Whole-brain mapping of monosynaptic afferent inputs to the CRH neurons in the medial prefrontal cortex of mice.

Corticotropin-releasing hormone (CRH) neurons are densely distributed in the medial prefrontal cortex (mPFC), which plays a crucial role in integrating and processing emotional and cognitive inputs from other brain regions. Therefore, it is important to know the neural afferent patterns of mPFCCRH neurons, which are still unclear. Here, we utilized a rabies virus-based monosynaptic retrograde tracing system to map the presynaptic afferents of the mPFCCRH neurons throughout the entire brain. The results show that the mPFCCRH neurons receive inputs from three main groups of brain regions: (1) the cortex, primarily the orbital cortex, somatomotor areas, and anterior cingulate cortex; (2) the thalamus, primarily the anteromedial nucleus, mediodorsal thalamic nucleus, and central medial thalamic nucleus; and (3) other brain regions, primarily the basolateral amygdala, hippocampus, and dorsal raphe nucleus. Taken together, our results are valuable for further investigations into the roles of the mPFCCRH neurons in normal and neurological disease states. These investigations can shed light on various aspects such as cognitive processing, emotional modulation, motivation, sociability, and pain.

Photon blockade induced by two-photon absorption in cavity quantum electrodynamics.

Photon blockade (PB) is an important quantum phenomenon in cavity quantum electrodynamics (QED). Here, we investigate the PB effect in the simplest cavity QED systems (one cavity containing first a single atom and then two atoms), where only the atoms are weakly driven. Via the analytical calculation and numerical simulation, we show that the strong PB can be generated even with the weak-coupling regime at the total resonance. This blockade is ascribed to the two-photon absorption, which is fundamentally different from the conventional and unconventional blockade mechanisms. Therefore, our study provides an alternative approach to produce the PB in the atom-driven cavity QED system.

Gain-of-Function KIDINS220 Variants Disrupt Neuronal Development and Cause Cerebral Palsy.

BACKGROUND: Kinase D-interacting substrate of 220 kDa (KIDINS220) is a multifunctional scaffolding protein essential for neuronal development. It has been implicated in neurological diseases with either autosomal dominant (AD) or autosomal recessive (AR) inheritance patterns. The molecular mechanisms underlying the AR/AD dual nature of KIDINS220 remain elusive, posing challenges to genetic interpretation and clinical interventions. Moreover, increased KIDINS220 exhibited neurotoxicity, but its role in neurodevelopment remains unclear. OBJECTIVE: The aim was to investigate the genotype-phenotype correlations of KIDINS220 and elucidate its pathophysiological role in neuronal development. METHODS: Whole-exome sequencing was performed in a four-generation family with cerebral palsy. CRISPR/Cas9 was used to generate KIDINS220 mutant cell lines. In utero electroporation was employed to investigate the effect of KIDINS220 variants on neurogenesis in vivo. RESULTS: We identified in KIDINS220 a pathogenic nonsense variant (c.4177C > T, p.Q1393*) that associated with AD cerebral palsy. We demonstrated that the nonsense variants located in the terminal exon of KIDINS220 are gain-of-function (GoF) variants, which enable the mRNA to escape nonsense-mediated decay and produce a truncated yet functional KIDINS220 protein. The truncated protein exhibited significant resistance to calpain and consequently accumulated within cells, resulting in the hyperactivation of Rac1 and defects in neuronal development. CONCLUSIONS: Our findings demonstrate that the location of variants within KIDINS220 plays a crucial role in determining inheritance patterns and corresponding clinical outcomes. The proposed interaction between Rac1 and KIDINS220 provides new insights into the pathogenesis of cerebral palsy, implying potential therapeutic perspectives. © 2023 International Parkinson and Movement Disorder Society.

Identification of ARF genes in Juglans Sigillata Dode and analysis of their expression patterns under drought stress.

BACKGROUND: Auxin response factor (ARF), a transcription factors that controls the expression of genes responsive to auxin, plays a key role in the regulation of plant growth and development. Analyses aimed at identifying ARF family genes and characterizing their functions in Juglans sigillata Dode are lacking. METHODS AND RESULTS: We used bioinformatic approaches to identify members of the J. sigillata ARF gene family and analyze their evolutionary relationships, collinearity, cis-acting elements, and tissue-specific expression patterns. The expression patterns of ARF gene family members under natural drought conditions were also analyzed. The J. sigillata ARF gene family contained 31 members, which were unevenly distributed across 16 chromosomes. We constructed a phylogenetic tree of JsARF genes and other plant ARF genes. Cis-acting elements in the promoters of JsARF were predicted. JsARF28 showed higher expressions in both the roots and leaves. A heat map of the transcriptome data of the cluster analysis under drought stress indicated that JsARF3/9/11/17/20/26 are responsive to drought. The expression of the 11 ARF genes varied under PEG treatment and JsARF18 and JsARF20 were significantly up-regulated. CONCLUSIONS: The interactions between abiotic stresses and plant hormones are supported by our cumulative data, which also offers a theoretical groundwork for comprehending the ARF mechanism and drought resistance in J. sigillata.

A robust method for simultaneous determination of eight B vitamins in human serum by liquid chromatography tandem mass spectrometry.

The level of vitamin B group in human serum is an important index of human health. Among B vitamins, cyanocobalamin in serum is unstable and its content is extremely low. Rapid and simultaneous detection of multiple B vitamins including cyanocobalamin is a challenge. Herein, we have developed a rapid and stable method that can realize the determination of thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin simultaneously in 6 min. The method was established based on protein precipitation with methanol and then chromatographic separation was achieved using Waters acquity ultra-high-performance liquid chromatography high strength silica T3 column, which was stable and sensitive especially for cyanocobalamin. Limit of quantification, precision, trueness, and matrix effect were validated according to the European Medicines Agency and United States Food and Drug guidelines and Clinical and Laboratory Standards Institute guidelines on bioanalytical method. The limit of quantification for thiamine, riboflavin, nicotinamide, pantothenic acid, pyridoxic acid, biotin, 5-methyltetrahydrofolate, and cyanocobalamin was 0.4, 0.4, 0.8, 2.0, 0.4, 0.1, 0.4, and 0.04 ng/mL separately, respectively. Intra- and interday precisions were 1.1%-12.4% and 2.0%-13.5%, respectively. The relative errors were between 0.3% and 13.3%, and the matrix effects were between 2.6% and 10.4%.

ABA-responsive AREB1/ABI3-1/ABI5 cascade regulates IAA oxidase gene SlDAO2 to inhibit hypocotyl elongation in tomato.

Hypocotyl elongation is dramatically influenced by environmental factors and phytohormones. Indole-3-acetic acid (IAA) plays a prominent role in hypocotyl elongation, whereas abscisic acid (ABA) is regarded as an inhibitor through repressing IAA synthesis and signalling. However, the regulatory role of ABA in local IAA deactivation remains largely uncharacterized. In this study, we confirmed the antagonistic interplay of ABA and IAA during the hypocotyl elongation of tomato (Solanum lycopersicum) seedlings. We identified an IAA oxidase enzyme DIOXYGENASE FOR AUXIN OXIDATION2 (SlDAO2), and its expression was induced by both external and internal ABA signals in tomato hypocotyls. Moreover, the overexpression of SlDAO2 led to a reduced sensitivity to IAA, and the knockout of SlDAO2 alleviated the inhibitory effect of ABA on hypocotyl elongation. Furthermore, an ABA-responsive regulatory SlAREB1/SlABI3-1/SlABI5 cascade was identified to act upstream of SlDAO2 and to precisely control its expression. SlAREB1 directly bound to the ABRE present in the SlDAO2 promoter to activate SlDAO2 expression, and SlABI3-1 enhanced while SlABI5 inhibited the activation ability of SlAREB1 by directly interacting with SlAREB1. Our findings revealed that ABA might induce local IAA oxidation and deactivation via SlDAO2 to modulate IAA homoeostasis and thereby repress hypocotyl elongation in tomato.

The biogenesis, biological functions and modification of Circular RNAs.

Circular RNAs are covalently closed and non-coding in eukaryotes, which have tissue- specific and temporal-specific expression patterns whose biogenesis is regulated by transcription and splicing. Most circular RNAs are localized in the cytoplasm. The sequences and protein-binding elements of circular RNAs facilitate circular RNAs in exerting biological functions through complementary base pairing, regulating protein function or self-translation. Recent studies have revealed that N6-Methyladenosine (m6A), a prevalent post-transcriptional modification, can affect the translation, localization, and degradation of circular RNAs. Cutting-edge research into circular RNAs have benefitted from the development of high-throughput sequencing technology. Furthermore, the expansion of novel research methods has promoted progress into circular RNA research.

Efficacy of high-fidelity simulation in advanced life support training: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Simulation is an increasingly used novel method for the education of medical professionals. This study aimed to systematically review the efficacy of high-fidelity (HF) simulation compared with low-fidelity (LF) simulation or no simulation in advanced life support (ALS) training. METHODS: A comprehensive search of the PubMed, Chinese Biomedicine Database, Embase, CENTRAL, ISI, and China Knowledge Resource Integrated Database was performed to identify randomized controlled trials (RCTs) that evaluated the use of HF simulation in ALS training. Quality assessment was based on the Cochrane Handbook for Systematic Reviews of Interventions version 5.0.1. The primary outcome was the improvement of knowledge and skill performance. The secondary outcomes included the participants' confidence and satisfaction at the course conclusion, skill performance at one year, skill performance in actual resuscitation, and patient outcomes. Data were synthesized using the RevMan 5.4 software. RESULTS: Altogether, 25 RCTs with a total of 1,987 trainees were included in the meta-analysis. In the intervention group, 998 participants used HF manikins, whereas 989 participants received LF simulation-based or traditional training (classical training without simulation). Pooled data from the RCTs demonstrated a benefit in improvement of knowledge [standardized mean difference (SMD) = 0.38; 95% confidence interval (CI): 0.18-0.59, P = 0.0003, I2 = 70%] and skill performance (SMD = 0.63; 95% CI: 0.21-1.04, P = 0.003, I2 = 92%) for HF simulation when compared with LF simulation and traditional training. The subgroup analysis revealed a greater benefit in knowledge with HF simulation compared with traditional training at the course conclusion (SMD = 0.51; 95% CI: 0.20-0.83, P = 0.003, I2 = 61%). Studies measuring knowledge at three months, skill performance at one year, teamwork behaviors, participants' satisfaction and confidence demonstrated no significant benefit for HF simulation. CONCLUSIONS: Learners using HF simulation more significantly benefited from the ALS training in terms of knowledge and skill performance at the course conclusion. However, further research is necessary to enhance long-term retention of knowledge and skill in actual resuscitation and patient's outcomes.

[Phenotypic and genetic analysis of a child with partial trisomy 7q].

OBJECTIVE: To define the nature and origin of a chromosomal aberration in a child with unexplained growth and development retardation, and to analyze its genotype-phenotype correlation. METHODS: A child who had presented at the Affiliated Children's Hospital of Zhengzhou University on July 9, 2019 was selected as the study subject. Chromosomal karyotypes of the child and her parents were determined with routine G-banding analysis. Their genomic DNA was also analyzed with single nucleotide polymorphism array (SNP array). RESULTS: Karyotyping analysis combined with SNP array suggested that the chromosomal karyotype of the child was 46,XX,dup(7)(q34q36.3), whilst no karyotypic abnormality was found in either of her parents. SNP array has identified a de novo 20.6 Mb duplication at 7q34q36.3 [arr[hg19] 7q34q36.3(138335828_158923941)×3] in the child. CONCLUSION: The partial trisomy 7q carried by the child was rated as a de novo pathogenic variant. SNP array can clarify the nature and origin of chromosomal aberrations. Analysis of the correlation between genotype and phenotype can facilitate the clinical diagnosis and genetic counseling.

[Clinical characteristics and genetic analysis of two children with Tuberous sclerosis complex].

OBJECTIVE: To explore the clinical characteristics and genetic variants in two children with Tuberous sclerosis complex (TSC). METHODS: Two children who had presented at the Children's Hospital Affiliated to Zhengzhou University respectively in June 2020 and July 2021 were selected as the study subjects. Clinical data of the children were collected, and potential pathogenic variants were screened by whole exome sequencing (WES). Candidate variants were verified by Sanger sequencing of their family members. RESULTS: Child 1 was a 7-month-and-29-day-old male, and child 2 was a 2-year-and-6-month-old male. Both children had shown symptoms of epileptic seizures and multiple hypomelanotic macules. Genetic testing revealed that both children had harbored de novo variants of the TSC2 gene, namely c.3239_3240insA and c.3330delC, which were unreported previously. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were rated as pathogenic (PVS1+PS2+PM2_Supporting). CONCLUSION: This study has uncovered the genetic etiology for two children with TSC. Above findings have also enriched the phenotypic and mutational spectrum of TSC in the Chinese population.

[Analysis of CLCN4 gene variant in a child with Raynaud-Claes syndrome].

OBJECTIVE: To analyze the clinical phenotype and genetic variant in a child with Raynaud-Claes syndrome (RCS). METHODS: A child who was diagnosed with RCS at the Children's Hospital Affiliated to Zhengzhou University for delayed language and motor development in August 2022 was selected as the study subject. Clinical data of the child were collected, and potential genetic variant was detected by next-generation sequencing and Sanger sequencing. The pathogenicity of the candidate variant was analyzed. RESULTS: The child, a 4-year-and-4-month-old male, has manifested global developmental delay, speech disorders, special facial features and behavioral abnormalities. Genetic testing revealed that he has harbored a hemizygous c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene, which was not detected in either of his parents. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was rated as pathogenic (PVS1+PS2+PM2_Supporting). CONCLUSION: The c.1174C>T (p.Gln392Ter) variant of the CLCN4 gene probably underlay the PCS in this child. Above finding has expanded the mutational spectrum of the CLCN4 gene and enabled genetic counseling and prenatal diagnosis for his family.

[Protective Effect of Mesenchymal Stem Cell Transplantation on Intestinal Injury in Septic Mice and Its Mechanism].

Objective: To explore the protective effect of placenta-derived mesenchymal stem cells (P-MSCs) transplantation on intestinal injury in septic mice and its mechanism. Methods: A total of 24 mice were randomly assigned to 3 groups, a sham operation group, a sepsis group that underwent cecal ligation and puncture (CLP) procedure, and a group that received CLP and P-MSCs treatment. Hereinafter, the three groups are referred to as the Sham group, the CLP group, and the CLP+P-MSCs group. For the mice in the Sham group, the abdomen was cut open and the cecum was exposed and then placed back in the abdomen. CLP was performed in the other two groups to establish the sepsis model. Mice in the Sham and the CLP groups received 0.1 mL of 0.9% NaCl injection in the tail vein 1 hour after operation, while mice in the CLP+P-MSCs group received 2×10 5 P-MSCs infusion 1 hour after operation. Intestinal and blood specimens were collected from the mice in each group 24 hours after P-MSCs transplantation. Hematoxylin and eosin (HE) staining of the intestinal tissue was performed for pathological evaluation. The serum concentrations of D-lactic acid, diamine oxidase (DAO), endotoxin, interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6, IL-10, and transforming growth factor (TGF)-ß were determined by enzyme linked immunosorbent assay (ELISA). The gene expression of the relevant inflammatory factors in the small intestinal tissue was determined by real-time fluorescence polymerase chain reaction. The expression of zonula occludens protein-1 (ZO-1) and occludin protein in the intestine was determined by Western blot, the infiltration of intestinal macrophages was determined by immunohistochemical method, and the polarization of macrophages was determined by immunofluorescence. Results: The exogenous transplantation of P-MSCs could form colonies in the injured intestines of septic mice. Compared with those of the CLP group, the intestinal injury of the CLP+P-MSCs group was significantly alleviated, the serum concentrations of D-lactic acid, DAO, endotoxin, IL-1ß, IL-6, and TNF-α were significantly decreased ( P<0.05), while the serum concentrations of IL-10 and TGF-ß were significantly increased ( P<0.05), the expression levels of IL-1 ß, TNF-α and IL-6 genes in the intestinal tissue were significantly decreased ( P<0.05), while the expression levels of IL-10 and TGF-ß genes were significantly increased ( P<0.05), and the expression of ZO-1 and occludin proteins in the intestine was also significantly increased ( P<0.05). In addition, the distribution of macrophages in the intestinal tissue of the CLP+P-MSCs group decreased significantly and the macrophages showed a tendency for M2 polarization. Conclusion: Exogenous transplantation of P-MSCs can significantly reduce inflammatory injury and improve the intestinal barrier function in septic mice with intestinal injury. Reduction in the infiltration of macrophages and promotion of the polarization of macrophages from M1 to M2 may be the mechanisms underlying the reduction of inflammation.

Lumi-HOF@Tb as Probes for Multiple Ratiometric Fluorescence and Chemiluminescence Sensing of α-Glucosidase.

The innovative assembly of luminescent hydrogen-bonded organic frameworks (HOFs) into multifunctional optical sensors is of great significance for developing advanced materials. Herein, we report a facile room-temperature synthesis strategy for the luminol HOF modified by Tb3+ (Lumi-HOF@Tb) and featuring sensitive chemiluminescence and fluorescence characteristics. Lumi-HOF@Tb is further pioneered as a dual-signal sensor for selective detection of α-glucosidase, a type of enzyme that plays a crucial role in the digestion of carbohydrates, and screening of its inhibitors. The sensor is constructed by combining the dual optical characteristics of luminol from the HOF and lanthanide ion assistance. From the hydrolysis of α-glucosidase and the 4-nitrophenyl-α-d-glucopyranoside (pNGP) substrate emerges the fluorescent luminol-p-nitrophenol (pNP) complex at 466 nm and changes the inner filter absorption to recover Tb3+ characteristic fluorescence at 546 nm; luminol also produces a chemiluminescence signal driven by H2O2 from additional glucose oxidase-catalyzed hydrolysis of α-d-glucose. Fluorescence and chemiluminescence assays for α-glucosidase activity have therefore been established and exhibit detection limits as low as 0.04 and 0.005 U L-1, respectively. This study not only presents the possibility of Ln3+-HOF-based sensors as intelligent optical materials by integration of fluorescence and chemiluminescence techniques but also demonstrates great potential for future applications in biosensing.

The evaluation of preventive and control measures on congenital syphilis in Guangdong Province, China: a time series modeling study.

PURPOSE: To evaluate the effectiveness of preventive and control measures for congenital syphilis (CS) implemented since 2012 in Guangdong Province, China, and assess the epidemic trend in the near future. METHODS: The interrupted time series analysis was conducted to compare changes in slope and level of CS notification rate from 2005 to 2020 in Guangdong Province and its three regions with different economic developmental levels. The ARIMA model was established to predict the new CS case number of Guangdong Province in 2021. RESULTS: A total of 12,687 CS cases were reported from 2005 to 2020. The CS notification rate of the province had been increasing until 2012 (128.55 cases per 100,000 live births) and then been decreasing constantly, hitting the lowest point in 2020 (5.76 cases per 100,000 live births). The severe epidemic cluster shifted from the developed region to underdeveloped ones over time. The effectiveness of the measures was proved by the significant change in the slope of the notification rate which was found in both of the provinces (- 18.18, 95% CI - 25.63 to - 10.75) and two less-developed regions (- 10.49, 95% CI - 13.13 to - 7.86 and - 32.89, 95% CI - 41.67 to - 24.10, respectively). In the developed region where the notification rate had already been decreasing in the pre-implementation period, implementing these measures also aided in hastening the rate of descent. The CS case number in 2021 was predicted to be 48, indicating a low-level epidemic. CONCLUSIONS: The preventive and control measures have assisted Guangdong Province to control CS effectively, of which the supportive ones ensured a successful implementation. For resource-limited countries where CS is still endemic, especially guaranteeing the support in financial subsidy, professional training, supervision and so on might trigger the effectiveness of other measures and eventually make significant and sustainable progress.

Randomised controlled trial: effect of metformin add-on therapy on functional cure in entecavir-treated patients with chronic hepatitis B.

INTRODUCTION AND OBJECTIVES: Hepatitis B surface antigen (HBsAg) clearance, indicating functional cure or resolved chronic hepatitis B (CHB), remains difficult to achieve via nucleos(t)ide analogue monotherapy. We investigated whether metformin add-on therapy could help achieve this goal in entecavir-treated patients with hepatitis B e antigen (HBeAg)-negative CHB. PATIENTS AND METHODS: Patients with HBeAg-negative CHB who met eligibility criteria (entecavir treatment for > 12 months, HBsAg < 1000 IU/mL) were randomly assigned (1:1) to receive 24 weeks of either metformin (1000 mg, oral, once a day) or placebo (oral, once a day) add-on therapy. The group allocation was blinded for both patients and investigators. Efficacy and safety analyses were based on the intention-to-treat set. The primary outcome, serum HBsAg level (IU/mL) at weeks 24 and 36, was analysed using mixed models. RESULTS: Sixty eligible patients were randomly assigned to the metformin (n = 29) and placebo (n = 31) groups. There was no substantial between-group difference in the HBsAg level at week 24 (adjusted mean difference 0.05, 95% confidence interval -0.04 to 0.13, p = 0.278) or week 36 (0.06, -0.03 to 0.15, p = 0.187), and no significant effect of group-by-time interaction on the HBsAg level throughout the trial (p = 0.814). The occurrence of total adverse events between the two groups was comparable (9 [31.0%] of 29 vs. 5 [16.1%] of 31, p = 0.227) and no patient experienced serious adverse events during the study. CONCLUSION: Although it was safe, metformin add-on therapy did not accelerate HBsAg clearance in entecavir-treated patients with HBeAg-negative CHB.

Sandwich-type electrochemical immunosensor based on nitrogen-doped porous carbon and nanoporous trimetallic nanozyme (PdAgCu) for determination of prostate specific antigen.

A sandwich-type electrochemical immunosensor was designed for the ultrasensitive detection of prostate-specific antigen (PSA), using Au nanoparticles (Au NPs) modified nitrogen-doped porous carbon (NPC) as sensor platform and trimetallic PdAgCu mesoporous nanospheres (PdAgCu MNSs) as enzyme-mimicking labels. NPC was prepared by a facile one-step pyrolysis strategy of biomimetic phylloid zeolite imidazole framework (ZIF-L) nanosheets. Through this strategy, the graphitization of the microcrystalline structure enhanced the electrical conductivity, while its enlarged specific surface area and abundant pore volume can enrich H2O2 to improve the catalytic efficiency. Moreover, Au NPs were used to modify NPC without cross-linking agents to further optimize electron transport while capturing primary antibodies, improving stability and sensitivity of the immunosensor. PdAgCu MNSs with uniform size, cylindrical open mesoporous channels, and continuous crystal frame structure were self-assembling synthesized by electrostatic adsorption and ascorbic acid (AA) co-reduction with amphiphilic dioctadecyldimethylammonium chloride (DODAC) as surfactant-cum-micelle, whose unique structure maximizes the use of polyatoms to expose catalytic sites, exhibiting good biocompatibility and electrocatalytic ability. Under the optimal conditions, the immunosensor showed superior sensitivity, a wide dynamic detection range (10 fg mL-1 ~ 100 ng mL-1) and a low limit of detection (LOD, 3.29 fg mL-1). This work provides a convenient strategy for the clinical detection of PSA.

Enhancing cross-Kerr coupling via mechanical parametric amplification.

We present a proposal to enhance the cross-Kerr coupling between the cavity and the mechanical oscillator significantly. Specifically, the periodic modulation of the mechanical spring constant induces strong mechanical parametric amplification, which leads to the cross-Kerr nonlinear enhancement. Also, we discuss its application in photon-phonon blockade and phonon-number measurement. We find that under the strong cross-Kerr coupling condition, not only the photon-phonon blockade effect is dramatically enhanced but also different phonon number is clearly distinguished. Our results offer an alternative approach to perform quantum manipulation between photon and phonon.

Enhancement of charge sensitivity by nonlinear optomechanics.

Quantum estimation of electrical charge is investigated by using nonlinear optomechanical interaction. Due to the light-matter decoupling at one mechanical period, we need to consider only the cavity state, meaning that no direct access to the oscillator state is required. It is shown that the charge sensitivity can be greatly improved by enhancing optomechanical coupling. Further, we find that our theoretical result can surpass the sensitivity obtained from electrical measurements.

PP2A-C may be a promising candidate for postmortem interval estimation.

Determining the postmortem interval (PMI) is an important task in forensic pathology. However, a reliable means of determining the PMI between 24 h and approximately 7 days after death has not yet been established. A previous study demonstrated that subunit A of protein phosphatase 2A (PP2A-A) is a promising candidate to estimate the PMI during the first 96 h. However, more detailed work is still needed to investigate PP2A's function in PMI estimation. PP2A is a serine/threonine phosphatase consisting of three subunits (PP2A-A, PP2A-B, and PP2A-C), and its activation is reflected by Tyr-307 phosphorylation of the catalytic subunit (P-PP2A-C). In this study, we speculated that the other two subunits of PP2A and the activation of PP2A may play different roles in estimating the PMI. For this purpose, mice were euthanized and stored at different temperatures (4, 15, and 25 °C). At each temperature, the musculus vastus lateralis was collected at different time points (0, 24, 48, and 96 h) to investigate the degradation of PP2A-B, PP2A-C, and P-PP2A-C (Tyr-307). Homocysteine (Hcy) was used to establish a hyperhomocysteinemia animal model to explore the effects of plasma Hcy on PMI estimation. The data showed not only that PP2A-C was more stable than PP2A-B, but also that it was not affected by homocysteine (Hcy). These characteristics make PP2A-C a promising candidate for short-term (24 h to 48 h) PMI estimation.

Dual-mode imaging of copper transporter 1 in HepG2 cells by hyphenating confocal laser scanning microscopy with laser ablation ICPMS.

Copper transporter 1 (CTR1) is a transport protein involved in copper and cisplatin uptake. The visualization of cellular CTR1 migration and its redistribution is highly important in copper/cisplatin exposure/transport. However, to the best of our knowledge, this is a highly challenging task. Herein, a dual-mode imaging strategy for CTR1 is developed by hyphenating confocal laser scanning microscopy (CLSM) and laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) with a fluorescent/elemental bifunctional tag conjugated with anti-CTR1 antibody. The tag consists of rhodamine B and zirconium metal-organic frameworks (Zr-MOF) for CLSM fluorescence imaging and LA-ICPMS element imaging for a same group of HepG2 cells in a designated visual zone. This dual-mode imaging strategy facilitates visualization of CTR1 migration and meanwhile provides information of CTR1 redistribution in HepG2 cells by uptake of divalent copper or cisplatin. The present dual-mode imaging strategy provides in-depth information for the elucidation of CTR1 involved biological processes. Graphical abstract.