Direct Oxidative Cyclization of 3-Arylpropionic Acids to 3,4-Dihydrocoumarins: Reinvestigation of the Reaction Mechanism.

Instigated by olfactory analysis of odorant molecules, the constitutions of 3,4-dihydrocoumarins prepared by PIFA-based oxidative cyclizations of 3-arylpropionic acids were revised by means of 2D NMR and X-ray analysis. Supported by computational analysis, the migratory mechanism of intermediate spirolactonic cations has been amended: 1,2-alkyl shifts instead of 1,2-carboxylic shifts were selectively obtained.

How to Choose the Appropriate Posterior Slope Angle Can Lead to Good Knee Joint Function Recovery in Total Knee Arthroplasty?

Objective: In this study, we aim to examine the effects of osteotomy under varying posterior slope angles on knee joint function recovery following knee arthroplasty. Methods: We conducted a retrospective analysis from September 2015 to September 2018 on 240 patients who underwent knee arthroplasty three years previously. The study participants were categorized based on changes in the angle of the posterior slope before and after surgery: Group 1, > 5°; Group 2, 3°-5°; Group 3, 0°-3°; Group 4, -3°-0°; Group 5, < -3°. All participants were affected with knee osteoarthritis. The Knee Society Clinical Rating System (KSS) knee function score, Western Ontario and McMaster Universities Arthritis Index (WOMAC) knee function score, Visual Analogue Scale (VAS) pain score, and postoperative complications were measured 3 years after surgery. Results: The level of pain experienced by the patients decreased significantly than before, with pain scores ranging from 1.0-3.0, and there was a statistical difference between groups (H = 93.400, P < 0.001). The KSS score increased, with group 5 having the lowest median score of 78.0 and group 2 having the highest median score of 97.0, and there was a statistical difference between groups (H = 164.460, P < 0.001). The WOMAC score was reduced, with the median score being 24.0, 11.0, 14.0, 20.0, and 26.0, in the five groups, respectively. Group 5 had the highest score, while Group 2 had the lowest score, and there was a statistically significant difference between groups (H = 164.223, P < 0.001). No symptoms such as periprosthetic femoral fracture, prosthetic loosening, or pad wear were detected in patients postoperatively. Conclusion: Osteotomy at various posterior slope angles in total knee arthroplasty impacts postoperative knee function rehabilitation. An excessive increase or decrease in angle can have an impact on the postoperative recovery of knee function.

An investigation into whether changes in the posterior tibial slope affect the outcome of cruciate-retaining total knee arthroplasty by affecting tibiofemoral articular contact kinematics.

Aims: The outcomes of total knee arthroplasty (TKA) are affected by many factors. This study aims to evaluate whether changes in the posterior tibial slope (PTS) affect patients' outcomes after cruciate-retaining TKA by affecting tibiofemoral articular contact kinematics. It was hypothesized that changes in PTS affect the outcomes of PCR TKA by affecting tibiofemoral articular contact kinematics. Methods: A total of 60 knees (30 patients) that underwent posterior cruciate-retaining TKA (with the same size prosthesis) for medial osteoarthritis were assessed preoperatively and one year postoperatively. Before and after TKA, changes in the PTS, as seen on lateral radiographs, were noted. The knees were placed in groups according to these PTS changes (preoperative value - postoperative value): group 1 >3° change and group 2 ≤3° change. Knee kinematics were observed under mid-flexion weight-bearing conditions and were compared between the two groups using the two-dimensional/three-dimensional registration technique. Pain was measured using the visual analog scale, and knee function was assessed using the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and the Knee Society Score (KSS). Results: Group 2 experienced paradoxical anterior motion of the medial femoral condyle postoperatively, but group 1 did not. A comparison of the results of the TKA between the two groups showed a significant difference in pain using the visual analog scale, and knee function of the KSS and the WOMAC (P < 0.05). The postoperative results were better in group 1 than in group 2. Conclusions: These results suggest that achieving a greater change in the PTS improves outcomes in patients undergoing posterior cruciate-retaining TKA because it reduces the paradoxical motion of the medial femoral condyle.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Pou4f1-Tbr1 transcriptional cascade controls the formation of Jam2-expressing retinal ganglion cells.

More than 40 retinal ganglion cell (RGC) subtypes have been categorized in mouse based on their morphologies, functions, and molecular features. Among these diverse subtypes, orientation-selective Jam2-expressing RGCs (J-RGCs) has two unique morphologic characteristics: the ventral-facing dendritic arbor and the OFF-sublaminae stratified terminal dendrites in the inner plexiform layer. Previously, we have discovered that T-box transcription factor T-brain 1 (Tbr1) is expressed in J-RGCs. We further found that Tbr1 is essential for the expression of Jam2, and Tbr1 regulates the formation and the dendritic morphogenesis of J-RGCs. However, Tbr1 begins to express in terminally differentiated RGCs around perinatal stage, suggesting that it is unlikely involved in the initial fate determination for J-RGC and other upstream transcription factors must control Tbr1 expression and J-RGC formation. Using the Cleavage Under Targets and Tagmentation technique, we discovered that Pou4f1 binds to Tbr1 on the evolutionary conserved exon 6 and an intergenic region downstream of the 3'UTR, and on a region flanking the promoter and the first exon of Jam2. We showed that Pou4f1 is required for the expression of Tbr1 and Jam2, indicating Pou4f1 as a direct upstream regulator of Tbr1 and Jam2. Most interestingly, the Pou4f1-bound element in exon 6 of Tbr1 possesses high-level enhancer activity, capable of directing reporter gene expression in J-RGCs. Together, these data revealed a Pou4f1-Tbr1-Jam2 genetic hierarchy as a critical pathway in the formation of J-RGC subtype.

Differential Susceptibility of Retinal Neurons to the Loss of Mitochondrial Biogenesis Factor Nrf1.

The retina, the accessible part of the central nervous system, has served as a model system to study the relationship between energy utilization and metabolite supply. When the metabolite supply cannot match the energy demand, retinal neurons are at risk of death. As the powerhouse of eukaryotic cells, mitochondria play a pivotal role in generating ATP, produce precursors for macromolecules, maintain the redox homeostasis, and function as waste management centers for various types of metabolic intermediates. Mitochondrial dysfunction has been implicated in the pathologies of a number of degenerative retinal diseases. It is well known that photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and susceptibility to oxidative stress. However, it is unclear how defective mitochondria affect other retinal neurons. Nuclear respiratory factor 1 (Nrf1) is the major transcriptional regulator of mitochondrial biogenesis, and loss of Nrf1 leads to defective mitochondria biogenesis and eventually cell death. Here, we investigated how different retinal neurons respond to the loss of Nrf1. We provide in vivo evidence that the disruption of Nrf1-mediated mitochondrial biogenesis results in a slow, progressive degeneration of all retinal cell types examined, although they present different sensitivity to the deletion of Nrf1, which implicates differential energy demand and utilization, as well as tolerance to mitochondria defects in different neuronal cells. Furthermore, transcriptome analysis on rod-specific Nrf1 deletion uncovered a previously unknown role of Nrf1 in maintaining genome stability.

NRF1 association with AUTS2-Polycomb mediates specific gene activation in the brain.

The heterogeneous family of complexes comprising Polycomb repressive complex 1 (PRC1) is instrumental for establishing facultative heterochromatin that is repressive to transcription. However, two PRC1 species, ncPRC1.3 and ncPRC1.5, are known to comprise novel components, AUTS2, P300, and CK2, that convert this repressive function to that of transcription activation. Here, we report that individuals harboring mutations in the HX repeat domain of AUTS2 exhibit defects in AUTS2 and P300 interaction as well as a developmental disorder reflective of Rubinstein-Taybi syndrome, which is mainly associated with a heterozygous pathogenic variant in CREBBP/EP300. Moreover, the absence of AUTS2 or mutation in its HX repeat domain gives rise to misregulation of a subset of developmental genes and curtails motor neuron differentiation of mouse embryonic stem cells. The transcription factor nuclear respiratory factor 1 (NRF1) has a novel and integral role in this neurodevelopmental process, being required for ncPRC1.3 recruitment to chromatin.

A Temporal Activity of CA1 Neurons Underlying Short-Term Memory for Social Recognition Altered in PTEN Mouse Models of Autism Spectrum Disorder.

Memory-guided social recognition identifies someone from previous encounters or experiences, but the mechanisms of social memory remain unclear. Here, we find that a short-term memory from experiencing a stranger mouse lasting under 30 min interval is essential for subsequent social recognition in mice, but that interval prolonged to hours by replacing the stranger mouse with a familiar littermate. Optogenetic silencing of dorsal CA1 neuronal activity during trials or inter-trial intervals disrupted short-term memory-guided social recognition, without affecting the ability of being sociable or long-term memory-guided social recognition. Postnatal knockdown or knockout of autism spectrum disorder (ASD)-associated phosphatase and tensin homolog (PTEN) gene in dorsal hippocampal CA1 similarly impaired neuronal firing rate in vitro and altered firing pattern during social recognition. These PTEN mice showed deficits in social recognition with stranger mouse rather than littermate and exhibited impairment in T-maze spontaneous alternation task for testing short-term spatial memory. Thus, we suggest that a temporal activity of dorsal CA1 neurons may underlie formation of short-term memory to be critical for organizing subsequent social recognition but that is possibly disrupted in ASD.

Characterization of Tbr2-expressing retinal ganglion cells.

The mammalian retina contains more than 40 retinal ganglion cell (RGC) subtypes based on their unique morphologies, functions, and molecular profiles. Among them, intrinsically photosensitive RGCs (ipRGCs) are the first specified RGC type emerging from a common retinal progenitor pool during development. Previous work has shown that T-box transcription factor T-brain 2 (Tbr2) is essential for the formation and maintenance of ipRGCs, and that Tbr2-expressing RGCs activate Opn4 expression upon native ipRGC ablation, suggesting that Tbr2+ RGCs contain a reservoir for ipRGCs. However, the identity of Tbr2+ RGCs has not been fully vetted. Here, using genetic sparse labeling and single cell recording, we showed that Tbr2-expressing retinal neurons include RGCs and a subset of GABAergic displaced amacrine cells (dACs). Most Tbr2+ RGCs are intrinsically photosensitive and morphologically resemble native ipRGCs with identical retinofugal projections. Tbr2+ RGCs also include a unique and rare Pou4f1-expressing OFF RGC subtype. Using a loss-of-function strategy, we have further demonstrated that Tbr2 is essential for the survival of these RGCs and dACs, as well as maintaining the expression of Opn4. These data set a strong foundation to study how Tbr2 regulates ipRGC development and survival, as well as the expression of molecular machinery regulating intrinsic photosensitivity.

Molecular and functional architecture of the mouse photoreceptor network.

Mouse photoreceptors are electrically coupled via gap junctions, but the relative importance of rod/rod, cone/cone, or rod/cone coupling is unknown. Furthermore, while connexin36 (Cx36) is expressed by cones, the identity of the rod connexin has been controversial. We report that FACS-sorted rods and cones both express Cx36 but no other connexins. We created rod- and cone-specific Cx36 knockout mice to dissect the photoreceptor network. In the wild type, Cx36 plaques at rod/cone contacts accounted for more than 95% of photoreceptor labeling and paired recordings showed the transjunctional conductance between rods and cones was ~300 pS. When Cx36 was eliminated on one side of the gap junction, in either conditional knockout, Cx36 labeling and rod/cone coupling were almost abolished. We could not detect direct rod/rod coupling, and cone/cone coupling was minor. Rod/cone coupling is so prevalent that indirect rod/cone/rod coupling via the network may account for previous reports of rod coupling.

Ultrasensitive RNAscope In Situ Hybridization System on Embryonic and Adult Mouse Retinas.

In situ hybridization (ISH) techniques provide important information regarding gene expression in cells and tissues. Especially, ISH details complex spatial RNA expression in highly heterogeneous tissues, such as developing and mature central nervous systems, where rare genes involved in many fundamental developmental or biological events are expressed. Although several techniques have been developed to detect low levels of RNA expression, there are still problematic issues caused by a low signal-to-noise ratio after signal amplification. RNAscope is a recently developed ISH technique with high sensitivity and low background. RNAscope utilizes a unique probe system (double Z probe) to amplify signal from rare RNAs. Additionally, the double Z probe enables a significant reduction in nonspecific signal amplification. Here we report detailed procedures of the brown-color RNAscope ISH on embryonic and adult mouse retinas.

Genetically Directed Sparse Labeling System for Anatomical Studies of Retinal Ganglion Cells.

The stereotypic dendritic morphology is one of the landmark characteristics for classifying retinal ganglion cell (RGC) subtypes. These unique dendritic morphologies and their corresponding stratification level in the inner plexiform layer are indicators of their physiological function and presynaptic connection with other neurons. Mis-patterned dendritic morphologies underlie many neurological disease conditions. To streamline the morphological analysis of RGCs, here, we describe a simple protocol using Cre-/lox-dependent genetically directed sparse labeling strategy on flat-mounted retinas to inspect dendritic morphology of specific RGC subtypes.

Regulation of lifespan by neural excitation and REST.

The mechanisms that extend lifespan in humans are poorly understood. Here we show that extended longevity in humans is associated with a distinct transcriptome signature in the cerebral cortex that is characterized by downregulation of genes related to neural excitation and synaptic function. In Caenorhabditis elegans, neural excitation increases with age and inhibition of excitation globally, or in glutamatergic or cholinergic neurons, increases longevity. Furthermore, longevity is dynamically regulated by the excitatory-inhibitory balance of neural circuits. The transcription factor REST is upregulated in humans with extended longevity and represses excitation-related genes. Notably, REST-deficient mice exhibit increased cortical activity and neuronal excitability during ageing. Similarly, loss-of-function mutations in the C. elegans REST orthologue genes spr-3 and spr-4 elevate neural excitation and reduce the lifespan of long-lived daf-2 mutants. In wild-type worms, overexpression of spr-4 suppresses excitation and extends lifespan. REST, SPR-3, SPR-4 and reduced excitation activate the longevity-associated transcription factors FOXO1 and DAF-16 in mammals and worms, respectively. These findings reveal a conserved mechanism of ageing that is mediated by neural circuit activity and regulated by REST.

Essential Roles of Tbr1 in the Formation and Maintenance of the Orientation-Selective J-RGCs and a Group of OFF-Sustained RGCs in Mouse.

In the mouse retina, more than 30 retinal ganglion cell (RGC) subtypes have been classified based on a combined metric of morphological and functional characteristics. RGCs arise from a common pool of retinal progenitor cells during embryonic stages and differentiate into mature subtypes in adult retinas. However, the cellular and molecular mechanisms controlling formation and maturation of such remarkable cellular diversity remain unknown. Here, we demonstrate that T-box transcription factor T-brain 1 (Tbr1) is expressed in two groups of morphologically and functionally distinct RGCs: the orientation-selective J-RGCs and a group of OFF-sustained RGCs with symmetrical dendritic arbors. When Tbr1 is genetically ablated during retinal development, these two RGC groups cannot develop. Ectopically expressing Tbr1 in M4 ipRGCs during development alters dendritic branching and density but not the inner plexiform layer stratification level. Our data indicate that Tbr1 plays critical roles in regulating the formation and dendritic morphogenesis of specific RGC types.

Essential roles of mitochondrial biogenesis regulator Nrf1 in retinal development and homeostasis.

BACKGROUND: Mitochondrial dysfunction has been implicated in the pathologies of a number of retinal degenerative diseases in both the outer and inner retina. In the outer retina, photoreceptors are particularly vulnerable to mutations affecting mitochondrial function due to their high energy demand and sensitivity to oxidative stress. However, it is unclear how defective mitochondrial biogenesis affects neural development and contributes to neural degeneration. In this report, we investigated the in vivo function of nuclear respiratory factor 1 (Nrf1), a major transcriptional regulator of mitochondrial biogenesis in both proliferating retinal progenitor cells (RPCs) and postmitotic rod photoreceptor cells (PRs). METHODS: We used mouse genetic techniques to generate RPC-specific and rod PR-specific Nrf1 conditional knockout mouse models. We then applied a comprehensive set of tools, including histopathological and molecular analyses, RNA-seq, and electroretinography on these mouse lines to study Nrf1-regulated genes and Nrf1's roles in both developing retinas and differentiated rod PRs. For all comparisons between genotypes, a two-tailed two-sample student's t-test was used. Results were considered significant when P < 0.05. RESULTS: We uncovered essential roles of Nrf1 in cell proliferation in RPCs, cell migration and survival of newly specified retinal ganglion cells (RGCs), neurite outgrowth in retinal explants, reconfiguration of metabolic pathways in RPCs, and mitochondrial morphology, position, and function in rod PRs. CONCLUSIONS: Our findings provide in vivo evidence that Nrf1 and Nrf1-mediated pathways have context-dependent and cell-state-specific functions during neural development, and disruption of Nrf1-mediated mitochondrial biogenesis in rod PRs results in impaired mitochondria and a slow, progressive degeneration of rod PRs. These results offer new insights into the roles of Nrf1 in retinal development and neuronal homeostasis and the differential sensitivities of diverse neuronal tissues and cell types of dysfunctional mitochondria. Moreover, the conditional Nrf1 allele we have generated provides the opportunity to develop novel mouse models to understand how defective mitochondrial biogenesis contributes to the pathologies and disease progression of several neurodegenerative diseases, including glaucoma, age-related macular degeneration, Parkinson's diseases, and Huntington's disease.

Substituting mouse transcription factor Pou4f2 with a sea urchin orthologue restores retinal ganglion cell development.

Pou domain transcription factor Pou4f2 is essential for the development of retinal ganglion cells (RGCs) in the vertebrate retina. A distant orthologue of Pou4f2 exists in the genome of the sea urchin (class Echinoidea) Strongylocentrotus purpuratus (SpPou4f1/2), yet the photosensory structure of sea urchins is strikingly different from that of the mammalian retina. Sea urchins have no obvious eyes, but have photoreceptors clustered around their tube feet disc. The mechanisms that are associated with the development and function of photoreception in sea urchins are largely unexplored. As an initial approach to better understand the sea urchin photosensory structure and relate it to the mammalian retina, we asked whether SpPou4f1/2 could support RGC development in the absence of Pou4f2. To answer this question, we replaced genomic Pou4f2 with an SpPou4f1/2 cDNA. In Pou4f2-null mice, retinas expressing SpPou4f1/2 were outwardly identical to those of wild-type mice. SpPou4f1/2 retinas exhibited dark-adapted electroretinogram scotopic threshold responses, indicating functionally active RGCs. During retinal development, SpPou4f1/2 activated RGC-specific genes and in S. purpuratus, SpPou4f2 was expressed in photoreceptor cells of tube feet in a pattern distinct from Opsin4 and Pax6. Our results suggest that SpPou4f1/2 and Pou4f2 share conserved components of a gene network for photosensory development and they maintain their conserved intrinsic functions despite vast morphological differences in mouse and sea urchin photosensory structures.

REST represses a subset of the pancreatic endocrine differentiation program.

To contribute to devise successful beta-cell differentiation strategies for the cure of Type 1 diabetes we sought to uncover barriers that restrict endocrine fate acquisition by studying the role of the transcriptional repressor REST in the developing pancreas. Rest expression is prevented in neurons and in endocrine cells, which is necessary for their normal function. During development, REST represses a subset of genes in the neuronal differentiation program and Rest is down-regulated as neurons differentiate. Here, we investigate the role of REST in the differentiation of pancreatic endocrine cells, which are molecularly close to neurons. We show that Rest is widely expressed in pancreas progenitors and that it is down-regulated in differentiated endocrine cells. Sustained expression of REST in Pdx1(+) progenitors impairs the differentiation of endocrine-committed Neurog3(+) progenitors, decreases beta and alpha cell mass by E18.5, and triggers diabetes in adulthood. Conditional inactivation of Rest in Pdx1(+) progenitors is not sufficient to trigger endocrine differentiation but up-regulates a subset of differentiation genes. Our results show that the transcriptional repressor REST is active in pancreas progenitors where it gates the activation of part of the beta cell differentiation program.

Chronic constant light-induced hippocampal late-phase long-term potentiation impairment in vitro is attenuated by antagonist of D1/D5 receptors.

Previous study reported that chronic constant light exposure caused hippocampus-dependent long-term memory deficit. However, the underlying cellular mechanism of this impairment is still unclear. Multiple lines of evidence indicated that long-term potentiation (LTP) is a cellular model for memory formation. Here we found that, by recording of field excitatory postsynaptic potential (fEPSP) in vitro, chronic constant light (CCL, 3 weeks) exposure impaired the late long-term potentiation (L-LTP), but not early long-term potentiation (E-LTP) and basal transmission in Schaffer collateral (SC)-CA1 synapses of hippocampal slices from rats. Because L-LTP depends on D1/D5 receptors, we examined whether interference of D1/D5 receptors can modulate L-LTP of CCL rats. Bath application of D1/D5 receptors antagonist SCH23390 (1µM) blocked L-LTP in control rats and attenuated the impaired L-LTP in CCL rats. In contrast, pre-incubation of D1/D5 receptors agonist SKF38393 (25µM) occluded further L-LTP in control rats while exacerbated the L-LTP impairment in CCL rats. These results suggested that CCL-induced L-LTP impairment can be modulated by D1/D5 receptors. Our findings may contribute to the further understanding of synaptic plasticity mechanism underlying hippocampal long-term memory impairment induced by circadian rhythm disruption.

Type I interferons regulate eomesodermin expression and the development of unconventional memory CD8(+) T cells.

CD8(+) T-cell memory phenotype and function are acquired after antigen-driven activation. Memory-like cells may also arise in absence of antigenic exposure in the thymus or in the periphery. Eomesodermin (Eomes) is a key transcription factor for the development of these unconventional memory cells. Herein, we show that type I interferon signalling in CD8(+) T cells directly activates Eomes gene expression. Consistent with this observation, the phenotype, function and age-dependent expansion of 'virtual memory' CD8(+) T cells are strongly affected in absence of type I interferon signalling. In addition, type I interferons induce a sustained expansion of 'virtual memory' CD8(+) T cells in an Eomes-dependent fashion. We further show that the development of 'innate thymic' CD8(+) T cells is dependent on the same pathway. In conclusion, we demonstrate that type I interferon signalling in CD8(+) T cells drives Eomes expression and thereby regulates the function and homeostasis of memory-like CD8(+) T cells.