Recent progress of methods for cuproptosis detection.

Varying from other identified cell death pathways, cuproptosis is a new type of regulated cell death characterized by excess Cu ions, abnormal aggregation of lipoylated proteins in TCA cycle, loss of Fe-S cluster proteins, upregulation of HSP70, leading to proteotoxic and oxidative stress. Cuproptosis is highly concerned by scientific community and as the field of cuproptosis further develops, remarkable progress has been made in the verification and mechanism of cuproptosis, and methods used to detect cuproptosis have been continuously improved. According to the characteristic changes of cuproptosis, techniques based on cell death verification, Cu content, morphology, molecular biology of protein levels of cuproptosis-related molecules and biochemical pathways of cuproptosis-related enzyme activity and metabolites of oxidative stress, lipoic acid, TCA cycle, Fe-S cluster proteins, oxidative phosphorylation, cell respiration intensity have been subject to cuproptosis verification and research. In order to further deepen the understanding of detecting cuproptosis, the principle and application of common cuproptosis detection methods are reviewed and categorized in cellular phenomena and molecular mechanism in terms of cell death, Cu content, morphology, molecular biology, biochemical pathways with a flow chart. All the indicating results have been displayed in response to the markers of cuproptosis, their advantages and limitations are summaried, and comparison of cuproptosis and ferroptosis detection is performed in this study. Our collection of methods for cuproptosis detection will provide a great basis for cuproptosis verification and research in the future.

A nomogram incorporating Psoas muscle index for predicting tumor recurrence after liver transplantation: A retrospective study in an Eastern Asian population.

Background and aims: Tumor recurrence significantly affects the prognostic outcomes for liver cancer patients following liver transplantation. However, existing predictive models often neglect the inclusion of body composition indicators. Hence, this research aimed to investigate the significance of the psoas muscle index (PMI) in evaluating the post-transplant prognosis of liver cancer. Methods: A retrospective analysis was conducted on liver cancer patients who underwent liver transplantation surgery. Imaging analysis was performed using CT data to calculate PMI based on the left and right psoas muscle areas. Subsequently, the patients were categorized into PMI-Low and PMI-High groups using the established cut-off values. Univariate and multivariate analyses were performed using Cox proportional hazards regression to assess the correlation between PMI and clinical outcomes, and a nomogram was constructed accordingly. Results: Among the 225 patients included in the analysis, the PMI-High group exhibited significantly improved overall survival (P < 0.001) and disease-free survival (DFS, P < 0.001) rates compared to the PMI-Low group. PMI exhibited a positive correlation with body mass index (R = 0.25, P < 0.001), but no significant correlations were observed. In the multivariate analysis, PMI (HR = 4.596, P < 0.001), MELD score (HR = 1.591, P = 0.038), and Hangzhou criteria (HR = 2.557, P < 0.001) emerged as significant predictors of DFS. The constructed nomogram, incorporating these predictors, demonstrated outstanding predictive performance. Decision curve analysis revealed the superiority of the nomogram over conventional methods. Conclusions: PMI serves as a valuable prognostic factor for tumor recurrence in liver cancer patients after liver transplantation. The established nomogram is pivotal in delivering personalized predictions of DFS.

Versatile, reusable and highly sensitive SERS-based point-of-care testing microplatform for reliable ATP detection.

The advancement in miniaturized Raman spectrometers, coupled with the single-molecule-level sensitivity and unique fingerprint identification capability of surface-enhanced Raman scattering (SERS), offers great potential for point-of-care testing (POCT). Despite this, accurately quantifying analyte molecules, particularly in complex samples with limited sample volumes, remains difficult. Herein, we present a versatile and reusable SERS microplatform for highly sensitive and reliable quantitative detection of adenosine triphosphate (ATP) in biological fluids. The platform utilizes gold-Prussian blue core-shell nanoparticles modified with polyethyleneimine (Au@PB@PEI NPs), embedded within gold nanoparticle-immobilized capillary-based silica monolithic materials. PB acts as an internal standard, while PEI enhances molecular capture. The periodic, bimodal porous structure of the silica monolithic materials provides uniform and abundant sites for nanoparticle attachment, facilitating rapid liquid permeation, intense SERS enhancement, and efficient enrichment. The platform regulates ATP capture and release through magnesium ions in the liquid phase, eliminating matrix interferences and enabling platform reuse. Integrating efficient molecular enrichment, separation, an interference-free internal standard, a liquid flow channel, and a detection chamber, our platform offers simplicity in operation, exceptional sensitivity and accuracy, and rapid analysis (∼10 min). Employing PB as an internal calibration standard, ratiometric Raman signals (I732/I2123) facilitate precise ATP quantification, achieving a remarkable limit of detection down to 0.62 pM. Furthermore, this platform has been proven to be highly reproducible and validated for ATP quantification in both mouse cerebrospinal fluid and human serum, underscoring its immense potential for POCT applications.

Ultrasound-assisted nucleation and growth of hydroxyl-protected and ligand-free Cs3Cu2X5 nanocrystals with bright luminescence.

The commercial applications of lead halide perovskites are hindered by their negative environmental impact and inherent instability. Consequently, developing environmentally friendly copper-based perovskite materials is crucial for future solid-state lighting and display applications. In this study, an ultrafast high-power ultrasonic synthesis strategy was utilized to achieve uniform nucleation and growth of Cs3Cu2X5 (X = Cl, Br, I) nanocrystals (NCs) that possess remarkable luminescence properties, hydroxyl protection, and ligand-free characteristics. These Cs3Cu2X5 NCs exhibited a tunable spectral range spanning from 446 to 525 nm, accompanied by photoluminescence quantum yields (PLQYs) varying from 0.2% to 79.2%. The spectral attributes of the NCs were effectively controlled by modulating the halide type and composition. It is worth noting that density functional theory (DFT) calculations offer valuable insights into the synthesis of NCs and the selection of suitable alcohol solvents. Moreover, we successfully fabricated an efficient and stable white light-emitting diode (WLED) with a high luminous efficiency of 23 lm W-1 and CIE color coordinates of (0.3266, 0.3487). Our work provides a new strategy to synthesize Cs3Cu2X5 NCs and holds promise for their potential application in display and lighting devices.

Unveiling the Pharmacological Role of Human Deubiquitinating Enzymes in Temozolomide Response of Glioblastoma Cells.

Temozolomide (TMZ) stands as the primary chemotherapeutic drug utilized in clinical glioma treatment, particularly for high-grade glioblastoma (GBM). However, the emergence of TMZ resistance in GBM poses a significant hurdle to its clinical efficacy. Our objective was to elucidate the role of deubiquitinating enzymes (DUBs) in GBM cell resistance to TMZ. We employed the broad-spectrum DUBs inhibitor G5 to investigate the function of DUBs in TMZ cytotoxicity against GBM cells. Eighty-two GBM cell lines with specified DUBs knockout were generated and subjected to CCK-8 assays to assess cell proliferation and TMZ resistance. Furthermore, the association between DUBs and TMZ resistance in GBM cells, along with the modulation of autophagic flux, was examined. The pan-DUBs inhibitor G5 demonstrated the ability to induce cell death and enhance TMZ toxicity in GBM cells. Subsequently, we identified potential DUBs involved in regulating GBM cell proliferation and TMZ resistance. The impact of DUBs knockout on TMZ cytotoxicity was found to be associated with their regulation of TMZ-induced autophagy. In summary, our study provides primary insights into the role of DUBs in GBM cell proliferation and TMZ resistance, and contributes to a deeper understanding of the complex function of DUBs genes underlying TMZ resistance in GBM cells.

Applications, advancements, and challenges of 3D bioprinting in organ transplantation.

To date, organ transplantation remains an effective method for treating end-stage diseases of various organs. In recent years, despite the continuous development of organ transplantation technology, a variety of problems restricting its progress have emerged one after another, and the shortage of donors is at the top of the list. Bioprinting is a very useful tool that has huge application potential in many fields of life science and biotechnology, among which its use in medicine occupies a large area. With the development of bioprinting, advances in medicine have focused on printing cells and tissues for tissue regeneration and reconstruction of viable human organs, such as the heart, kidneys, and bones. In recent years, with the development of organ transplantation, three-dimensional (3D) bioprinting has played an increasingly important role in this field, giving rise to many unsolved problems, including a shortage of organ donors. This review respectively introduces the development of 3D bioprinting as well as its working principles and main applications in the medical field, especially in the applications, and advancements and challenges of 3D bioprinting in organ transplantation. With the continuous update and progress of printing technology and its deeper integration with the medical field, many obstacles will have new solutions, including tissue repair and regeneration, organ reconstruction, etc., especially in the field of organ transplantation. 3D printing technology will provide a better solution to the problem of donor shortage.

Successful liver transplantation with ctDNA clearance after PD­1 inhibitor plus FOLFOX­HAIC treatment in HCC: A case report.

Liver transplantation (LT) is the primary treatment for patients with early-stage hepatocellular carcinoma (HCC). However, the 5-year survival rate after LT remains low for patients with advanced HCC. Recently, combining programmed cell death protein-1 (PD-1) inhibitors with hepatic arterial infusion chemotherapy (HAIC) has achieved promising outcomes in advanced HCC treatment. However, there is a lack of sufficient clinical data demonstrating its effectiveness as a pre-LT down-staging treatment. The current study presented a case of advanced HCC beyond the Milan criteria who underwent LT and achieved a favorable outcome following PD-1 inhibitor combined with FOLFOX-HAIC therapy. Of note, due to treatment-induced tumor necrosis, precise post-treatment tumor size evaluation became challenging. To address this, circulating tumor DNA (ct-DNA) clearance was used as the LT criterion. After three cycles of Pembrolizumab and FOLFOX-HAIC therapy, the patient's serum ctDNA became undetectable and serum α-fetoprotein levels returned to normal. Magnetic resonance imaging results also revealed a significant reduction in liver tumor size post down-staging treatment. Subsequent to LT, serum ctDNA was monitored every two months, consistently yielding diminished results. There were no clinical signs of recurrence 19 months post-LT. These findings suggest that Pembrolizumab in combination with FOLFOX-HAIC may serve as a potential down-staging strategy prior to LT. In addition, ctDNA clearance may be considered a viable biomarker for LT eligibility.

[Application of single base editing technique in pig genetic improvement: a review].

Traditional pig breeding has a long cycle and high cost, and there is an urgent need to use new technologies to revitalize the pig breeding industry. The recently emerged CRISPR/Cas9 genome editing technique shows great potential in pig genetic improvement, and has since become a research hotspot. Base editor is a new base editing technology developed based on the CRISPR/Cas9 system, which can achieve targeted mutation of a single base. CRISPR/Cas9 technology is easy to operate and simple to design, but it can lead to DNA double strand breaks, unstable gene structures, and random insertion and deletion of genes, which greatly restricts the application of this technique. Different from CRISPR/Cas9 technique, the single base editing technique does not produce double strand breaks. Therefore, it has higher accuracy and safety for genome editing, and is expected to advance the pig genetic breeding applications. This review summarized the working principle and shortcomings of CRISPR/Cas9 technique, the development and advantages of single base editing, the principles and application characteristics of different base editors and their applications in pig genetic improvement, with the aim to facilitate genome editing-assisted genetic breeding of pig.

L-carnitine promotes liver regeneration after hepatectomy by enhancing lipid metabolism.

BACKGROUND: Lipid metabolism plays an important role in liver regeneration, but its regulation still requires further research. In this study, lipid metabolites involved in mouse liver regeneration at different time points were sequenced and analyzed to study their influence on liver regeneration and its mechanism. METHODS: Our experiment was divided into two parts. The first part examined lipid metabolites during liver regeneration in mice. In this part, lipid metabolites were sequentially analyzed in the livers of 70% mouse hepatectomy models at 0, 1, 3and 7 days after operation to find the changes of lipid metabolites in the process of liver regeneration. We screened L-carnitine as our research object through metabolite detection. Therefore, in the second part, we analyzed the effects of carnitine on mouse liver regeneration and lipid metabolism during liver regeneration. We divided the mouse into four groups: control group (70% hepatectomy group); L-carnitine group (before operation) (L-carnitine were given before operation); L-carnitine group (after operation)(L-carnitine were given after operation) and L-carnitine + perhexiline maleate (before operation) group. Weighing was performed at 24 h, 36 and 48 h in each group, and oil red staining, HE staining and MPO staining were performed. Tunnel fluorescence staining, Ki67 staining and serological examination. RESULTS: Sequencing analysis of lipid metabolites in 70% of mouse livers at different time points after hepatectomy showed significant changes in carnitine metabolites. The results showed that, compared with the control group the mouse in L-carnitine group (before operation) at 3 time points, the number of fat drops in oil red staining was decreased, the number of Ki67 positive cells was increased, the number of MPO positive cells was decreased, the number of Tunnel fluorescence positive cells was decreased, and the liver weight was increased. Serum enzymes were decreased. Compared with control group, L-carnitine group (after operation) showed similar trends in all indexes at 36 and 48 h as L-carnitine group (before operation). L-carnitine + perhexiline maleate (before operation) group compared with control group, the number of fat drops increased, the number of Ki67 positive cells decreased, and the number of MPO positive cells increased at 3 time points. The number of Tunnel fluorescent positive cells increased and serum enzyme increased. However, both liver weights increased. CONCLUSION: L-carnitine can promote liver cell regeneration by promoting lipid metabolism and reduce aseptic inflammation caused by excessive lipid accumulation.

The tree shrew as a new animal model for the study of periodontitis.

AIM: Periodontitis is an inflammatory, infectious disease of polymicrobial origin that can damage tooth-supporting bone and tissue. Tree shrews, evolutionarily closer to humans than commonly used rodent models, have been increasingly used as biomedical models. However, a tree shrew periodontitis model has not yet been established. MATERIALS AND METHODS: Periodontitis was induced in male tree shrews/Sprague-Dawley rats by nylon thread ligature placement around the lower first molars. Thereafter, morphometric and histological analyses were performed. The distance from the cemento-enamel junction to the alveolar bone crest was measured using micro-computed tomography. Periodontal pathological tissue damage, inflammation and osteoclastogenesis were assessed using haematoxylin and eosin staining and quantitative immunohistochemistry, respectively. RESULTS: Post-operatively, gingival swelling, redness and spontaneous bleeding were observed in tree shrews but not in rats. After peaking, bone resorption decreased gradually until plateauing in tree shrews. Contrastingly, rapid and near-complete bone loss was observed in rats. Inflammatory infiltrates were observed 1 week post operation in both models. However, only the tree shrew model transitioned from acute to chronic inflammation. CONCLUSIONS: Our study revealed that a ligature-induced tree shrew model of periodontitis partly reproduced the pathological features of human periodontitis and provided theoretical support for using tree shrews as a potential model for human periodontitis.

Elevated prelimbic cortex-to-basolateral amygdala circuit activity mediates comorbid anxiety-like behaviors associated with chronic pain.

Chronic pain can cause both hyperalgesia and anxiety symptoms. However, how the two components are encoded in the brain remains unclear. The prelimbic cortex (PrL), a critical brain region for both nociceptive and emotional modulations, serves as an ideal medium for comparing how the two components are encoded. We report that PrL neurons projecting to the basolateral amygdala (PrLBLA) and those projecting to the ventrolateral periaqueductal gray (PrLl/vlPAG) were segregated and displayed elevated and reduced neuronal activity, respectively, during pain chronicity. Consistently, optogenetic suppression of the PrL-BLA circuit reversed anxiety-like behaviors, whereas activation of the PrL-l/vlPAG circuit attenuated hyperalgesia in mice with chronic pain. Moreover, mechanistic studies indicated that elevated TNF-α/TNFR1 signaling in the PrL caused increased insertion of GluA1 receptors into PrLBLA neurons and contributed to anxiety-like behaviors in mice with chronic pain. Together, these results provide insights into the circuit and molecular mechanisms in the PrL for controlling pain-related hyperalgesia and anxiety-like behaviors.

Astrocytic lactate dehydrogenase A regulates neuronal excitability and depressive-like behaviors through lactate homeostasis in mice.

Alterations in energy metabolism are associated with depression. However, the role of glycolysis in the pathogenesis of depression and the underlying molecular mechanisms remain unexplored. Through an unbiased proteomic screen coupled with biochemical verifications, we show that the levels of glycolysis and lactate dehydrogenase A (LDHA), a glycolytic enzyme that catalyzes L-lactate production, are reduced in the dorsomedial prefrontal cortex (dmPFC) of stress-susceptible mice in chronic social defeat stress (CSDS) model. Conditional knockout of LDHA from the brain promotes depressive-like behaviors in both male and female mice, accompanied with reduced L-lactate levels and decreased neuronal excitability in the dmPFC. Moreover, these phenotypes could be duplicated by knockdown of LDHA in the dmPFC or specifically in astrocytes. In contrast, overexpression of LDHA reverses these phenotypic changes in CSDS-susceptible mice. Mechanistic studies demonstrate that L-lactate promotes neuronal excitability through monocarboxylic acid transporter 2 (MCT2) and by inhibiting large-conductance Ca2+-activated potassium (BK) channel. Together, these results reveal a role of LDHA in maintaining neuronal excitability to prevent depressive-like behaviors.

USP25 deficiency promotes T cell dysfunction and transplant acceptance via mitochondrial dynamics.

BACKGROUND: During organ transplantation, pharmacologic drugs targeting T cell activation signal to inhibit T cell-mediated allo-rejection are insufficient and not durable to suppress chronic rejection. Recent advances highlight an exhausted or dysfunctional status of T cells, which favor transplant acceptance. METHODS: The models of MHC-mismatched (BALB/c to C57BL/6 or USP25 KO mice) heterotopic heart transplantation and skin transplantation were utilized to evaluate the regulatory effects of ubiquitin-specific protease 25(USP25) deficiency in vivo. The consequences of USP25 deficiency on murine T-cell proliferation, activation, cytokine secretion, mixed lymphocyte reaction (MLR) and energy metabolism were investigated in vitro. The signaling pathway of T cells in knock out mice was detected by Western blotting and Co-IP. RESULTS: We found T cells were dysfunctional inUSP25KO mice. Due to T cell dysfunction, skin and heart graft had a longer survival. In these dysfunctional T cells, mitochondria number and cristae condensation were decreased. Impaired mitochondrial mass and function favored to allo-graft acceptance. Furthermore, USP25 interacted with ATP5A and ATP5B to promote their stability. CONCLUSIONS: Our data suggest that USP25 is a potential target to induce T cell dysfunction and allo-graft tolerance. And USP25 mediated mitochondrial homeostasis may contribute to reverse T cell exhaustion or dysfunction in tumor and chronic infection.

Bioinspired polydopamine-mediated metal-organic framework click-grafting aptamers functionalized fabric for highly-specific recognition of microcystin-leucine arginine.

Fabricating functional electrospun nanofiber coating for highly selective extraction of microcystin-LR (MC-LR) was of significant importance for water-safety monitoring. Herein, a novel MOF@aptamer functionalized nanofabric was presented via a facile and reliable strategy integrating polydopamine (PDA) mediation and thiol-ene chemistry and applied for specific recognition of the MC-LR model analyte. Using polydopamine (PDA) as the mediating layer, vinyl-UiO-66 MOF was grown in situ, followed by post-synthetic modification (PSM) of Zr4+ with vinyl phosphate and rapid UV-initiated click reaction of aptamers. Uniform deposition of Zr-based MOF (vinyl-UiO-66) on the nanofibers was directly produced, and the tedious co-electrospinning process was abandoned to prevent the aggregation and encapsulation of MOF. Via an efficient "thiol-ene" chemistry, massive thiol-terminated aptamers were grafted on MOF within one step under friendly conditions, rather than the time-consuming nanoparticle adsorption or unfriendly covalent chemical reactions. As a result, the robust MOF@aptamer-coated nano-fabrics were obtained, and a highly selective performance towards MC-LR was illustrated with a limit of detection (LOD) at 0.002 ng/mL, good precision (CV<8.3%), good repeatability (2.2∼6.0%) when coupled with LC-MS. Almost 1∼2 orders of magnitude higher detection sensitivity was exhibited than that of the common non-specific SPE/SPME fiber reported so far. Applied to water samples, the good matrix-resistance ability, and acceptable recovery yields were achieved with high specificity. This strategy might provide a rapid and friendly protocol to efficiently fabricate MOF@aptamer functionalized nano-fabrics through electrospinning and interfacial "thiol-ene" chemistry for highly-selective microextraction.

Green synthesis of highly stable CsPbBr3 perovskite nanocrystals using natural deep eutectic solvents as solvents and surface ligands.

Perovskite nanocrystals (PNCs) have attracted widespread attention as promising materials for the optoelectronic field due to their remarkable photophysical properties and structural tunability. However, their poor stability and the use of toxic organic solvents in the preparation process have severely restricted their practical applications. Herein, a facile, rapid and toxic organic solvent-free synthesis strategy of CsPbBr3 PNCs was developed for the first time via the ligand-assisted reprecipitation (LARP) method using natural deep eutectic solvents (NADESs) as solvents and surface ligands. In this method, the NADESs not only functioned as solvents for green synthesis, but also served simultaneously as surface ligands of CsPbBr3 PNCs to significantly improve their optical properties and stability. The as-synthesized CsPbBr3 PNCs exhibited high photoluminescence quantum yield (PLQY, ∼96.8%), narrow full width at half-maximum (FWHM, ∼18.8 nm) and a high stability that retained 82.9% of PL intensity after 70 days. This work provides a new strategy for the green synthesis of PNCs, which promises feasibility for the industrial large-scale synthesis of high-quality PNCs.

Ratiometric fluorescence sensing of temperature based on perovskite nanocrystals and rhodamine B doped electrospun fibers.

Sensing temperature (T) has gained great attention since T is the most important parameter in daily life, scientific research and industry. A ratiometric fluorescence T sensor is fabricated by doping MAPbBr3 perovskite nanocrystals (PNCs) and rhodamine B (RhB) into a polyacrylonitrile (PAN) matrix and the composite materials are electrospun into optical fibers. The fibers show characteristic emissions at 521 and 587 nm under UV irradiation (λ ex = 365 nm). Both emission intensities gradually increased with elevating T, accompanied with a fluorescence color change from green to yellow. There is a linear relationship between fluorescence intensity ratio (I 521/I 587) and T in the range of 30-45 °C. The T response sensitivity is as high as 4.38% °C-1 at 45 °C.

Comparative study of dedifferentiated fat cell and adipose-derived stromal cell sheets for periodontal tissue regeneration: In vivo and in vitro evidence.

AIM: To compare the efficacy of adipocyte-derived dedifferentiated fat (DFAT) cell and adipose-derived stromal cell (ADSC) sheets for regenerative treatment of intra-bony periodontal defects. MATERIALS AND METHODS: DFAT cells were obtained using the ceiling culture method and were compared with ADSCs using Cell Counting Kit-8, colony formation assay, surface antigen identification, and multilineage differentiation assays. DFAT and ADSC sheets were prepared in a cell sheet culture medium. The biological characteristics of DFAT cell and ADSC sheets were compared using haematoxylin and eosin staining, quantitative reverse transcription polymerase chain reaction, and immunofluorescence staining. Micro-computed tomography and histological staining were used to compare the effects of the two cell sheets on the repair of periodontal intra-bony defects in rats. RESULTS: DFAT cells and ADSCs demonstrated mesenchymal stem cell characteristics. Both cell types were CD29-, CD90-, and CD146-positive and CD31-, CD34-, and CD45-negative. DFAT cells and ADSCs exhibited similar osteogenic and adipogenic differentiation capabilities and colony formation ability. DFAT cells displayed stronger proliferation capabilities compared with ADSCs. Compared with the ADSC sheets, DFAT cell sheets exhibited a higher expression of periodontal-related genes and proteins and greater ability to regenerate periodontal tissue. CONCLUSIONS: Our findings suggest that DFAT cell sheets are an ideal seed cell source and form of cell delivery for periodontal intra-bony defects.

Circadian misalignment leads to changes in cortisol rhythms, blood biochemical variables and serum miRNA profiles.

The circadian clock plays a critical role in synchronizing the inner molecular, metabolic and physiological processes to environmental cues that cycle with a period of 24 h. Non-24 h and shift schedules are commonly used in maritime operations, and both of which can disturb circadian rhythms. In this study, we first conducted an experiment in which the volunteers followed a 3-d rotary schedule with consecutive shift in sleep time (rotatory schedule), and analyzed the changes in salivary cortisol rhythms and blood variables. Next we conducted another experiment in which the volunteers followed an 8 h-on and 4-h off schedule (non-24-h schedule) to compare the changes in blood/serum variables. The rotatory schedule led to elevated levels of serum cortisol during the early stage, and the phase became delayed during the early and late stages. Interestingly, both of the schedules caused comprehensive changes in blood/serum biochemical variables and increased phosphate levels. Furthermore, transcriptomic analysis of the plasma miRNAs from the volunteers following the rotatory schedule identified a subset of serum miRNAs targeting genes involved in circadian rhythms, sleep homeostasis, phosphate transport and multiple important physiological processes. Overexpression of miRNAs targeting the phosphate transport associated genes, SLC20A1 and SLC20A2, showed altered expression due to rotary schedule resulted in attenuated cellular levels of phosphate, which might account for the changed levels in serum phosphate. These findings would further our understanding of the deleterious effects of shift schedules and help to optimize and enhance the performances and welfare of personnel working on similar schedules.

The ketogenic diet increases Neuregulin 1 expression via elevating histone acetylation and its anti-seizure effect requires ErbB4 kinase activity.

BACKGROUND: The ketogenic diet (KD)has been considered an effective treatment for epilepsy, whereas its underlying mechanisms remain obscure. We have previously reported that the KD feeding increased Neuregulin 1 (NRG1) expression in the hippocampus; disruption of NRG1 signaling by genetically deleting its receptor-ErbB4 abolished KD's effects on inhibitory synaptic activity and seizures. However, it is still unclear about the mechanisms underlying the effect of KD on NRG1 expression and whether the effects of KD require ErbB4 kinase activity. METHODS: The effects of the KD on NRG1 expression were assessed via western blotting and real-time PCR. Acetylation level at the Nrg1 promoter locus was examined using the chromatin immunoprecipitation technique. Kainic acid (KA)-induced acute seizure model was utilized to examine the effects of KD and histone deacetylase inhibitor-TSA on seizures. Synaptic activities in the hippocampus were recorded with the technique of electrophysiology. The obligatory role of ErbB4 kinase activity in KD's effects on seizures and inhibitory synaptic activity was evaluated by using ErbB kinase antagonist and transgenic mouse-T796G. RESULTS: We report that KD specifically increases Type I NRG1 expression in the hippocampus. Using the chromatin immunoprecipitation technique, we observe increased acetylated-histone occupancy at the Nrg1 promoter locus of KD-fed mice. Treatment of TSA dramatically elevates NRG1 expression and diminishes the difference between the effects of the control diet (CD) and KD. These data indicate that KD increases NRG1 expression via up-regulating histone acetylation. Moreover, both pharmacological and genetic inhibitions of ErbB4 kinase activity significantly block the KD's effects on inhibitory synaptic activity and seizure, suggesting an essential role of ErbB4 kinase activity. CONCLUSION: These results strengthen our understanding of the role of NRG1/ErbB4 signaling in KD and shed light on novel therapeutic interventions for epilepsy.

Quantitative Fluorescent in situ Hybridization Reveals Differential Transcription Profile Sharpening of Endocytic Proteins in Cochlear Hair Cells Upon Maturation.

The organ of Corti (OC) comprises two types of sensory cells: outer hair cells (OHCs) and inner hair cells (IHCs). While both are mechanotransducers, OHCs serve as cochlear amplifiers, whereas IHCs transform sound into transmitter release. Reliable sound encoding is ensured by indefatigable exocytosis of synaptic vesicles associated with efficient replenishment of the vesicle pool. Vesicle reformation requires retrieval of vesicle membrane from the hair cell's membrane via endocytosis. So far, the protein machinery for endocytosis in pre-mature and terminally differentiated hair cells has only partially been deciphered. Here, we studied three endocytic proteins, dynamin-1, dynamin-3, and endophilin-A1, by assessing their transcription profiles in pre-mature and mature mouse OCs. State-of-the-art RNAscope® fluorescent in situ hybridization (FISH) of whole-mount OCs was used for quantification of target mRNAs on single-cell level. We found that pre-mature IHCs contained more mRNA transcripts of dnm1 (encoding dynamin-1) and sh3gl2 (endophilin-A1), but less of dnm3 (dynamin-3) than OHCs. These differential transcription profiles between OHCs and IHCs were sharpened upon maturation. It is noteworthy that low but heterogeneous signal numbers were found between individual negative controls, which highlights the importance of corresponding analyses in RNAscope® assays. Complementary immunolabeling revealed strong expression of dynamin-1 in the soma of mature IHCs, which was much weaker in pre-mature IHCs. By contrast, dynamin-3 was predominantly found in the soma and at the border of the cuticular plates of pre-mature and mature OHCs. In summary, using quantitative RNAscope® FISH and immunohistochemistry on whole-mount tissue of both pre-mature and mature OCs, we disclosed the cellular upregulation of endocytic proteins at the level of transcription/translation during terminal differentiation of the OC. Dynamin-1 and endophilin-A1 likely contribute to the strengthening of the endocytic machinery in IHCs after the onset of hearing, whereas expression of dynamin-3 at the cuticular plate of pre-mature and mature OHCs suggests its possible involvement in activity-independent apical endocytosis.