based method for production of sgRNAs in vitro requires a 5' GG motif for efficient initiation. The resulting transcripts bear a 5' GG motif, which significantly constrains the number of targetable sites in the silkworm genome. RESULTS: In this study, we used the T7 promoter to add two supernumerary G residues to the 5' end of conventional (perfectly matched) 20-nucleotide sgRNA targeting sequences. We then asked if sgRNAs with this structure can generate mutations even if the genomic target does not contain corresponding GG residues. As expected, 5' GG mismatches depress the mutagenic activity of sgRNAs, and a single 5' G mismatch has a relatively minor effect. However, tests involving six sgRNAs targeting two genes show that the mismatches do not eliminate mutagenesis in vivo, and the efficiencies remain at useable levels. One sgRNA with a 5' GG mismatch at its target performed mutagenesis more efficiently than a conventional sgRNA with 5' matched GG residues at a second target within the same gene. Mutations generated by sgRNAs with 5' GG mismatches are also heritable. We successfully obtained null mutants with detectable phenotypes from sib-mated mosaics after one generation. CONCLUSIONS: In summary, our method improves the utility and flexibility of the ribonucleoprotein-based CRISPR/Cas9 system in silkworm.

Data mining in clinical big data: the frequently used databases, steps, and methodological models.

Many high quality studies have emerged from public databases, such as Surveillance, Epidemiology, and End Results (SEER), National Health and Nutrition Examination Survey (NHANES), The Cancer Genome Atlas (TCGA), and Medical Information Mart for Intensive Care (MIMIC); however, these data are often characterized by a high degree of dimensional heterogeneity, timeliness, scarcity, irregularity, and other characteristics, resulting in the value of these data not being fully utilized. Data-mining technology has been a frontier field in medical research, as it demonstrates excellent performance in evaluating patient risks and assisting clinical decision-making in building disease-prediction models. Therefore, data mining has unique advantages in clinical big-data research, especially in large-scale medical public databases. This article introduced the main medical public database and described the steps, tasks, and models of data mining in simple language. Additionally, we described data-mining methods along with their practical applications. The goal of this work was to aid clinical researchers in gaining a clear and intuitive understanding of the application of data-mining technology on clinical big-data in order to promote the production of research results that are beneficial to doctors and patients.

Risk factor for vital signs elevation during percutaneous microwave ablation of liver tumor under deep sedation.

PURPOSE: To evaluate the frequency and the degree of vital signs elevation, as well as to elucidate the risk factors for elevation of cardiopulmonary parameters. MATERIALS AND METHODS: We retrospectively evaluated the medical records of 101 patients who received microwave ablation (MWA) under deep sedation with propofol. Univariate analysis followed by multivariate linear regression analysis was performed to determine the risk factors associated with the elevation of cardiopulmonary parameters. RESULTS: The heart rate (HR), mean blood pressure (BP) and respiratory rate (RR) were elevated in 53.5%, 45.5% and 30.7%. Hyperhemodynamic state (mean BP or HR increased > 30% of the baseline) and high RR (RR > 20 times/min) were detected in 23.8% and 13.9%. Age ≤ 50 years was signifiant for mean BP and HR elevation (p = 0.032; p = 0.027), ablation zone abutting the parietal peritoneum (p = 0.001; p = 0.001; p < 0.001) and the diaphragm (p = 0.001) were risk factors for BP and RR elevation. CONCLUSIONS: Elevations in HR and BP are common. Risk factors for vital signs elevation include ablation zone abutting the parietal peritoneum and the diaphragm, as well as young age. These findings help devise strategies for anesthetic management.

Large-Scale Synthesis of Functionalized Nanowires to Construct Nanoelectrodes for Intracellular Sensing.

A strategy for one-pot and large-scale synthesis of functionalized core-shell nanowires (NWs) to high-efficiently construct single nanowire electrodes is proposed. Based on the polymerization reaction between 3,4-ethylenedioxythiophene (EDOT) and noble metal cations, manifold noble metal nanoparticles-polyEDOT (PEDOT) nanocomposites can be uniformly modified on the surface of any nonconductive NWs. This provides a facile and versatile approach to produce massive number of core-shell NWs with excellent conductivity, adjustable size, and well-designed properties. Nanoelectrodes manufactured with such core-shell NWs exhibit excellent electrochemical performance and mechanical stability as well as favorable antifouling properties, which are demonstrated by in situ intracellular monitoring of biological molecules (nitric oxide) and unraveling its relevant unclear signaling pathway inside single living cells.

Quantitative Nano-amperometric Measurement of Intravesicular Glutamate Content and its Sub-Quantal Release by Living Neurons.

Quantitative measurements of intravesicular glutamate (Glu) and of transient exocytotic release contents directly from individual living neurons are highly desired for understanding the mechanisms (full or sub-quantal release?) of synaptic transmission and plasticity. However, this could not be achieved so far due to the lack of adequate experimental strategies relying on selective and sensitive Glu nanosensors. Herein, we introduce a novel electrochemical Glu nanobiosensor based on a single SiC nanowire that can selectively measure in real-time Glu fluxes released via exocytosis by large Glu vesicles (ca. 125 nm diameter) present in single hippocampal axonal varicosities as well as their intravesicular content before exocytosis. These measurements revealed a sub-quantal release mode in living hippocampal neurons, viz., only ca. one third to one half of intravesicular Glu molecules are released by individual vesicles during exocytotic events. Importantly, this fraction remained practically the same when hippocampal neurons were pretreated with L-Glu-precursor L-glutamine, while it significantly increased after zinc treatment, although in both cases the intravesicular contents were drastically affected.

Correction: Plasticizer and catalyst co-functionalized PEDOT:PSS enables stretchable electrochemical sensing of living cells.

[This corrects the article DOI: 10.1039/D1SC04138J.].

Stretchable Electrode Based on Au@Pt Nanotube Networks for Real-Time Monitoring of ROS Signaling in Endothelial Mechanotransduction.

Vascular endothelial cells (ECs) are natively exposed to dynamic cyclic stretch and respond to it by the production of vasoactive molecules. Among them, reactive oxygen species (ROS) are closely implicated to the endothelial function and vascular homeostasis. However, the dynamic monitoring of ROS release during endothelial mechanotransduction remains a steep challenge. Herein, we developed a stretchable electrochemical sensor by decoration of uniform and ultrasmall platinum nanoparticles (Pt NPs) on gold nanotube (Au NT) networks (denoted as Au@Pt NTs). The orchestrated structure exhibited prominent electrocatalytic property toward the oxidation of hydrogen peroxide (H2O2) (as the most stable ROS) while maintaining excellent mechanical compliance of Au NT networks. Moreover, the favorable biocompatibility of Au NTs and Pt NPs promoted the adhesion and proliferation of ECs cultured thereon. These allowed in situ inducing ECs mechanotransduction and synchronously real-time monitoring of H2O2 release. Further investigation revealed that the production of H2O2 was positively correlated with the applied mechanical strains and could be boosted by other coexisting pathogenic factors. This indicates the great prospect of our proposed sensor in exploring ROS-related signaling for the deep understanding of cell mechanotransduction and vascular disorder.