Abstract:In vertebrate embryonic development, the segmentation clock controls the cyclic formation of somites through presomitic mesoderm (PSM) cells. Somites are paired segmented structures along the anterior-posterior axis that eventually develop into vertebrae and ribs. Disruptions in the segmentation clock leads to defects in somitogenesis, resulting in congenital spinal diseases. The major patterning modules that are involved in segmentation clock is the clock and wavefront, which primarily relies on signaling gradients and cyclic oscillation. Mesodermal differentiation is regulated by combinatorial gradient system that involves the activity of the fibroblast growth factor (FGF), the Wnt/β-catenin, and the retinoic acid (RA) signaling pathways. The antagonistic gradients of these signals set a position of the determination front. In the tail bud and posterior mesoderm, FGF and Wnt signaling prevent cell maturation and the molecular oscillators start to express. The molecular oscillators rely on negative feedback loops to maintain their oscillatory expression patterns. As the cells move anteriorly, FGF signaling gradually decays and RA signaling began to strengthen. Meanwhile, the molecular oscillators propagate anteriorly with wave pattern. At the determination front, low levels of FGF signaling and high levels of RA signaling eliminate differentiation inhibition and initiate molecular oscillators to activate cyclic genes, such as Mesp2, leading to the formation of repetitive structures in somites. Advancements in live reporter and 2D culture systems have revealed that coupling delays in cell communication can maintain the synchronous segmentation clock between adjacent cells. Studies have shown that these coupling delays are controlled by Lfng gene, it can adjust coupling delays to fit in-phase oscillations by increasing the time required for intercellular DLL1-Notch signaling. To sum up, the dual homeostasis of opposing signaling gradients determines the segment boundaries, the distance traveled by a molecular oscillator in one oscillation cycle determines the somite size, and the delayed coupling in intercellular signaling regulates the synchronization of clock oscillations. These three factors interact with each other to form a segmentation clock network coordinating somitogenesis. Recent studies have revealed that the intercellular coupling delay mechanism is a major factor influencing the maintenance of oscillation synchronization. Intercellular coupling delay errors, such as increased or decreased delay time, can desynchronizing intercellular oscillations and resulting in somite fusion. However, the mechanisms governing how intercellular communication becomes involved in oscillation synchronization remains unclear. Congenital scoliosis (CS) is a result of anomalous development of the vertebrate which associate with somitogenesis malformation. We observed that deficiency or overdose of vitamin A intake in gestation may lead to CS. While the deep mechanism of how RA signaling regulates oscillation synchronization still need to be detected. With the rapid development of 3D culture systems, researchers have successfully recapitulated the formation of somite-like structures with antero-posterior identity and indicated that the rate of metabolism is directly proportional to that of development. In summary, deconstructing the segmentation clock in vitro facilitates the dissection of regulation networks of the segmentation clock and offers an excellent proxy for studying the metabolic regulation of somitogenesis speed across species and the mechanisms underlying the formation of bilateral symmetry. It also creates a platform for exploring dysregulation mechanisms involved in the development of pathological somite defects.

Abstract:Cells not only contain membrane-bound organelles (MBOs), but also membraneless organelles (MLOs) formed by condensation of many biomacromolecules. Examples include RNA-protein granules such as nucleoli and PML nuclear bodies (PML-NBs) in the nucleus, as well as stress granules and P-bodies in the cytoplasm. Phase separation is the basic organizing principle of the form of the condensates or membraneless organelles (MLOs) of biomacromolecules including proteins and nucleic acids. In particular, liquid-liquid phase separation (LLPS) compartmentalises and concentrates biological macromolecules into liquid condensates. It has been found that phase separation of biomacromolecules requires some typical intrinsic characteristics, such as intrinsically disordered regions, modular domains and multivalent interactions. The phase separation of biomacromolecules plays a key role in many important cell activities. In recent years, the phase separation of biomacromolecules phase has become a focus of research in gene transcriptional regulation. Transcriptional regulatory elements such as RNA polymerases, transcription factors (TFs), and super enhancers (SEs) all play important roles through phase separation. Our group has previously reported for the first time that long-term inactivation or absence of assembly factors leads to the formation of condensates of RNA polymerase II (RNAPII) subunits in the cytoplasm, and this process is reversible, suggesting a novel regulatory model of eukaryotic transcription machinery. The phase separation of biomacromolecules provides a biophysical understanding for the rapid transmission of transcriptional signals by a large number of TFs. Moreover, phase separation during transcriptional regulation is closely related to the occurrence of cancer. For example, the activation of oncogenes is usually associated with the formation of phase separation condensates at the SEs. In this review, the intrinsic characteristics of the formation of biomacromolecules phase separation and the important role of phase separation in transcriptional regulation are reviewed, which will provide reference for understanding basic cell activities and gene regulation in cancer.

Abstract:The World Health Organization has declared that the outbreak of coronavirus disease 2019 (COVID-19) is a global pandemic. As mutations occurred in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the global epidemic still needs further concern. Worryingly, the effectiveness and neutralizing activity of existing antibodies and vaccines against SARS-CoV-2 variants is declining. There is an urgent need to find an effective antiviral medication with broad-spectrum inhibitory effects on novel coronavirus mutant strains against the SARS-CoV-2 infection. Neutralizing antibodies play an important role in the prevention and treatment of COVID-19. The interaction of spike-receptor-binding domain (Spike-RBD) of SARS-CoV-2 and human angiotensin-converting enzyme 2 (ACE2) is the first and critical step of SARS-CoV-2 infection. Hence, the SARS-CoV-2 Spike-RBD is a hot target for neutralizing antibodies development. Evusheld, the combination of Tixagevimab and Cilgavimab monoclonal antibodies (mAbs) targeting Spike-RBD exhibits neutralizing activity against BA.2.12.1, BA.4 and BA.5, which could be used as pre-exposure prophylaxis against SARS-CoV-2 infection. The nucleocapsid (N) protein is a conservative and high-abundance structural protein of SARS-CoV-2. The nCoV396 monoclonal antibody, isolated from the blood of convalescent COVID-19 patients against the N protein of SARS-CoV-2. This mAb not only showed neutralizing activity but also inhibits hyperactivation of complement and lung injury induced by N protein. The mAb 3E8 targeting ACE2 showed broadly neutralizing activity against SARS-CoV-2 and D614G, B.1.1.7, B.1.351, B.1.617.1 and P.1 variants in vitro and in vivo, but did not impact the biological activity of ACE2. Compared with neutralizing antibodies, small molecule inhibitors have several advantages, such as broad-spectrum inhibitory effect, low cost, and simple administration methods. Several small-molecule inhibitors disrupt viral binding by targeting the ACE2 and N-terminal domain (NTD) of SARS-CoV-2 spike protein. Known drugs such as chloroquine and hydroxychloroquine could also block the infection of SARS-CoV-2 by interacting with residue Lys353 in the peptidase domain of ACE2. The transmembrane protease serine 2 (TMPRSS2) inhibitors Camostat mesylate and Proxalutamide inhibit infection by blocking TMPRSS2 mediates viral membrane fusion. The main protease inhibitor Paxlovid and RNA-dependent RNA polymerase inhibitor Azvudine have been approved for treatment of COVID-19 patients. This review summarizes the current research status of neutralizing antibodies and small molecule inhibitors and prospects for their application. We expect to provide more valuable information for further studies in this field.

Abstract:Mitophagy, a highly precise form of autophagy, plays a pivotal role in maintaining cellular homeostasis by selectively targeting and eliminating damaged mitochondria through a process known as mitophagy. Within this tightly regulated mechanism, dysfunctional mitochondria are specifically delivered to lysosomes for degradation. Disruptions in mitophagy have been implicated in a diverse range of pathological conditions, spanning diseases of the nervous system, cardiovascular system, cancer, aging, and metabolic syndrome. The elucidation of mitophagy’s impact on cardiovascular disorders, liver diseases, metabolic syndromes, immune dysfunctions, inflammatory conditions, and cancer has significantly advanced our understanding of the complex pathogenesis underlying these conditions. These studies have shed light on the intricate connections between dysfunctional mitophagy and disease progression. Among the disorders associated with mitochondrial dysfunction, insulin resistance (IR) stands out as a prominent condition linked to metabolic disorders. IR is characterized by a diminished response to normal levels of insulin, necessitating higher insulin levels to trigger a typical physiological reaction. Hyperinsulinemia and metabolic disturbances often coexist with IR, primarily due to defects in insulin signal transduction. Oxidative stress, stemming from mitochondrial dysfunction, exerts dual effects in the context of IR. Initially, it disrupts insulin signaling pathways and subtly contributes to the development of IR. Additionally, by inducing mitochondrial damage and autophagy, oxidative stress indirectly impedes insulin signaling pathways. Consequently, mitophagy acts as a protective mechanism, encapsulating damaged or dysfunctional mitochondria through the autophagy-lysosome pathway. This efficient process eliminates excessive oxidative stress reactive. The intricate interplay between mitochondrial function, oxidative stress, mitophagy, and IR represents a captivating field of investigation in the realm of metabolic disorders. By unraveling the underlying complexities and comprehending the intricate relationships between these intertwined processes, researchers strive toward uncovering novel therapeutic strategies. With a particular focus on mitochondrial quality control and the maintenance of redox homeostasis, these interventions hold tremendous potential in mitigating IR and enhancing overall metabolic health. Emerging evidence from a myriad of studies has shed light on the active involvement of mitophagy in the pathogenesis of metabolic disorders. Notably, interventions such as exercise, drug therapies, and natural products have been documented to induce mitophagy, thereby exerting beneficial effects on metabolic health through the activation of diverse signaling pathways. Several pivotal signaling molecules, including AMPK, PINK1/Parkin, BNIP3/Nix, and FUNDC1, have been identified as key regulators of mitophagy and have been implicated in the favorable outcomes observed in metabolic disorders. Of particular interest is the unique role of PINK1/Parkin in mitophagy compared to other proteins involved in this process. PINK1/Parkin exerts influence on mitophagy through the ubiquitination of outer mitochondrial membrane proteins. Conversely, BNIP3/Nix and FUNDC1 modulate mitophagy through their interaction with LC3, while also displaying certain interrelationships with each other. In this comprehensive review, our objective is to investigate the intricate interplay between mitophagy and IR, elucidating the relevant signaling pathways and exploring the treatment strategies that have garnered attention in recent years. By assimilating and integrating these findings, we aim to establish a comprehensive understanding of the multifaceted roles and intricate mechanisms by which mitophagy influences IR. This endeavor, in turn, seeks to provide novel insights and serve as a catalyst for further research in the pursuit of innovative treatments targeting IR.

Abstract:The CRISPR/Cas system consists of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (Cas). The system forms an adaptive immune system in archaea and bacteria. The inherent defense mechanism enables these microorganisms to protect themselves against the invasion of foreign genetic material. The system functions of immune response including three main stages: adaptation, expression/maturation, and interference, each stage needs specific Cas proteins encoded by Cas gene located near the CRISPR sequences, along with other auxiliary proteins. In 2015, Zhang et al. reported Cas12a (Cpf1) as a member of the Class II type V CRISPR/Cas12a system, which possesses endonuclease activity. This finding holds great promise for its application in the field of biotechnology. In 2018, Doudna’s team first applied the CRISPR/Cas12a system for detecting HPV nucleic acid. The system comprises the following essential components in vitro detection: Cas12a, the crRNA sequence complementary to the target DNA, the PAM sequence, and the ssDNA reporter. Cas12a possesses a typical RuvC domain, displaying a canonical bilobed architecture that consists of a recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe contains the REC1 and REC2 domains, and the NUC lobe includes RuvC, PAM-interacting (PI), Wedge (WED), and bridge helix (BH) domains. The mature crRNA for Cas12a has a length of 42-44 nt, consists of repeat sequence (19/ 20 nt) and spacer sequence (23-25 nt). The crRNA spacer sequence has been found to require a length of 18 nt to achieve complete cleavage activity in vitro. Additionally, mutation in the bases of crRNA can indeed affect the activity of Cas12a. The PAM sequence plays a critical role in the recognition and degradation of DNA by the CRISPR/Cas system, enabling the system to distinguish between self and non-self genomic materials. Cas12a can effectively target the spacer sequence downstream of a T-rich PAM sequence at the 5" end. LbCas12a and AsCas12a both recognize the PAM sequences of 5"-TTTN-3", while FnCas12a recognizes the PAM sequences of 5"-TTN-3". All of these PAM sequences are located upstream on the non-template strand (NTS) at the 5" end. Cas12a (Cpf1), guided by the crRNA, binds to the target DNA by recognizing the PAM sequence. It exhibits the ability to induce arbitrary cleavage of ssDNA within the system while cleaving the target ssDNA or dsDNA. According to this feature, an array of nucleic acid detection methods has been developed for tumor detection and infection diagnostics, such as the DETECTR (RPA-CRISPR/Cas12a method) and HOLMES (PCR-CRISPR/Cas12a method) in 2018. Then, in 2019, Cas12aVDet (one-step detection method), where Cas12a protein was immobilized on the upper wall of the reaction tube. This not only prevented contamination from opening the tube but also reduced the detection reaction time. In 2021, the dWS-CRISPR (digital warm-start CRISPR) was developed as a one-pot detection method. It serves as an accurate approach for quantitatively detecting SARS-CoV-2 in clinical specimens. With the innovation of scientific technology, the high-sensitivity signal transduction technology has also been integrated with the CRISPR/Cas12a system, enabling direct detection of nucleic acids, and eliminating the need for nucleic acid amplification steps. Here, we elaborated the detection principles of CRISPR/Cas12a in in vitro detection. We discussed the different stages leading to the catalytic pathway of target DNA, and the practical applications of Cas12a in nucleic acid detection. These findings revealed a target interference mechanism that originates from the binding of Cas12a-guided RNA complex to complementary DNA sequences within PAM-dependent (dsDNA) regions. The crRNA-DNA binding activates Cas12a, enabling site-specific dsDNA cleavage and non-specific ssDNA trans-cleavage. The release of Cas12a ssDNase activity provides a novel approach to enhance the sensitivity and specificity of molecular diagnostic applications. Before these CRISPR/Cas12a-based nucleic acid detection methods can be introduced into clinical use, substantial work is still required to ensure the accuracy of diagnosis. Nevertheless, we believe that these innovative detection tools based on CRISPR/Cas will revolutionize future diagnostic technologies, particularly offering significant assistance in pathogen infection diagnosis for developing countries with relatively poor healthcare conditions and high prevalence of infectious diseases.

Abstract:Chimeric RNA is a fusion transcript comprising of exon fragments from different genes. There are three splicing types: chromosome rearrangements, trans-splicing, cis-splicing, and the recently mentioned circular chimeric RNA. The traditional methods for the detection of chimeric RNA includes chromosome karyotype analysis, FISH, DNA microarray, etc., but their specificity, sensitivity and accuracy for the detection of chimeric RNA are poorly understood. With the development of sequencing technology, second-generation sequencing technology has shown strong data processing capabilities and can detect chimeric RNA through high-throughput sequence analysis. Currently, detection methods making use of high-throughput sequencing datasets includes FusionCatcher, SOAPfuse, EricScript, etc. For validation of the detected chimeric RNA, the commonly used methods include PCR, RPA, agarose gel electrophoresis, sanger sequencing, etc. The development of newly introduced techniques has led to the discovery of different novel chimeric RNA, the third and fourth generation sequencing has also been developed and nearly mature, and the sequencing technology taking PacBio as an example has also brought a new dawn to the discovery of chimeric RNA, but each of them has its advantages and disadvantages, mainly focusing on its cost, false positive rate, detection time, etc. This paper basically describes various different techniques that can be utilized for the detection and validation of chimeric RNA.

Abstract:Circular RNAs (circRNAs) are a kind of non-coding RNA (ncRNA) with covalent closed-loop structure. They have attracted more and more attention because of their high stability, evolutionary conservatism, and tissue expression specificity. It has shown that circRNAs are involved in the development of a variety of diseases including malignant tumors recently. Nasopharyngeal carcinoma (NPC) is a malignant tumor that occurs in the nasopharynx and has a unique ethnic and geographical distribution in South China and Southeast Asia. Epstein-Barr virus (EBV) infection is closely related to the development of NPC. Radiotherapy and chemotherapy are the mainstays of treatment for NPC. But tumor recurrence or distant metastasis is the leading cause of death in patients with NPC. Several studies have shown that circRNAs, as gene expression regulators, play an important role in NPC and affect the progression of NPC. This review mainly summarized the research status of abnormally expressed circRNAs in NPC and EBV-encoded circRNAs. We also discussed the possibility of circRNAs as a therapeutic target, diagnostic and prognostic marker for NPC.

Abstract:Glycosylation is one of the most important reactions in living organisms as it results in the formation of glycoconjugates with diverse biological functions. Sugar nucleotides are structurally composed of sugar and nucleoside diphosphate or monophosphate, which are widespread within a variety of biological cells. As glycosyl donors for the transglycosyl reactions catalyzed by Leloir-type glycosyltransferases, sugar nucleotides are essential for the synthesis of glycans and glycoconjugates. However, high costs and limited availability of nucleotide sugars prevent applications of biocatalytic cascades on an industrial scale. Therefore, attentions on synthetic strategies of sugar nucleotides have been increasing to achieve their wide applications in various fields. The 9 common sugar nucleotides in mammals have been fully studied with large-scale synthesis through chemical, enzymatic (chemo-enzymatic) and cell factory strategies. In addition to common sugar nucleotides, many rare sugar nucleotides are present in plants and bacteria. Although unnatural sugar nucleotides cannot be synthesized in organisms, they have great potential in research as substrates for glycosyltransferases in carbohydrate synthesis, as enzyme inhibitors in biochemical studies, and as components of glycoconjugate biosynthesis. Therefore, increasing attention has been paid to explore the efficient synthesis of unnatural sugar nucleotides. Currently, strategies for chemical synthesis of sugar nucleotides have been greatly improved, such as the use of effective catalysts for forming pyrophosphate bonds and the development of entirely new synthesis protocols. Multiple sugar nucleotides, especially unnatural sugar nucleotides, are synthesized chemically. However, chemical synthesis requires tedious protection and deprotection steps, resulting in complex steps, high cost and low yield. In contrast, enzymatic (chemo-enzymatic) and cell factory methods have significant advantages such as high yield, easy operation and easy process scale-up in the preparation of sugar nucleotides. Hence, they are prominent strategies for sugar nucleotide preparation. Herein, the biosynthesis and application of sugar nucleotides are reviewed, mainly focusing on the 9 sugar nucleotides common in mammals. The early strategies for enzymatic synthesis of sugar nucleotides generally used de novo synthesis pathway. With the discoveries of enzymes involved in salvage pathway of sugar nucleotide synthesis and the development of one-pot multienzyme (OPME) method, the synthesis of sugar nucleotides was greatly simplified. Cell factory method employs the microbial living cells as a “processing plant” by engineering their metabolic pathways through genetic engineering technology. The cell factory method has high yield, and has been applied for efficient synthesis of several sugar nucleotides. Moreover, the strategy of gram-scale synthesis of multiple rare sugar nucleotides by cascade reactions from common sugar nucleotides using sugar nucleotides synthases cloned from different sources was illustrated. In recent years, the synthesis cost of sugar nucleotides has been further reduced through various ways, such as regeneration of nucleotides, regeneration of organic cofactors, and application of immobilized enzyme technology. Furthermore, through the continuous improvement of sugar nucleotide purification process, the use of high concentration of multi-enzyme cascade and rapid non-chromatographic purification process, the synthesis of multiple sugar nucleotides and their derivatives from monosaccharides was achieved, which gradually broke the limitations of the existing strategy. With the efficient synthesis of sugar nucleotides, their applications in various fields have been increasingly explored, including the synthesis of glycans and glycoconjugates, biochemical characterization of glycosyltransferases and bioorthogonal labeling strategies, which are of great significance to the research of biochemistry, glycobiology and the development of related pharmaceutical products.

Abstract:When skin injuries are healing, complex wound environments can be easily created, which can result in wound infection, excessive inflammation caused by neutrophil accumulation and inflammatory factors, and excessive reactive oxygen species, resulting in high levels of oxidative stress. As a result of these factors, cell membranes, proteins, DNA, etc. may become damaged, which adversely affects the repair function of normal cells around the wound, resulting in the formation of chronic wounds. The effectiveness of wound dressings as a treatment is well known. They can offer temporary skin damage protection, prevent or control wound infection, create an environment that is conducive to mending skin damage, and speed wound healing. Traditional dressings like gauze, cotton balls, and bandages, however, have the drawbacks of having no antimicrobial properties, having weak adhesive properties, having poor mechanical properties, being susceptible to inflammation, obstructing angiogenesis, needing frequent replacement, and being unable to create an environment that is conducive to wound healing. As an innovative bandage, self-assembled hydrogel has great water absorption, high water retention, superior biocompatibility, biodegradability and three-dimensional (3D) structure. With properties including hemostasis, antibacterial, anti-inflammatory, and antioxidant, the synthesized raw material itself and the loaded active compounds have a wide range of potential applications in the treatment of skin injuries and wound healing. This research begins by examining and discussing the mechanism of cross-linking in self-assembled hydrogels. The cross-linking modes include non-covalent consisting of physical interaction forces such as electrostatic interactions, π-stacking, van der Waals forces, hydrophobic interactions, and metal-ligand bonds, covalent cross-linking formed by dynamic covalent bonding such as disulfide bonding and Schiff bases. And hybrid cross-linking with mixed physical forces and dynamic covalent bonding. The next part describes the special structure and excellent functions of self-assembled hydrogels, which include an extracellular matrix-like structure, the removal of exogenous microorganisms, and the mitigation of inflammation and oxidative stress. It goes on to explain the benefits of using self-assembled hydrogels as dressings for skin injuries. These dressings are capable of controlling cell proliferation, loading active ingredients, achieving hemostasis and coagulation, hastening wound healing, and controlling the regeneration of the injured area. The development of self-assembly hydrogels as dressings is summarized in the last section. The transition from purely non-covalent or covalent cross-linking to hybrid cross-linking with multiple networks, from one-strategy action to multi-strategy synergy in exerting antimicrobial, anti-inflammatory, and antioxidant effects and from single-function to multi-functioning in a single product. Additionally, it is predicted that future developments in self-assembled hydrogels will focus on creating biomimetic gels with multi-strategy associations linkage from naturally self-assembling biomolecules peptides, lipids, proteins and polysaccharides; improving the properties and cross-linking of raw materials to enhance the storage capabilities of hydrogels and cross-linking techniques, realizing the recycling of hydrogels; conducting additional research and exploration into the cross-linking process of hydrogels; and realizing the gel’s controllable rate of degradation. Furthermore, combining 3D printing and 3D microscopic imaging technology to design and build one-to-one specialized gel dressings; using computer simulation and virtual reality to eliminate the time factor, resulting in self-assembled hydrogels that perfectly fit the ideal dressing.

Abstract:Lipoprotein(a) (Lp(a)) is a complex circulating lipoprotein, and increasing evidence has demonstrated its role as a risk factor for atherosclerotic cardiovascular disease and as a possible therapeutic target. Proprotein converting enzyme proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitor significantly decreases the circulating level of Lp(a) and reduces the risk of cardiovascular events. Based on the research results in recent years, this review will systematically summarize the relevant mechanisms of PCSK9 inhibitor reducing Lp(a) synthesis and promoting its degradation. The mechanisms are influenced by whether statins used in combination and baseline levels of Lp(a). PCSK9 inhibitors decrease Lp(a) levels mainly by reducing Lp(a) synthesis. However, the importance of low-density lipoprotein receptor (LDLR) mediated enhancing Lp(a) degradation gradually increases when the LDL level decreases. Meanwhile, many other receptor pathways may also exist, including very low-density lipoprotein (VLDL) receptor, LDL receptor-related protein 1, CD36, toll-like receptor 2, scavenger receptor B1 and plasminogen receptor. At present, further studies are still needed to explore the mechanisms by which PCSK9 inhibitors reduce Lp(a) level, such as inhibition of Lp(a) synthesis and intracellular assembly, and LDLR-mediated Lp(a) degradation. In addition, whether the reduction of Lp(a) level by PCSK9 inhibitor is related to age, gender and race and whether the dose-effect relationship of reducing Lp(a) is influenced by background lipid level, all of which require in-depth exploration. In short, the cellular and molecular mechanisms underlying the regulation of Lp(a) synthesis and degradation is not completely clear. It is worth carrying out relevant research to provide a theoretical basis for better clinical application of such drugs.

Abstract:Pupil size, as a window into the minds of others, plays a crucial role in social interaction. While previous studies have focused on the influence of non-social factors, such as the physical properties of stimuli, on pupil diameter, recent research has emphasized the significant connection between social information processing and pupil size. In this comprehensive review, we aim to explore how the processing of social stimuli (e.g., face, biological motion) and their emotional characteristics affect pupil size. In essence, pupil size is believed to reflect an individual’s perception of social stimuli. It goes beyond simple physical properties and encompasses the processing of complex social information, including social contexts and interactions. The modulation of pupil size in response to social stimuli is believed to be driven by two key mechanisms: emotional arousal and social attention. When individuals encounter emotionally charged social cues, their pupils tend to dilate, indicating heightened emotional engagement. Similarly, the dilation of pupils when individuals focus on specific social cues suggests an increased allocation of cognitive resources to process relevant social information. Furthermore, the connection between pupil size and social information processing has provided intriguing findings in individuals with autism spectrum disorder (ASD). Known for their significant social deficits, individuals with ASD exhibited abnormal pupillary responses when presented with social stimuli. These findings raise the possibility of utilizing pupillary responses as a potential index for identifying individuals with ASD at a relatively younger age. Moreover, the incorporation of pupillary response measurements in the diagnosis holds great promise in transcending the limitations of the minimum diagnostic age. This can have important implications both in terms of theoretical understanding and practical applications related to the diagnosis and intervention of ASD.

Abstract:Methamphetamine (METH) is a powerful stimulant drug that can cause addiction and serious health problems. It is one of the most widely abused drugs in the world. However, the mechanisms of how METH affects the brain and leads to addiction are still unclear, and there are no effective treatments for METH addiction in clinical practice. Therefore, it is important to explore the new addiction mechanisms and treatment strategies of METH. METH addiction is a complex and chronic brain disorder that involves multiple brain regions and neurotransmitter systems. Neurotransmitters are chemical messengers that transmit signals between neurons (nerve cells) in the brain. Some of the main neurotransmitters involved in METH addiction are dopamine (DA), glutamate (Glu), norepinephrine (NE), and serotonin (SNRIS). These neurotransmitters regulate various aspects of brain function, such as reward, reinforcement, motivation, cognition, emotion, and behavior. When a person takes METH, it causes a surge of these neurotransmitters in the brain, especially in the prefrontal cortex (mPFC), ventral tegmental area (VTA), and nucleus accumbens (NAc). These brain regions form a circuit called the mesocorticolimbic system, which is responsible for mediating the rewarding and reinforcing effects of drugs and natural stimuli. The increased levels of neurotransmitters in this circuit make the person feel euphoric, alert, confident, and energetic. However, repeated or chronic use of METH can also cause negative effects, such as anxiety, paranoia, psychosis, depression, and cognitive impairment. The effects of METH on the brain are not only due to the changes in neurotransmitter levels, but also to the changes in gene expression. Gene expression is the process by which genes are turned on or off to produce proteins that perform various functions in the cells. Gene expression can be influenced by environmental factors, such as drugs, stress, diet, etc. One way that environmental factors can affect gene expression is through epigenetic mechanisms. Epigenetics is a branch of genetics that studies the heritable changes in gene expression that are not caused by changes in DNA sequence. Epigenetic mechanisms include histone modifications, DNA methylation, and non-coding RNA regulation. These mechanisms can modulate the chromatin structure and accessibility, thereby affecting the transcriptional activity of genes. Chromatin is a complex of DNA and proteins that forms the chromosomes in the nucleus of the cell. The chromatin structure can be altered by adding or removing chemical groups to histones (proteins that wrap around DNA) or DNA itself. These chemical groups can either activate or repress gene expression by changing the affinity of transcription factors (proteins that bind to DNA and initiate transcription) or other regulatory molecules. Non-coding RNAs are RNA molecules that do not code for proteins but can regulate gene expression by interacting with DNA, RNA, or proteins. Epigenetic mechanisms provide a link between environmental stimuli and gene expression, and play an important role in various physiological and pathological processes, including drug addiction. Recent studies have shown that epigenetic mechanisms are involved in the regulation of neurotransmitter systems and neural plasticity in response to METH exposure. Neural plasticity is the ability of neurons to change their structure and function in response to experience or injury. Neural plasticity is essential for learning, memory, adaptation, and recovery. The expression of some genes related to METH addiction is altered by epigenetic modifications, such as histone acetylation, methylation, ubiquitination, and non-coding RNA regulation. These epigenetic changes may affect the synaptic function and morphology, neuronal connectivity, and circuitry formation in the brain regions implicated in METH addiction. Moreover, some epigenetic modifications may persist for a long time after METH withdrawal, suggesting that they may contribute to the development and maintenance of METH addiction. In this article, we review the current literature on the epigenetic mechanisms of METH addiction. We will first introduce METH and its pharmacological effects, and then discuss the epigenetic regulation of neurotransmitter systems and neural plasticity by METH. We will focus on the changes of histone, DNA, and RNA during METH addiction, and the possible causes and consequences of their relationship with METH addiction. We will also provide some perspectives on the potential applications of epigenetic interventions for METH addiction treatment.

Abstract:Food addiction refers to the individual dependence on certain specific foods (high-calorie foods) to the extent that it becomes difficult to control and manifests a series of addictive-like behavioral changes. Food addiction is an important factor in the development of human obesity and is also a core factor that most people cannot maintain weight loss or adhere to restrictive diets to maintain a healthy weight. A deeper understanding of food addiction and its neurobiological mechanisms will provide accurate targets for intervening in food addiction to improve obesity. Food addiction is characterized by compulsive, chronic and repetitive nature. The Yale Food Addiction Scale (YFAS), a scale specifically designed to assess food addiction, was developed in 2009 by modeling all the DSM-IV for substance dependence to be applicable to eating behavior. In 2016, Gearhardt developed the Yale Food Addiction Scale 2.0, which contains 35 survey questions, to align the YFAS scale with the diagnostic criteria for addictive disorders in the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders. One of the most valid and used animal models for food addiction is the mouse food self-administration model. The mouse food self-administration model was modified according to the rat cocaine addiction model, and the food addiction status of the animals was evaluated based on three behaviors: persistence of feeding response, feeding motivation, and compulsive feeding. Studies have shown that the neural circuits of the lateral hypothalamus-ventral tegmental area-nucleus accumbens and ventral tegmental area-prelimbic-nucleus accumbens are key neurobiological mechanisms that regulate food addiction. Dopaminergic neurons in the ventral tegmental area project to the nucleus accumbens (NAc) to facilitate food reinforcement, food reward, and food addiction. The corticotropin-releasing factor (CRF) secreted by the hypothalamus may mediate chronic stress-induced VTA-nucleus accumbens reward system dysfunction and promote food addiction in mice. Meanwhile, the nucleus accumbens receives glutamatergic projections from the prelimbic cortex, an integral part of the reward system. Specific inhibition of the PL-NAc neural circuit develops a food addiction-susceptible phenotype in mice. Furthermore, dopaminergic projections from the ventral tegmental area to the prelimbic cortex specifically inhibited the PL-NAc neural circuit to promote a food-addicted phenotype in mice. Additionally, neurotensin-positive neurons in the lateral septum (LS^Nts) project to the tuberal nucleus (TU) via GABA signaling to suppress hedonic feeding.

Abstract:Human-animal interaction has a long-standing tradition dating back to ancient times. With the rapid advancements in intelligent chips, wearable devices, and machine algorithms, the intelligent interaction between animals and electronic technology, facilitated by electronic devices and systems for communication, perception, and control, has become a reality. These electronic devices aim to implement an animal-centric working mode to enhance human understanding of animals and promote the development of animal intelligence and creativity. This article takes medium-sized and large animals as research objects, with the goal of developing their ability enhancement, and introduces the concept of “intelligent animal augmentation system (IAAS)”. This concept is used to describe the characteristics of such devices and provides a comprehensive overview of existing animal and computer interface solutions. In general, IAAS can be divided into implantable and non-implantable types, each composed of interface platforms, perception and interpretation, control and instruction components. Through various levels of enhancement systems and architectural patterns, intelligent interaction between humans and animals can be realized. Although existing IAAS still lack a complete independent interaction system architecture, they hold great promise and development space in the future. Not only can they be applied as substitutes for cutting-edge devices and transportation equipment, but they are also expected to achieve cross-species information interaction through intelligent interconnection. Additionally, IAAS can promote bidirectional interaction between humans and animals, playing a significant role in advancing animal ethics and ecological protection. Furthermore, the development of interaction models based on animal subjects can provide insightful research experiences for the design of human-computer interaction systems, thereby contributing to the more efficient realization of the ambitious goal of human-machine integration.

Abstract:Objective Acute myocardial infarction (AMI) is a highly prevalent and deadly disease globally, with its incidence continuing to rise in recent years. Timely reperfusion therapy is crucial for improving the prognosis of AMI patients. However, myocardial reperfusion can lead to irreversible myocardial ischemia/reperfusion (MI/R) injury, which is associated with adverse cardiovascular outcomes following AMI. Studies have shown that microRNAs (miRNAs) are abnormally expressed during MI/R injury and play an important role in the fate of cardiomyocytes. Effective preventive and therapeutic strategies against MI/R injury remain lacking in clinical practice, necessitating elucidation of the molecular mechanisms underlying MI/R onset and progression. This study investigated the role of microRNA-878 (miR-878) in the regulation of mitochondria-mediated apoptosis in MI/R injury.Methods The H9c2 cells were flushed with a gas mixture containing 1% O₂, 5% CO₂ and 94% N₂ for 3 h. Then the cells were incubated in complete culture medium under 5% CO₂ and 95% air for 6 h to mimic in vivo hypoxia/reoxygenation (H/R) injury. Cell viability were detected by CCK-8 assay. The concentrations of lactate dehydrogenase (LDH) were then measured.The level of apoptosis was analyzed by flow cytometry. The morphology of mitochondria was analyzed by immunofluorescence and laser confocal microscopy. The levels of mitochondrial reactive oxygen species (mtROS) were detected by immunofluorescence. Dual luciferase reporter gene assay was used to study the binding site of miR-878 and Pim1. RNA immunoprecipitation (RIP) assay was used to verify the binding relationship between miR-878 and Pim1. The gene expression levels were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot.Results The study found that compared with the control group, the expression of miR-878 in H/R-treated H9c2 cells was significantly increased ((1.00±0.25) vs (9.70±2.63), P<0.01). In H/R-induced cells, transfection of miR-878 inhibitor significantly increased cell viability ((46.67±3.00) vs (74.62±4.08), P<0.000 1), and decreased LDH release ((358.58±41.71) vs (179.09±15.59), P<0.000 1) and cell apoptosis rate ((43.41±0.72) vs (27.42±4.48), P<0.01). At the same time, downregulation of miR-878 expression significantly inhibited DRP1-mediated mitochondrial overdivision and mtROS production ((6.60±0.57) vs (4.32±0.91), P<0.000 1). The mechanism study showed that miR-878 could target and bind Pim1 and inhibit the expression level of Pim1 ((1.00±0.13) vs (0.38±0.03), P<0.01). Rescue experiments confirmed that down-regulation of Pim1 expression significantly reversed the anti-injury effect of miR-878 inhibitor in H9c2 cells (P<0.01), promoted mitochondrial overdivision and mtROS production ((1.00±0.12) vs (2.41±0.12), P<0.01), and decreased the expression level of p-DRP1 ((1.00±0.15) vs (0.59±0.06), P<0.05).Conclusion The present study demonstrates that miR-878 expression is upregulated in H9c2 cardiomyocytes subjected to H/R injury. Inhibition of miR-878 expression alleviates H/R-induced cardiomyocyte damage. Notably, downregulation of miR-878 significantly inhibits DRP1-mediated mitochondrial fission and mitigates mtROS production. Mechanistically, miR-878 targets and binds to the 3"-UTR of the Pim1 gene, thereby suppressing Pim1 protein expression. Collectively, these findings suggest that under H/R conditions, miR-878 promotes excessive mitochondrial fragmentation through DRP1 activation by targeting Pim1, ultimately contributing to cardiomyocyte injury. Modulation of the miR-878/Pim1 axis may represent a potential therapeutic strategy for mitigating MI/R-induced cardiac damage.

Abstract:Objective To study the effects of BMI1 on the proliferation and drug resistance of cervical cancer (CC) and endometrial cancer (EC) cells. In addition, the mechanism of paclitaxel (PTX) resistance induced by BMI1 was explored.Methods In this study, we utilized the GTEx, Cbioportal, TCGA, and CPTAC databases to comprehensively analyze the mutation rate as well as mRNA and protein expression profiles of BMI1 in CC and EC. Subsequently, immunohistochemistry (IHC) analysis was employed to evaluate the protein expression levels of BMI1 in 40 pairs of CC and 40 pairs of EC tissue samples. Western blot was conducted to investigate alterations in downstream factor protein levels upon BMI1 knockdown in CC and EC cells. Furthermore, functional experiments were performed to elucidate the role of BMI1 in CC and EC cells. Finally, we assessed the synergistic anti-growth effect by combining BMI1 knockdown with paclitaxel treatment in vitro.Results The Cbioportal database revealed that BMI1 amplification, misinterpretation, and splicing occurred in 1.5% of CC patients and 1.9% of EC patients. Mining the data from TCGA and CPTAC databases, high mRNA levels of BMI1 were associated with the pathological type of CC and lower overall survival, and high protein levels of BMI1 were related to EC’s pathological type and tumor grade. Furthermore, the BMI1 protein level is overexpressed in cancer tissues of CC and EC compared with normal tissues, as detected by IHC analysis. Besides, drug sensitivity experiments showed that overexpression of BMI1 resulted in decreased sensitivity of HeLa and HEC-1-A cells to a variety of anticancer drugs, including paclitaxel. In order to further analyze the relationship between BMI1 and paclitaxel resistance, Western blot was used to detect the changes in the protein levels of downstream factors of BMI1 in HeLa and HEC-1-A cells after BMI1 knockdown. The results showed that the level of anti-apoptotic factor Bcl-2 protein decreased, while that of pro-apoptotic factor BAX increased with BMI1 knockdown. Additionally, we showed that high expression of BMI1 promoted the proliferation and migration of CC and EC cells in vitro. Moreover, CC and EC cells with low BMI1 expression were more sensitive to the paclitaxel.Conclusion The expression of BMI1 is significantly upregulated in tumor tissues from patients with cervical and endometrial cancer, and silencing BMI1 makes CC and EC cells more sensitive to paclitaxel via enhancing pro-apoptotic regulation.

Abstract:Obiective Alzheimer’s disease (AD) is a degenerative disease of the central nervous system (CNS) caused by a variety of risk factors. There are various pathological changes, but apoptosis of the neurological meridian cells is one of the most important pathological bases. Hyperlipidemia is a high-risk factor for the development of AD, which can lead to increased levels of oxidized low-density lipoprotein (ox-LDL) in brain tissues. PCSK9 is a protease closely related to lipid metabolism, but studies have shown that it may be related to the development of AD. LRP1 is abundantly expressed in neuronal cells, and it is an important transporter for the clearance of Aβ. There is now a large amount of literature confirming that PCSK9 can induce the degradation of LRP1. PI3K/AKT is an important signaling pathway in vivo, which plays an important role in apoptosis, and there is now a large amount of literature confirming that LRP1 activates the PI3K/AKT pathway, which has an anti-apoptotic effect. So can PCSK9 affect the PI3K/AKT pathway through LRP1 and thus regulate neuronal apoptosis? This deserves further investigation.The aim of this study was to explore the role of PCSK9 in mediating ox-LDL pro-apoptotic neuronal cell death and its mechanism, and then further elaborate the mechanism of hyperlipidemia leading to neurodegenerative diseases such as AD.Methods Firstly, PC12 cells were treated with different concentrations of ox-LDL (0, 25, 50, 75 and 100 mg/L) for 24 h. Oil red O staining was used to detect lipid accumulation in PC12 cells, Hoechst33258 staining and flow cytometry to detect apoptosis in PC12 cells, ELISA to detect the content of Aβ secreted by PC12, Western blot to detect expression of SREBP2, PCSK9 and LRP1. Then PC12 cells were treated with 75 mg/L ox-LDL for different times (0, 6, 12, 24, 48 h), and Western blot were performed to detect the expression of SREBP2, PCSK9 and LRP1. Finally, after transfecting 100 nmol/L PCSK9 siRNA into PC12 cells for 48 h, PC12 cells were treated with 75 mg/L ox-LDL for 24 h, Hoechst33258 staining and flow cytometry to detect apoptosis rate of PC12 cells, and Western blot to detect PCSK9, LRP1, PI3K, AKT, P-PI3K , P-AKT, NF-κB, Bcl-2, Bax, Caspase-9 and Caspase-3 expression, and ELISA detected Aβ content secreted by PC12 cells.Results ox-LDL increased lipid accumulation and promoted apoptosis and Aβ secretion in PC12 cells, as well as increasing the expression of SREBP2 and PCSK9 and decreasing the expression of LRP1 in PC12 cells. pCsk9 siRNA could be inhibited through the PI3K/AKT pathway and the NF-κB-Bcl-2/Bax-Caspase-9/3 pathway to inhibit ox-LDL-induced apoptosis in PC12 cells while increasing Aβ secretion in PC12 cells.Conclusion ox-LDL plays a bidirectional regulatory role in ox-LDL-induced apoptosis of PC12 cells by inducing an increase in PCSK9 expression and a decrease in LRP1 expression in PC12 cells, which in turn affects different signaling pathways downstream.

Abstract:Objective This study aimed to observe the impact of sinomenine hydrochloride on the proliferation of fibroblasts and the mRNA expression of related genes in knee joint adhesion and contracture in rabbits. Additionally, we sought to explore its potential mechanisms in combating knee joint adhesion and contracture.Methods Fibroblasts were cultured in vitro, and experimental groups with varying concentrations of sinomenine hydrochloride were established alongside a control group. Cell proliferation was assessed using the CCK-8 assay. Changes in the mRNA expression of fibroblast-related genes following sinomenine hydrochloride treatment were evaluated using RT-qPCR. The impact of the drug on serum levels of inflammatory cytokines was determined using the ELISA method, and the expression of related proteins was assessed using Western blot.Results Sinomenine hydrochloride was found to inhibit fibroblast viability, with viability decreasing as the concentration of sinomenine hydrochloride increased. The effects of sinomenine hydrochloride in all experimental groups were highly significant (P<0.05). At the mRNA expression level, compared to the control group, sinomenine hydrochloride led to a significant downregulation of inflammatory cytokines in all groups (P<0.05). Additionally, the expression levels of apoptosis-related proteins significantly increased, while Bcl-2 mRNA expression decreased (P<0.05). The mRNA expression levels of the PI3K/mTOR/AKT3 signaling pathway also decreased (P<0.05). At the protein expression level, in comparison to the control group, the levels of inflammatory cytokines IL-6, IL-8, IL-1β, and TGF-β were significantly downregulated in the middle and high-dose sinomenine hydrochloride groups (P<0.05). The expression levels of cleaved-PARP, cleaved caspase-3/7, and Bax increased and were positively correlated with the dose, while the expression levels of the anti-apoptotic protein Bcl-2 and the PI3K/AKT3/mTOR signaling pathway were negatively correlated with the dose. Sinomenine hydrochloride exhibited a significant inhibitory effect on the viability of rabbit knee joint fibroblasts, which may be associated with the downregulation of inflammatory cytokines IL-6, IL-8, and IL-1β, promotion of apoptosis-related proteins cleaved-PARP, cleaved caspase-3/7, and Bax, suppression of Bcl-2 expression, and inhibition of gene expression in the downstream PI3K/AKT3/mTOR signaling pathway.Conclusion Sinomenine hydrochloride can inhibit the inflammatory response of fibroblasts in adhesive knee joints and accelerate fibroblast apoptosis. This mechanism may offer a novel approach to improving and treating knee joint adhesion.

Abstract:Objective This study aimed to develop a novel method for encapsulating oocytes in sodium alginate hydrogel using microfluidics, then to vitrify these encapsulated oocytes in a single-step process with low concentrations of cryoprotectants.Methods We utilized a flow-focusing microfluidic chip to generate sodium alginate hydrogel microspheres. The influence of various parameters, including throat structure, cross-linking method, sodium alginate concentrations, and flow rate ratios on the stability diameter, and coefficient of variation of microspheres were examined. To further investigate the cold-resistance of these microspheres, we used cryomicroscopy to observe changes in volume and morphology of microspheres during cooling and warming processes. We used microfluidic chip to encapsulate oocytes in sodium alginate hydrogel microspheres, the empty rate of microspheres and loss rate of oocytes were determined. After releasing from microspheres and parthenogenetic activation with cytochalasin B and strontium chloride, the survival, cleavage and blastocyst rates were evaluated during in vitro maturation. Finally, oocytes encapsulated in sodium alginate microspheres were vitrified with low concentrations of cryoprotectants. We compared the survival and development capability of the oocytes with the Cryotop method.Results When the throat of the microfluidic chip measures 300 μm in length and 120 μm in width, microspheres can be uniformly formed at the throat of the chip. Sodium alginate generates microspheres with a wide size distribution when cross-linking outside the chip, while internal cross-linking within the chip results in more uniform microspheres. The stability of microsphere formation is significantly improved with the use of a three-channel internal cross-linking chip. At a flow rate of 2 μl/min and with 1% sodium alginate, the microfluidic chip can consistently and uniformly produce microspheres. Under flow rate ratios of 10, 15, and 20, the average microsphere diameters are 262.71 μm, 193.63 μm, and 156.63 μm, respectively. The sodium alginate hydrogel microspheres maintained their volume and structural integrity during the cooling and warming processes. Using a three-channel internal cross-linking microfluidic chip to encapsulate oocytes, at a flow rate ratio of 10, the empty rate is 32.28%, and the cell loss rate is 11.09%. After encapsulation and subsequent release, the oocyte survival rate (96.99%), cleavage rate (88.71%), and blastocyst formation rate (26.29%) showed no significant differences compared to the fresh group. After the microspheres were vitrified using a low concentration of cryoprotectant (10% DMSO+10% ehylene glycol (EG)+0.5 mol/L trehalose), the survival rate, cleavage rate, and blastocyst rate were 92.48%, 70.80%, and 20.42%, respectively. No significant difference was observed when compared to the Cryotop method using a higher concentration of cryoprotectant solution (15% DMSO+15% EG+0.5 mol/L trehalose).Conclusion We designed and fabricated a microfluidic system with three-channel internal cross-linking chips used for oocyte vitrification preservation. The microfluidic system can generate oocytes-loaded sodium alginate hydrogel microspheres with uniform size, low empty rate, and good cold-resistance. The method successfully reduced the concentration of cryoprotectants in a single-step vitrification process, the developmental capability of oocytes during in vitro maturation were comparable with Cryotop method. Unlike the Cryotop method, the oocytes encapsulated in hydrogel does not come into contact with liquid nitrogen, eliminating the risk of cross-contamination. This study provides a novel approach to oocyte vitrification.