Executive compensation controls and corporate cash holdings.

As a crucial component of internal corporate governance, remuneration controls possess the potential to influence the cash holdings of firms. However, identifying the causal relationship between these controls and such holdings presents a considerable challenge. To address this research gap, this paper leverages the implementation of China's Guidance on Further Regulating the Remuneration Management of Heads of Central Enterprises as a quasi-natural experiment to investigate the relationship between executive remuneration controls and firms' cash holdings, utilizing a double-difference approach. Based on an analysis of a sample of listed companies from 2007-2012, the results indicate that firms subject to regulated executive compensation exhibit lower cash holdings. To ensure the robustness of these findings, various statistical techniques such as parallel trend tests, variable replacement, propensity score matching, and placebo tests were employed. Additionally, a mechanism test was conducted, whereby the mediating effect of executive compensation controls on firms' cash holdings was examined, revealing a reduction in internal agency costs. Finally, the analysis of heterogeneity demonstrated that the impact of executive compensation controls on firms' cash holdings was more pronounced in companies with high-quality internal controls, stronger management oversight, and lower information asymmetry.

Quantitative proteomic and phosphoproteomic analyses of the hippocampus reveal the involvement of NMDAR1 signaling in repetitive mild traumatic brain injury.

The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment. This cognitive impairment is thought to result specifically from damage to the hippocampus. In this study, we detected cognitive impairment in mice 6 weeks after repetitive mild traumatic brain injury using the novel object recognition test and the Morris water maze test. Immunofluorescence staining showed that p-tau expression was increased in the hippocampus after repetitive mild traumatic brain injury. Golgi staining showed a significant decrease in the total density of neuronal dendritic spines in the hippocampus, as well as in the density of mature dendritic spines. To investigate the specific molecular mechanisms underlying cognitive impairment due to hippocampal damage, we performed proteomic and phosphoproteomic analyses of the hippocampus with and without repetitive mild traumatic brain injury. The differentially expressed proteins were mainly enriched in inflammation, immunity, and coagulation, suggesting that non-neuronal cells are involved in the pathological changes that occur in the hippocampus in the chronic stage after repetitive mild traumatic brain injury. In contrast, differentially expressed phosphorylated proteins were mainly enriched in pathways related to neuronal function and structure, which is more consistent with neurodegeneration. We identified N-methyl-D-aspartate receptor 1 as a hub molecule involved in the response to repetitive mild traumatic brain injury , and western blotting showed that, while N-methyl-D-aspartate receptor 1 expression was not altered in the hippocampus after repetitive mild traumatic brain injury, its phosphorylation level was significantly increased, which is consistent with the omics results. Administration of GRP78608, an N-methyl-D-aspartate receptor 1 antagonist, to the hippocampus markedly improved repetitive mild traumatic brain injury-induced cognitive impairment. In conclusion, our findings suggest that N-methyl-D-aspartate receptor 1 signaling in the hippocampus is involved in cognitive impairment in the chronic stage after repetitive mild traumatic brain injury and may be a potential target for intervention and treatment.

Cingulate cGMP-dependent protein kinase I facilitates chronic pain and pain-related anxiety and depression.

ABSTRACT: Patients with chronic pain often experience exaggerated pain response and aversive emotion, such as anxiety and depression. Central plasticity in the anterior cingulate cortex (ACC) is assumed to be a critical interface for pain perception and emotion, which has been reported to involve activation of NMDA receptors. Numerous studies have documented the key significance of cGMP-dependent protein kinase I (PKG-I) as a crucial downstream target for the NMDA receptor-NO-cGMP signaling cascade in regulating neuronal plasticity and pain hypersensitivity in specific regions of pain pathway, ie, dorsal root ganglion or spinal dorsal horn. Despite this, whether and how PKG-I in the ACC contributes to cingulate plasticity and comorbidity of chronic pain and aversive emotion has remained elusive. Here, we uncovered a crucial role of cingulate PKG-I in chronic pain and comorbid anxiety and depression. Chronic pain caused by tissue inflammation or nerve injury led to upregulation of PKG-I expression at both mRNA and protein levels in the ACC. Knockdown of ACC-PKG-I relieved pain hypersensitivity as well as pain-associated anxiety and depression. Further mechanistic analysis revealed that PKG-I might act to phosphorylate TRPC3 and TRPC6, leading to enhancement of calcium influx and neuronal hyperexcitability as well as synaptic potentiation, which results in the exaggerated pain response and comorbid anxiety and depression. We believe this study sheds new light on the functional capability of ACC-PKG-I in modulating chronic pain as well as pain-associated anxiety and depression. Hence, cingulate PKG-I may represent a new therapeutic target against chronic pain and pain-related anxiety and depression.

Extracellular matrix protein laminin ß1 regulates pain sensitivity and anxiodepression-like behaviors in mice.

Patients with neuropathic pain often experience comorbid psychiatric disorders. Cellular plasticity in the anterior cingulate cortex (ACC) is assumed to be a critical interface for pain perception and emotion. However, substantial efforts have thus far been focused on the intracellular mechanisms of plasticity rather than the extracellular alterations that might trigger and facilitate intracellular changes. Laminin, a key element of the extracellular matrix (ECM), consists of one α-, one ß-, and one γ-chain and is implicated in several pathophysiological processes. Here, we showed in mice that laminin ß1 (LAMB1) in the ACC was significantly downregulated upon peripheral neuropathy. Knockdown of LAMB1 in the ACC exacerbated pain sensitivity and induced anxiety and depression. Mechanistic analysis revealed that loss of LAMB1 caused actin dysregulation via interaction with integrin ß1 and the subsequent Src-dependent RhoA/LIMK/cofilin pathway, leading to increased presynaptic transmitter release probability and abnormal postsynaptic spine remodeling, which in turn orchestrated the structural and functional plasticity of pyramidal neurons and eventually resulted in pain hypersensitivity and anxiodepression. This study sheds new light on the functional capability of ECM LAMB1 in modulating pain plasticity and identifies a mechanism that conveys extracellular alterations to intracellular plasticity. Moreover, we identified cingulate LAMB1/integrin ß1 signaling as a promising therapeutic target for the treatment of neuropathic pain and associated anxiodepression.

Nociceptor-localized cGMP-dependent protein kinase I is a critical generator for central sensitization and neuropathic pain.

Patients with neuropathic pain often experience exaggerated pain and anxiety. Central sensitization has been linked with the maintenance of neuropathic pain and may become an autonomous pain generator. Conversely, emerging evidence accumulated that central sensitization is initiated and maintained by ongoing nociceptive primary afferent inputs. However, it remains elusive what mechanisms underlie this phenomenon and which peripheral candidate contributes to central sensitization that accounts for pain hypersensitivity and pain-related anxiety. Previous studies have implicated peripherally localized cGMP-dependent protein kinase I (PKG-I) in plasticity of nociceptors and spinal synaptic transmission as well as inflammatory hyperalgesia. However, whether peripheral PKG-I contributes to cortical plasticity and hence maintains nerve injury-induced pain hypersensitivity and anxiety is unknown. Here, we demonstrated significant upregulation of PKG-I in ipsilateral L3 dorsal root ganglia (DRG), no change in L4 DRG, and downregulation in L5 DRG upon spared nerve injury. Genetic ablation of PKG-I specifically in nociceptors or post-treatment with intervertebral foramen injection of PKG-I antagonist, KT5823, attenuated the development and maintenance of spared nerve injury-induced bilateral pain hypersensitivity and anxiety. Mechanistic analysis revealed that activation of PKG-I in nociceptors is responsible for synaptic potentiation in the anterior cingulate cortex upon peripheral neuropathy through presynaptic mechanisms involving brain-derived neurotropic factor signaling. Our results revealed that PKG-I expressed in nociceptors is a key determinant for cingulate synaptic plasticity after nerve injury, which contributes to the maintenance of pain hypersensitivity and anxiety. Thereby, this study presents a strong basis for opening up a novel therapeutic target, PKG-I, in nociceptors for treatment of comorbidity of neuropathic pain and anxiety with least side effects.

The Postembryonic Development of the Immunological Barrier in the Chicken Spleens.

The avian immune system improves with the development of the lymphoid organs. The chickens' spleen serves as the largest peripheral lymphoid organ, but little immunological research has been conducted on that spleen during postembryonic development. We investigated the blood-spleen barrier (BSB) by developing morphological architecture, resistance to the corpuscular antigen, immunocyte distribution, gene expression levels of TLR2/4 and cytokines in the spleens of hatched chickens of differing ages. Results demonstrated that the resistance of exogenous carbon particles of the BSB improved with the morphological and structural development of the chicken spleens. The cuboidal endothelial cells which lined the sheathed capillaries were gradually visible, and the discontinuous basement membrane was thickened during postembryonic development. There was an increased number of T and B cells and antigen-presenting cells in the chicken spleen between hatching and adulthood. The mRNA expression levels of TLR2/4, IL-2, IFN-γ, and TNF-α were higher two weeks after hatching, but these decreased and remain stable between 21 and 60 days. As the age increased, the BSB developed structurally and functionally. Our findings provide a better understanding of splenic immune function and the pathogenesis of avian immunology in infectious diseases.

The dynamic distribution of duck Tembusu virus in the spleen of infected shelducks.

BACKGROUND: Duck Tembusu virus (DTMUV) is a novel member of Flavivirus. The isolated and purified DTMUV strain XZ-2012 was used as a strain model, to intramuscularly inject the six-month egg-laying shelducks with the infective dose of 104TCID50. The dynamic distribution of the virus in spleen at different time post-infection (pi) was studied using RT-PCR, RT-qPCR, ELISA, immunofluorescence and transmission electron microscopy (TEM). RESULT: The results showed that the virus occurred in the spleen after 2 hpi and lasted up to 18 dpi. The registered viral load increased from 2 hpi to 3 dpi, and then it diminished from 6 dpi to 18 dpi with a slight rise at 12 dpi. From 2 hpi to 6 dpi the DTMUV particles were mostly distributed in the periellipsoidal lymphatic sheath (PELS) of spleen white pulp, few being found in the sheathed capillary. From 9 dpi to 18 dpi, the DTMUV particles were migrating into periarterial lymphatic sheaths (PALS) around the central artery through the red pulp. Under TEM, the virus particles could be observed mostly in lymphocytes and macrophages. CONCLUSION: It was suggested that DTMUV invaded lymphocytes and macrophages of the spleen at 2 hpi and replicated significantly from 1 dpi to 3 dpi, being eliminated from 9 dpi to 18 dpi. This is the first study on the dynamic distribution of DTMUV from invasion to elimination in duck spleen conducted by molecular and morphological methods. It could provide theoretical basis for the occurrence, development and detoxification of the virus in the organs of the immune system.

In vivo cellular and molecular study on duck spleen infected by duck Tembusu virus.

Duck Tembusu virus (DTMUV) is a novel member of flavivirus with the highest viral loads in the spleen. Six-month egg-laying shelducks were intramuscularly injected with DTMUV strain XZ-2012. Morphological analysis revealed the presence of vacuolar degeneration in the periellipsoidal lymphatic sheaths (PELS) of spleen white pulp following infection, especially from 12 hpi to 3 dpi. Ultrastructural images showed an obvious swelling of cells and their mitochondria and endoplasmic reticulum. Using RNA-seq analysis, the expression levels of RIG-I like receptors (RLRs), downstream IRF7 and proinflammatory cytokines IL-6 from RIG-I signaling pathway were non-apparently upregulated at 2 hpi and apparently at 3 dpi, while MHC-II expression was obviously downregulated at 2 hpi. The expression levels of downstream antiviral cytokines type-I IFNs, anti-inflammatory cytokines IL-10, cell adhesion molecules (CAMs), chemokines and their receptors associated with lymphocyte homing were significantly upregulated at 3 dpi. The population of lymphocyte was increased at 6 dpi. The immune function of spleen was recovered starting from 9 dpi. These findings of this study suggest that DTMUV invaded into the spleen via RIG-I signaling pathway and enhanced immune evasion by inhibiting MHC-II expression during the early stage of infection. Additionally, DTMUV induced PELS lesions through activating IL-6 expression. Furthermore, DTMUV increased the expression levels of RLRs, antiviral type-I IFNs, lymphocyte homing-related genes and proteins as well as the number of lymphocytes in the infected duck spleen. Taken altogether, this study provides new insights into the cellular and molecular mechanisms of DTMUV infection in duck spleen.

In vivo dynamic distribution of multivesicular bodies and exosomes in spleen of DTMUV infected duck.

Exosomes are vesicles secreted by the multivesicular bodies (MVBs), which have been shown to mediate immunity regulation and virus transmission. In this study, the dynamic distribution and function of the MVBs and their exosomes was investigated through morphological characterization and molecular analyses in duck spleens infected with duck Tembusu virus (DTMUV) at different times post infection (1hpi, 2hpi, 12hpi, 24hpi). CD63, the marker of MVBs and exosomes, was distributed in the sheathed capillaries and the periellipsoidal lymphatic sheaths (PELS) of the white pulp. The numbers of MVBs and their exosomes were dramatically increased at 2 hpi, and with the increasing infection time, the numbers of MVBs and their exosomes were gradually decreased. DTMUV proteins were associated with exosomes according to double label immunofluorescence results. Ultrastructural characterization by transmission electron microscopy revealed four developing stages of MVBs containing exosomes were detected in high endothelial cells of the sheathed capillaries, lymphocytes and the ellipsoid-associated macrophages in PELS. Free exosomes were observed in the extracellular matrix and the blood vessels. Genes and proteins related to the endocytosis pathway were obviously up-regulated at 2 hpi as confirmed by RT-qPCR and western blotting. We speculated that DTMUV mediates host invasion through the endocytosis pathway by utilizing MVBs and their exosomes. The in vivo distribution pattern of MVBs and their exosomes in DTMUV infected spleens is shown for the first time in this study. This report could lay the foundations for understanding the infection mechanism of DTMUV.

The novel histological evidence of the blood-spleen barrier in duck (Anas platyrhynchos).

To identify the existence and composition of the blood-spleen barrier (BSB) in ducks, the microanatomical structures of the duck spleen were investigated by light and transmission electron microscopy, silver staining, enzymatic histochemistry and intravenous injection of ink. The endothelial cells of the sheathed capillaries were cuboidal-shaped and surrounded by an ellipsoid consisting of reticular cells, similar to high endothelial venules (HEVs). After ink injection, carbon particles were initially restricted to the ellipsoid and later trapped in the periellipsoidal lymphatic sheaths (PELS), and then transferred to the periarteriolar lymphatic sheaths (PALS) and splenic nodules over time. Reticular fibers were primarily distributed at the basement membrane of the sheathed capillaries and the periphery of the PELS. Macrophages were primarily distributed at the border between red pulp and PELS. These results suggested that the BSB was present in the ellipsoid and PELS and consisted of the mechanical barrier composed of endothelial cells of the sheathed capillaries, reticular cells and reticular fibers and the biological barrier composed of ellipsoid-associated macrophages. In conclusion, the BSB was identified in the duck spleen for the first time, including cuboidal endothelial cells, ellipsoid-associated macrophages, reticular cells and fibers, and resisting circulating pathogen invasions. The study of BSB in ducks provides a theoretical foundation for the structural composition of the avian immune system.

Lipophagy contributes to long-term storage of spermatozoa in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis.

Spermatozoa are known to be stored in the epididymis of the Chinese soft-shelled turtle Pelodiscus sinensis for long periods after spermiation from the testes, but the molecular mechanisms underlying this storage are largely unknown. In this study, epididymal spermatozoa were investigated to determine the potential molecular mechanism for long-term sperm storage in P. sinensis. Transmission electron microscopy (TEM) and Oil red O staining indicated that unusually large cytoplasmic droplets containing lipid droplets (LDs) were attached to the epididymal spermatozoa. However, the content of LDs decreased gradually with the sperm storage. LDs were surrounded by autophagic vesicles and sequestered as degradative cargo within autophagosome. Immunofluorescence and western blotting demonstrated that autophagy in spermatozoa increased gradually with the storage time. Invitro studies found that spermatozoa obtained from soft-shelled turtles in January can survive more than 40 days at 4°C. Furthermore, immunofluorescence and TEM showed that autophagy was involved in the degradation of LDs with the extension of sperm incubation. Inhibition of autophagy with 3-methyladenine significantly suppressed LD degradation. Moreover, adipose triglyceride lipase was involved in the metabolism of LDs. These findings indicate that lipophagy was activated to maximise LD breakdown, which contributes to long-term sperm storage in the epididymis of P. sinensis.

Remodelling of mitochondria during spermiogenesis of Chinese soft-shelled turtle (Pelodiscus sinensis).

Mitochondria are vital cellular organelles that have the ability to change their shape under different conditions, such as in response to stress, disease, changes in metabolic rate, energy requirements and apoptosis. In the present study, we observed remodelling of mitochondria during spermiogenesis and its relationship with mitochondria-associated granules (MAG). At the beginning of spermiogenesis, mitochondria are characterised by their round shape. As spermiogenesis progresses, the round-shaped mitochondria change into elongated and then swollen mitochondria, subsequently forming a crescent-like shape and finally developing into onion-like shaped mitochondria. We also noted changes in mitochondrial size, location and patterns of cristae at different stages of spermiogenesis. Significant differences (P<0.0001) were found in the size of the different-shaped mitochondria. In early spermatids transitioning to the granular nucleus stage, the size of the mitochondria decreased, but increased subsequently during spermiogenesis. Changes in size and morphological variations were achieved through marked mitochondrial fusion. We also observed a non-membranous structure (MAG) closely associated with mitochondria that may stimulate or control fusion during mitochondrial remodelling. The end product of this sophisticated remodelling process in turtle spermatozoa is an onion-like mitochondrion. The acquisition of this kind of mitochondrial configuration is one strategy for long-term sperm storage in turtles.

Molecular and Cellular Mechanisms of Apoptosis during Dissociated Spermatogenesis.

Apoptosis is a tightly controlled process by which tissues eliminate unwanted cells. Spontaneous germ cell apoptosis in testis has been broadly investigated in mammals that have an associated spermatogenesis pattern. However, the mechanism of germ cell apoptosis in seasonally breeding reptiles following a dissociated spermatogenesis has remained enigmatic. In the present study, morphological evidence has clearly confirmed the dissociated spermatogenesis pattern in Pelodiscus sinensis. TUNEL and TEM analyses presented dynamic changes and ultrastructural characteristics of apoptotic germ cells during seasonal spermatogenesis, implying that apoptosis might be one of the key mechanisms to clear degraded germ cells. Furthermore, using RNA-Seq and digital gene expression (DGE) profiling, a large number of apoptosis-related differentially expressed genes (DEGs) at different phases of spermatogenesis were identified and characterized in the testis. DGE and RT-qPCR analysis revealed that the critical anti-apoptosis genes, such as Bcl-2, BAG1, and BAG5, showed up-regulated patterns during intermediate and late spermatogenesis. Moreover, the increases in mitochondrial transmembrane potential in July and October were detected by JC-1 staining. Notably, the low protein levels of pro-apoptotic cleaved caspase-3 and CytC in cytoplasm were detected by immunohistochemistry and western blot analyses, indicating that the CytC-Caspase model might be responsible for the effects of germ cell apoptosis on seasonal spermatogenesis. These results facilitate understanding the regulatory mechanisms of apoptosis during spermatogenesis and uncovering the biological process of the dissociated spermatogenesis system in reptiles.

Novel cellular evidence of lipophagy within the Sertoli cells during spermatogenesis in the turtle.

Spermatogenesis is a complex process producing haploid spermatozoa, and the formation of lipid droplets (LDs) within Sertoli cells is critical to maintaining normal spermatogenesis. However, the utilization of LDs within Sertoli cells is still largely unknown. In the present study, proliferation of spermatogonial cells had begun in May, whereas the meiotic cells occurred predominately in July and majority of spermiogenic cells were observed in the seminiferous tubules in October. However, TEM and Oil Red O staining demonstrated that a larger number of LDs had accumulated within the Sertoli cells in May compared to that in October. There were several LDs attached to the isolation membrane/phagophore, suggesting that the LDs may be a source of endogenous energy for the biogenesis of autophagosomes. The LDs were enclosed within the autophagosomes in May, whereas, autophagosomes and mitochondria were directly attached with large LDs within the Sertoli cells in October. Furthermore, immunohistochemistry results demonstrated the stronger localization of LC3 on the Sertoli cells in May than in October. This study is the first to provide clear evidence of the two different modes of lipophagy for lipid consumption within Sertoli cells, which is a key aspect of Sertoli germ cell communication during spermatogenesis.