Regulation of retinal amacrine cell generation by miR-216b and Foxn3.

The mammalian retina contains a complex mixture of different types of neurons. We find that microRNA miR-216b is preferentially expressed in postmitotic retinal amacrine cells in the mouse retina, and expression of miR-216a/b and miR-217 in retina depend in part on Ptf1a, a transcription factor required for amacrine cell differentiation. Surprisingly, ectopic expression of miR-216b directed the formation of additional amacrine cells and reduced bipolar neurons in the developing retina. We identify the Foxn3 mRNA as a retinal target of miR-216b by Argonaute PAR-CLIP and reporter analysis. Inhibition of Foxn3, a transcription factor, in the postnatal developing retina by RNAi increased the formation of amacrine cells and reduced bipolar cell formation. Foxn3 disruption by CRISPR in embryonic retinal explants also increased amacrine cell formation, whereas Foxn3 overexpression inhibited amacrine cell formation prior to Ptf1a expression. Co-expression of Foxn3 partially reversed the effects of ectopic miR-216b on retinal cell formation. Our results identify Foxn3 as a novel regulator of interneuron formation in the developing retina and suggest that miR-216b likely regulates Foxn3 and other genes in amacrine cells.

Filter bank second-order underdamped stochastic resonance analysis for implementing a short-term high-speed SSVEP detection.

OBJECTIVE: The progression of brain-computer interfaces (BCIs) has been propelled by breakthroughs in neuroscience, signal processing, and machine learning, marking it as a dynamic field of study over the past few decades. Nevertheless, the nonlinear and non-stationary characteristics of steady-state visual evoked potentials (SSVEPs), coupled with the incongruity between frequently employed linear techniques and nonlinear signal attributes, resulted in the subpar performance of mainstream non-training algorithms like canonical correlation analysis (CCA), multivariate synchronization index (MSI), and filter bank CCA (FBCCA) in short-term SSVEP detection. METHODS: To tackle this problem, the novel fusions of common filter bank analysis, CCA dimensionality reduction methods, USSR models, and MSI recognition models are used in SSVEP signal recognition. RESULTS: Unlike conventional linear techniques such as CCA, MSI, and FBCCA, the filter bank second-order underdamped stochastic resonance (FBUSSR) analysis demonstrates superior efficacy in the detection of short-term high-speed SSVEPs. CONCLUSION: This research enlists 32 subjects and uses a public dataset to assess the proposed approach, and the experimental outcomes indicate that the non-training method can attain greater recognition precision and stability. Furthermore, under the conditions of the newly proposed fusion method and light stimulation, the USSR model exhibits the most optimal enhancement effect. SIGNIFICANCE: The findings of this study underscore the expansive potential for the application of BCI systems in the realm of neuroscience and signal processing.

Development of On-DNA Cyclic Imide Synthesis for DNA Encoded Library Construction.

Here, we describe a novel method for the on-DNA synthesis of cyclic imides, an important class of molecules that includes several well-known medications. Significantly, the new method enabled on-DNA synthesis under mild conditions with high conversions and a broad functional group tolerance, utilizing ubiquitous bifunctional amines and bis-carboxylic acid, or alkyl halides, and therefore served as the linchpin for DNA encoded library (DEL) synthesis. The mechanism study of off-DNA and on-DNA chemical transformations revealed unique insights in contrast to conventional chemical transformation.

On-DNA Morita-Baylis-Hillman Reaction: Accessing Targeted Covalent Inhibitor Motifs in DNA-Encoded Libraries.

We herein present the first application of the on-DNA Morita-Baylis-Hillman (MBH) reaction for the creation of pharmaceutically relevant targeted covalent inhibitors (TCIs) with an α-hydroxyl Michael acceptor motif. Adapting a DNA-compatible organocatalytic process, this MBH reaction for covalent selection-capable DNA encoded library (DEL) synthesis grants access to densely functionalized and versatile precursors to explore novel chemical space for molecule recognition in drug discovery. Most importantly, this methodology sheds light on potentially unexpected reaction outcomes of the MBH reaction.

DNA Compatible Oxidization and Amidation of Terminal Alkynes.

Through a modified Kinugasa reaction, a novel method of amidation on terminal oligo alkyne conjugates by copper-promoted oxidation with nitrones has been developed. Unprotected bifunctional carboxylic acid-amine reagents can be transformed directly to the respective amide products under these edited Kinugasa reaction conditions. 3-Cycle DNA-encoded libraries (DELs) can be built in three steps of chemical conversion.

microRNA-mRNA Profile of Skeletal Muscle Differentiation and Relevance to Congenital Myotonic Dystrophy.

microRNAs (miRNAs) regulate messenger RNA (mRNA) abundance and translation during key developmental processes including muscle differentiation. Assessment of miRNA targets can provide insight into muscle biology and gene expression profiles altered by disease. mRNA and miRNA libraries were generated from C2C12 myoblasts during differentiation, and predicted miRNA targets were identified based on presence of miRNA binding sites and reciprocal expression. Seventeen miRNAs were differentially expressed at all time intervals (comparing days 0, 2, and 5) of differentiation. mRNA targets of differentially expressed miRNAs were enriched for functions related to calcium signaling and sarcomere formation. To evaluate this relationship in a disease state, we evaluated the miRNAs differentially expressed in human congenital myotonic dystrophy (CMD) myoblasts and compared with normal control. Seventy-four miRNAs were differentially expressed during healthy human myocyte maturation, of which only 12 were also up- or downregulated in CMD patient cells. The 62 miRNAs that were only differentially expressed in healthy cells were compared with differentiating C2C12 cells. Eighteen of the 62 were conserved in mouse and up- or down-regulated during mouse myoblast differentiation, and their C2C12 targets were enriched for functions related to muscle differentiation and contraction.

Organocatalytic Isomerization/Allylic Alkylation of O-Acylated Hemithioacetals and Their Application in Tandem Sequence to Access 2,7-Dioxabicyclo[2.2.1]heptan-3-one Derivatives.

A novel protocol for the efficient preparation of α-hydroxy allylic thioesters via a Lewis base-catalyzed tandem isomerization/allylic alkylation process is reported. The resulting allylic thioesters can serve as valuable scaffolds to undergo a stereoselective intramolecular cyclization to deliver 2,7-dioxabicyclo[2.2.1]heptan-3-one derivatives in a catalytically atom-economic fashion.

Synthesis and characterization of a novel near-infrared fluorescent probe for applications in imaging A549 cells.

OBJECTIVE: To design and synthesize a novel near-infrared (NIR) fluorescent probe based on indocyanine Green (ICG), that can be applied in imaging living cells. RESULTS: A highly fluorescent novel NIR fluorescent probe (IR-793) was synthesized in two steps. IR-793 had better fluorescence and optical stability than ICG. In addition, no obvious cytotoxicity effect of IR-793 was observed and cell viability was above 75% at the maximum concentration (120 nM). IR-793 also exhibited good performance in imaging living A549 cells. CONCLUSION: IR-793, a novel NIR fluorescent probe that is stable, low-cost, highly fluorescent and low cytotoxicity, has been designed and synthesized for imaging living cells.

Construction of Magnesium Phosphate Chemical Conversion Coatings with Different Microstructures on Titanium to Enhance Osteogenesis and Angiogenesis.

Titanium (Ti) and its alloys are widely used as hard tissue substitutes in dentistry and orthopedics, but their low bioactivity leads to undesirable osseointegration defects in the early osteogenic phase. Surface modification is an important approach to overcome these problems. In the present study, novel magnesium phosphate (MgP) coatings with controllable structures were fabricated on the surface of Ti using the phosphate chemical conversion (PCC) method. The effects of the microstructure on the physicochemical and biological properties of the coatings on Ti were researched. The results indicated that accelerators in PCC solution were important factors affecting the microstructure and properties of the MgP coatings. In addition, the coated Ti exhibited excellent hydrophilicity, high bonding strength, and good corrosion resistance. Moreover, the biological results showed that the MgP coatings could improve the spread, proliferation, and osteogenic differentiation of mouse osteoblast cells (MC3T3-E1) and vascular differentiation of human umbilical vein endothelial cells (HUVECs), indicating that the coated Ti samples had a great effect on promoting osteogenesis and angiogenesis. Overall, this study provided a new research idea for the surface modification of conventional Ti to enhance osteogenesis and angiogenesis in different bone types for potential biomedical applications.

Negative regulation of Yap during neuronal differentiation.

Regulated proliferation and cell cycle exit are essential aspects of neurogenesis. The Yap transcriptional coactivator controls proliferation in a variety of tissues during development, and this activity is negatively regulated by kinases in the Hippo signaling pathway. We find that Yap is expressed in mitotic mouse retinal progenitors and it is downregulated during neuronal differentiation. Forced expression of Yap prolongs proliferation in the postnatal mouse retina, whereas inhibition of Yap by RNA interference (RNAi) decreases proliferation and increases differentiation. We show Yap is subject to post-translational inhibition in the retina, and also downregulated at the level of mRNA expression. Using a cell culture model, we find that expression of the proneural basic helix-loop-helix (bHLH) transcription factors Neurog2 or Ascl1 downregulates Yap mRNA levels, and simultaneously inhibits Yap protein via activation of the Lats1 and/or Lats2 kinases. Conversely, overexpression of Yap prevents proneural bHLH proteins from initiating cell cycle exit. We propose that mutual inhibition between proneural bHLH proteins and Yap is an important regulator of proliferation and cell cycle exit during mammalian neurogenesis.

A novel untrained SSVEP-EEG feature enhancement method using canonical correlation analysis and underdamped second-order stochastic resonance.

Objective: Compared with the light-flashing paradigm, the ring-shaped motion checkerboard patterns avoid uncomfortable flicker or brightness modulation, improving the practical interactivity of brain-computer interface (BCI) applications. However, due to fewer harmonic responses and more concentrated frequency energy elicited by the ring-shaped checkerboard patterns, the mainstream untrained algorithms such as canonical correlation analysis (CCA) and filter bank canonical correlation analysis (FBCCA) methods have poor recognition performance and low information transmission rate (ITR). Methods: To address this issue, a novel untrained SSVEP-EEG feature enhancement method using CCA and underdamped second-order stochastic resonance (USSR) is proposed to extract electroencephalogram (EEG) features. Results: In contrast to typical unsupervised dimensionality reduction methods such as common average reference (CAR), principal component analysis (PCA), multidimensional scaling (MDS), and locally linear embedding (LLE), CCA exhibits higher adaptability for SSVEP rhythm components. Conclusion: This study recruits 42 subjects to evaluate the proposed method and experimental results show that the untrained method can achieve higher detection accuracy and robustness. Significance: This untrained method provides the possibility of applying a nonlinear model from one-dimensional signals to multi-dimensional signals.

Constructive on-DNA Thiol Aerial Oxidization for DNA-Encoded Library Synthesis.

A novel on-DNA oxidative disulfide formation method has been developed. Under ambient conditions, the methodology showcased wide applicability and swift implementation in routine DNA-encoded library synthesis to access pharmaceutically relevant motifs.

Steady-state auditory motion based potentials evoked by intermittent periodic virtual sound source and the effect of auditory noise on EEG enhancement.

Hearing is one of the most important human perception forms, and humans can capture the movement of sound in complex environments. On the basis of this phenomenon, this study explored the possibility of eliciting a steady-state brain response in an intermittent periodic motion sound source. In this study, a novel discrete continuous and orderly change of sound source positions stimulation paradigm was designed based on virtual sound using head-related transfer functions (HRTFs). And then the auditory motion stimulation paradigms with different noise levels were designed by changing the signal-to-noise ratio (SNR). The characteristics of brain response and the effects of different noises on brain response were studied by analyzing electroencephalogram (EEG) signals evoked by the proposed stimulation. Experimental results showed that the proposed paradigm could elicit a novel steady-state auditory evoked potential (AEP), i.e., steady-state motion auditory evoked potential (SSMAEP). And moderate noise could enhance SSMAEP amplitude and corresponding brain connectivity. This study enriches the types of AEPs and provides insights into the mechanism of brain processing of motion sound sources and the impact of noise on brain processing.

Constructive On-DNA Abramov Reaction and Pudovik Reaction for DEL Synthesis.

Organophosphonic compounds are distinctive among natural products in terms of stability and mimicry. Numerous synthetic organophosphonic compounds, including pamidronic acid, fosmidromycin, and zoledronic acid, are approved drugs. DNA encoded library technology (DELT) is a well-established platform for identifying small molecule recognition to target protein of interest (POI). Therefore, it is imperative to create an efficient procedure for the on-DNA synthesis of α-hydroxy phosphonates for DEL builds.

The effect of motion frequency and sound source frequency on steady-state auditory motion evoked potential.

The ability of humans to perceive motion sound sources is important for accurate response to the living environment. Periodic motion sound sources can elicit steady-state motion auditory evoked potential (SSMAEP). The purpose of this study was to investigate the effects of different motion frequencies and different frequencies of sound source on SSMAEP. The stimulation paradigms for simulating periodic motion of sound sources were designed utilizing head-related transfer function (HRTF) techniques in this study. The motion frequencies of the paradigm are set respectively to 1-10 Hz, 15 Hz, 20 Hz, 30 Hz, 40 Hz, 60 Hz, and 80 Hz. In addition, the frequencies of sound source of the paradigms were set to 500 Hz, 1000 Hz, 2000 Hz, 3000 Hz, and 4000 Hz at motion frequencies of 6 Hz and 40 Hz. Fourteen subjects with normal hearing were recruited for the study. SSMAEP was elicited by 500 Hz pure tone at motion frequencies of 1-10 Hz, 15 Hz, 20 Hz, 30 Hz, 40 Hz, 60 Hz, and 80 Hz. SSMAEP was strongest at motion frequencies of 6 Hz. Moreover, at 6 Hz motion frequency, the SSMAEP amplitude was largest at the tone frequency of 500 Hz and smallest at 4000 Hz. Whilst SSMAEP elicited by 4000 Hz pure tone was significantly the strongest at motion frequency of 40 Hz. SSMAEP can be elicited by periodic motion sound sources at motion frequencies up to 80 Hz. SSMAEP also has a strong response at lower frequency. Low-frequency pure tones are beneficial to enhance SSMAEP at low-frequency sound source motion, whilst high-frequency pure tones help to enhance SSMAEP at high-frequency sound source motion. The study provides new insight into the brain's perception of rhythmic auditory motion.

Simple and Practical DNA Quantification Method for DNA-Encoded Library Synthesis.

Over the past three decades, DNA-encoded library (DEL) technologies have become one of the most relevant strategies for hit-finding. Recent advances in synthetic methodologies for DNA-encoded libraries rendered the increased chemical space available, but it is unknown how every variety of chemistry affects DNA's integrity. Available assays to quantify DNA damage are restricted to electrophoresis, ligation efficiency, and mostly qPCR quantification and sequencing, which may contain predisposition and inconsistency. We developed an external standard method through LC-MS analysis to accurately quantify DNA damage throughout the chemical transformations. An assessment was conducted on on-DNA chemical reactions that are frequently employed in DEL synthesis, and these results were compared to traditional qPCR measurements. Our study provides a simple, practicable, and accurate measurement for DNA degradation during DEL synthesis. Our finding reveals substantial disagreement among the usual DNA-damaging assessment methods, which have been largely neglected so far.

Three new compounds isolated from the whole plants of Salsola collina pall.

One new alkaloid, 6, 7-dimethoxyisoquinoline-N-oxide (1), one new benzofuran derivative, 3,7-dimethyl-6-acetyl-8-benzofuranol (2) and one new lignan, salsolains A (3), along with seven known compounds (4-10), were isolated from the whole plant of Salsola collina Pall. Their structures were elucidated by extensive analysis of spectroscopic data (IR, UV, HR-ESI-MS, 1 D and 2 D NMR), and their absolute configurations were determined by the X-ray crystallography and ECD calculation. The activities of compounds 1-10 against inflammatory cytokines IL-6 and TNF-α levels on LPS-induced RAW 264.7 macrophages were assessed, especially, compound 5 (50 µM) exhibited the most significant anti-inflammatory activity with the secretion levels of IL-6 and TNF-α at 3.87% and 4.03%, respectively.

Solasodine suppress MCF7 breast cancer stem-like cells via targeting Hedgehog/Gli1.

BACKGROUND: Recently, a novel therapy to treat cancer has been to target cancer stem-like cells (CSCs). The aim of this study was to investigate the effect of solasodine, a steroidal alkaloid isolated from Solanum incanum L., on MCF7 CSCs and to understand the compound's underlying mechanism of action. METHOD: A tumorsphere formation assay was used to evaluate the effects of solasodine on the proliferation and self-renewal ability of MCF7 CSCs. The level of expression of proteins associated with cancer stemness markers and Hh signaling mediators was determined. The interaction between solasodine and Gli1 was calculated by molecular docking and further demonstrated by cellular thermal shift assay. RESULTS: Solasodine significantly decreased the proliferation of MCF7 tumorspheres and showed a stronger cytotoxicity on breast cancer cells with higher levels of Gli1 expression. The results showed that the levels of CD44 and ALDH1 expression were suppressed. Furthermore, expression of CD24 was enhanced by solasodine, via a mechanism that involved dampening Gli1 expression and blocking the nuclear translocation of this protein in MCF7 tumorspheres. Computational studies predicted that solasodine showed a high affinity with the Gli1 zinc finger domain that resulted from hydrogen-bonds to the THR243 and ASP216 amino acids residues. In addition, solasodine specifically bound with Gli1 and enhanced Gli1 protein stability in MCF7 cells. CONCLUSION: Here, our findings indicated that solasodine can directly suppresses Hh/Gli1 signaling, and is a novel anticancer candidate that targets CSCs.

Real-time optical imaging of the hypoxic status in hemangioma endothelial cells during propranolol therapy.

Infantile hemangioma (IH) is the most common microvascular tumor of infancy involving the area of head and neck. One of the most important independent risk factors of IH is the hypoxia microenvironment. Fluorescent chemosensor provides a noninvasive intervention, high spatiotemporal resolution, ultrasensitive response, and real-time feedback approach to reveal the hypoxic status of cells. Our research group developed an ultrasensitive fluorescent chemosensor, HNT-NTR, and investigated the potential ability of imaging the hypoxic status of hemangioma-derived endothelial cells (HemECs). In this study, we successfully visualized the propranolol (PRN) treatment in HemECs using NHT-NTR with "Turn-off" sensing method. This chemosensor exhibited high sensitivity and selectivity for optical imaging of hypoxic status with fast responsiveness, real-time feedback and durable photostability of the fluorescent signal. It was also confirmed that HNT-NTR could monitor nitroreductase in vivo. Paramountly, we expected this chemosensor to offer an available optical method for imaging of the hypoxic status and visualizing the therapeutic status of PRN therapy in IH with the hypoxia-imaging capability.

POSH is an intracellular signal transducer for the axon outgrowth inhibitor Nogo66.

Myelin-derived inhibitors limit axon outgrowth and plasticity during development and in the adult mammalian CNS. Nogo66, a functional domain of the myelin-derived inhibitor NogoA, signals through the PirB receptor to inhibit axon outgrowth. The signaling pathway mobilized by Nogo66 engagement of PirB is not well understood. We identify a critical role for the scaffold protein Plenty of SH3s (POSH) in relaying process outgrowth inhibition downstream of Nogo66 and PirB. Blocking the function of POSH, or two POSH-associated proteins, leucine zipper kinase (LZK) and Shroom3, with RNAi in cortical neurons leads to release from myelin and Nogo66 inhibition. We also observed autocrine inhibition of process outgrowth by NogoA, and suppression analysis with the POSH-associated kinase LZK demonstrated that LZK operates downstream of NogoA and PirB in a POSH-dependent manner. In addition, cerebellar granule neurons with an RNAi-mediated knockdown in POSH function were refractory to the inhibitory action of Nogo66, indicating that a POSH-dependent mechanism operates to inhibit axon outgrowth in different types of CNS neurons. These studies delineate an intracellular signaling pathway for process outgrowth inhibition by Nogo66, comprised of NogoA, PirB, POSH, LZK, and Shroom3, and implicate the POSH complex as a potential therapeutic target to enhance axon outgrowth and plasticity in the injured CNS.