Discovery of LLC355 as an Autophagy-Tethering Compound for the Degradation of Discoidin Domain Receptor 1.

Discoidin domain receptor 1 (DDR1) is a potential target for cancer drug discovery. Although several DDR1 kinase inhibitors have been developed, recent studies have revealed the critical roles of the noncatalytic functions of DDR1 in tumor progression, metastasis, and immune exclusion. Degradation of DDR1 presents an opportunity to block its noncatalytic functions. Here, we report the discovery of the DDR1 degrader LLC355 by employing autophagosome-tethering compound technology. Compound LLC355 efficiently degraded DDR1 protein with a DC50 value of 150.8 nM in non-small cell lung cancer NCI-H23 cells. Mechanistic studies revealed compound LLC355 to induce DDR1 degradation via lysosome-mediated autophagy. Importantly, compound LLC355 potently suppressed cancer cell tumorigenicity, migration, and invasion and significantly outperformed the corresponding inhibitor 1. These results underline the therapeutic advantage of targeting the noncatalytic function of DDR1 over inhibition of its kinase activity.

Distinct local and global functions of mouse Aß low-threshold mechanoreceptors in mechanical nociception.

The roles of Aß low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions of SplitCre labeled mouse Aß-LTMRs in this regard. Genetic ablation of SplitCre-Aß-LTMRs increased mechanical nociception but not thermosensation in both acute and chronic inflammatory pain conditions, indicating a modality-specific role in gating mechanical nociception. Local optogenetic activation of SplitCre-Aß-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a model, in which Aß-LTMRs play distinctive local and global roles in transmitting or alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a strategy of global activation plus local inhibition of Aß-LTMRs for treating mechanical hyperalgesia.

"MrgprA3 neurons selectively control myeloid-derived cytokines for IL-17 dependent cutaneous immunity".

Skin employs interdependent cellular networks to facilitate barrier integrity and host immunity through ill-defined mechanisms. This study demonstrates that manipulation of itch-sensing neurons bearing the Mas-related G protein-coupled receptor A3 (MrgprA3) drives IL-17+ γδ T cell expansion, epidermal thickening, and resistance to the human pathogen Schistosoma mansoni through mechanisms that require myeloid antigen presenting cells (APC). Activated MrgprA3 neurons instruct myeloid APCs to downregulate interleukin 33 (IL-33) and up-regulate TNFα partially through the neuropeptide calcitonin gene related peptide (CGRP). Strikingly, cell-intrinsic deletion of IL-33 in myeloid APC basally alters chromatin accessibility at inflammatory cytokine loci and promotes IL-17/23-dependent epidermal thickening, keratinocyte hyperplasia, and resistance to helminth infection. Our findings reveal a previously undescribed mechanism of intercellular cross-talk wherein "itch" neuron activation reshapes myeloid cytokine expression patterns to alter skin composition for cutaneous immunity against invasive pathogens.

Increase in hnRNPA1 Expression Suffices to Kill Motor Neurons in Transgenic Rats.

A dominant mutation in hnRNPA1 causes amyotrophic lateral sclerosis (ALS), but it is not known whether this mutation leads to motor neuron death through increased or decreased function. To elucidate the relationship between pathogenic hnRNPA1 mutation and its native function, we created novel transgenic rats that overexpressed wildtype rat hnRNPA1 exclusively in motor neurons. This targeted expression of wildtype hnRNPA1 caused severe motor neuron loss and subsequent denervation muscle atrophy in transgenic rats that recapitulated the characteristics of ALS. These findings demonstrate that the augmentation of hnRNPA1 expression suffices to trigger motor neuron degeneration and the manifestation of ALS-like phenotypes. It is reasonable to infer that an amplification of an as-yet undetermined hnRNPA1 function plays a pivotal role in the pathogenesis of familial ALS caused by pathogenic hnRNPA1 mutation.

Distinct Local and Global Functions of Aß Low-Threshold Mechanoreceptors in Mechanical Pain Transmission.

The roles of Aß low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions of SplitCre labeled Aß-LTMRs in this regard. Genetic ablation of SplitCre-Aß-LTMRs increased mechanical pain but not thermosensation in both acute and chronic inflammatory pain conditions, indicating their modality-specific role in gating mechanical pain transmission. Local optogenetic activation of SplitCre-Aß-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a new model, in which Aß-LTMRs play distinctive local and global roles in transmitting and alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a new strategy of global activation plus local inhibition of Aß-LTMRs for treating mechanical hyperalgesia.

Distinct Local and Global Functions of Aß Low-Threshold Mechanoreceptors in Mechanical Pain Transmission.

The roles of Aß low-threshold mechanoreceptors (LTMRs) in transmitting mechanical hyperalgesia and in alleviating chronic pain have been of great interest but remain contentious. Here we utilized intersectional genetic tools, optogenetics, and high-speed imaging to specifically examine functions of Split Cre labeled Aß-LTMRs in this regard. Genetic ablation of Split Cre -Aß-LTMRs increased mechanical pain but not thermosensation in both acute and chronic inflammatory pain conditions, indicating their modality-specific role in gating mechanical pain transmission. Local optogenetic activation of Split Cre -Aß-LTMRs triggered nociception after tissue inflammation, whereas their broad activation at the dorsal column still alleviated mechanical hypersensitivity of chronic inflammation. Taking all data into consideration, we propose a new model, in which Aß-LTMRs play distinctive local and global roles in transmitting and alleviating mechanical hyperalgesia of chronic pain, respectively. Our model suggests a new strategy of global activation plus local inhibition of Aß-LTMRs for treating mechanical hyperalgesia.

First report of Diaporthe sojae causing stem canker on blueberry in China.

Blueberry (Vaccinium corymbosum) plants are popular all over the world due to their high nutritional value and health benefits. In October 2020, blueberry stems (cv. O'Neal) displaying reddish brown necrotic lesions were observed from a blueberry field in Anqing (Anhui, China), with the incidence of approximately 90%. The affected plants were somewhat stunted that had smaller fruit, and in severe cases, partial or whole plant died. We randomly selected three sampling sites to collect stems with the symptoms. Samples at the margin between diseased and healthy tissues were taken out, cut into 5 mm pieces in length，and then mixed them together. Twenty small samples were surface-sterilized, and plated onto potato dextrose agar (PDA). The plates were incubated at 25°C in the dark until fungal colonies were observed. After subculturing single hyphal tips, 9 out of 12 fungal isolates with similar morphologies were obtained. The representative isolate, LMKY12 was selected for further identification. The colonies on PDA showed white, fluffy aerial mycelia with 7.9  0.2 mm (n=5) diameter after inoculation in darkness at 25°C for one week. The colony darkens in color with age, yellowish pigmentation in reverse were observed. After 15 days of incubation, dark brown, irregular hard particles (fruiting bodies in sexual stage) accumulated on the surface of the colonies. Asci were 8-spored, sessile, club-like, hyaline, and 35-46 x 6-9 µm (n=30) in size. The ascospores were oval or spindle shaped, two-celled, constricted at division, and containing four guttulates with larger guttules at centre and smaller one at ends, measured 9-11 x 2-4 um (n=50). No sporulation observed on blueberry stems after inoculated 30 days. In order to induce the production of conidiophores, mycelial plugs were placed on blueberry leaves and cultured in darkness at 25°C. There are two types of conidia observed after 20 days of inoculation. Alpha conidia were aseptate, hyaline, smooth, ovate to ellipsoidal, often biguttulate, measured 5.33-7.26 x 1.65-2.53 µm (n=50). Beta conidia were hyaline, linear, measured 12.60-17.91 x 0.81-1.38 µm (n=30). The morphological characteristics matched the previous description of D. sojae (Udayanga et al. 2015; Guo et al. 2020). To confirm the identification, the mycelial genomic DNA of LMKY12 was extracted as a template. The rDNA internal transcribed spacer (ITS), translation elongation factor 1-α gene (TEF1-α), and calmodulin (CAL) were amplified and sequenced using primers ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R, and CAL-228F/CAL-737R (Carbone and Kohn 1999), respectively. BLAST analysis revealed that the ITS (ON545758), CAL (OP886852), and TEF1-α (OP886853) sequences were 100% (527/527 base pairs), 99.21% (504/508 base pairs), and 99.41% (336/338 base pairs) similar to the strain FAU636 of D. sojae (KJ590718, KJ612115, KJ590761), respectively. Phylogenetic analysis based on concatenated sequences of ITS, TEF1-α, and CAL using MEGA 7.0 by maximum likelihood attributed the isolate LMKY12 to the D. sojae clade. Pathogenicity tests were performed on blueberry cv. O'Neal using detached stems (n=8) in laboratory, one-year-old potted plants (n=4) in greenhouse. Inoculations were done by placing mycelial plugs (7 mm in diameter) taken from a 7-day-old PDA culture on wounded stems. Inoculations with uncolonized agar plugs served as negative controls. Reddish dark brown lesions similar to the symptoms were observed on all inoculated stems 7 days after inoculation. No symptoms developed on control stems. Reisolations were successfully made from all the inoculated stems, and the pathogen was confirmed by the presence of pycnidia, alpha conidia and beta conidia. To our knowledge, this is the first report of D. sojae causing blueberry stem canker in China.

Glutamate in primary afferents is required for itch transmission.

Whether glutamate or itch-selective neurotransmitters are used to confer itch specificity is still under debate. We focused on an itch-selective population of primary afferents expressing MRGPRA3, which highly expresses Vglut2 and the neuropeptide neuromedin B (Nmb), to investigate this question. Optogenetic stimulation of MRGPRA3+ afferents triggers scratching and other itch-related avoidance behaviors. Using a combination of optogenetics, spinal cord slice recordings, Vglut2 conditional knockout mice, and behavior assays, we showed that glutamate is essential for MRGPRA3+ afferents to transmit itch. We further demonstrated that MRGPRA3+ afferents form monosynaptic connections with both NMBR+ and NMBR- neurons and that NMB and glutamate together can enhance the activity of NMBR+ spinal DH neurons. Moreover, Nmb in MRGPRA3+ afferents and NMBR+ DH neurons are required for chloroquine-induced scratching. Together, our results establish a new model in which glutamate is an essential neurotransmitter in primary afferents for itch transmission, whereas NMB signaling enhances its activities.

Magnesium-based materials in orthopaedics: material properties and animal models.

As a new generation of medical metal materials, degradable magnesium-based materials have excellent mechanical properties and osteogenic promoting ability, making them promising materials for the treatment of refractory bone diseases. Animal models can be used to understand and evaluate the performance of materials in complex physiological environments, providing relevant data for preclinical evaluation of implants and laying the foundation for subsequent clinical studies. To date, many researchers have studied the biocompatibility, degradability and osteogenesis of magnesium-based materials, but there is a lack of review regarding the effects of magnesium-based materials in vivo. In view of the growing interest in these materials, this review briefly describes the properties of magnesium-based materials and focuses on the safety and efficacy of magnesium-based materials in vivo. Various animal models including rats, rabbits, dogs and pigs are covered to better understand and evaluate the progress and future of magnesium-based materials. This literature analysis reveals that the magnesium-based materials have good biocompatibility and osteogenic activity, thus causing no adverse reaction around the implants in vivo, and that they exhibit a beneficial effect in the process of bone repair. In addition, the degradation rate in vivo can also be improved by means of alloying and coating. These encouraging results show a promising future for the use of magnesium-based materials in musculoskeletal disorders.

Detergent-insoluble inclusion constitutes the first pathology in PFN1 transgenic rats.

Mutation of profilin 1 (PFN1) can cause amyotrophic lateral sclerosis (ALS). To assess how PFN1 mutation causes the disease, we created transgenic rats with human genomic DNA that harbors both the coding and the regulatory sequences of the human PFN1 gene. Selected transgenic lines expressed human PFN1 with or without the pathogenic mutation C71G at a moderate and a comparable level and in the similar pattern of spatial and temporal expression to rat endogenous PFN1. The artificial effects of arbitrary transgene expression commonly observed in cDNA transgenic animals were minimized in PFN1 transgenic rats. Expression of the mutant, but not the wild type, human PFN1 in rats recapitulated the cardinal features of ALS including the progressive loss of motor neurons and the subsequent denervation atrophy of skeletal muscles. Detergent-insoluble PFN1 inclusions were detected as the first pathology in otherwise asymptomatic transgenic rats expressing mutant human PFN1. The findings suggest that protein aggregation is involved in the neurodegeneration of ALS associated with PFN1 mutation. The resulting rat model is useful to mechanistic study on the ALS.

MRGPRX4 is a bile acid receptor for human cholestatic itch.

Patients with liver diseases often suffer from chronic itch, yet the pruritogen(s) and receptor(s) remain largely elusive. Here, we identify bile acids as natural ligands for MRGPRX4. MRGPRX4 is expressed in human dorsal root ganglion (hDRG) neurons and co-expresses with itch receptor HRH1. Bile acids elicited Ca2+ responses in cultured hDRG neurons, and bile acids or a MRGPRX4 specific agonist induced itch in human subjects. However, a specific agonist for another bile acid receptor TGR5 failed to induce itch in human subjects and we find that human TGR5 is not expressed in hDRG neurons. Finally, we show positive correlation between cholestatic itch and plasma bile acids level in itchy patients and the elevated bile acids is sufficient to activate MRGPRX4. Taken together, our data strongly suggest that MRGPRX4 is a novel bile acid receptor that likely underlies cholestatic itch in human, providing a promising new drug target for anti-itch therapies.

Increased Ubqln2 expression causes neuron death in transgenic rats.

Pathogenic mutation of ubiquilin 2 (UBQLN2) causes neurodegeneration in amyotrophic lateral sclerosis and frontotemporal lobar degeneration. How UBQLN2 mutations cause the diseases is not clear. While over-expression of UBQLN2 with pathogenic mutation causes neuron death in rodent models, deletion of the Ubqln2 in rats has no effect on neuronal function. Previous findings in animal models suggest that UBQLN2 mutations cause the diseases mainly through a gain rather than a loss of functions. To examine whether the toxic gain in UBQLN2 mutation is related to the enhancement of UBQLN2 functions, we created new transgenic rats over-expressing wild-type human UBQLN2. Considering that human UBQLN2 may not function properly in the rat genome, we also created transgenic rats over-expressing rat's own Ubqln2. When over-expressed in rats, both human and rat wild-type Ubqln2 caused neuronal death and spatial learning deficits, the pathologies that were indistinguishable from those observed in mutant UBQLN2 transgenic rats. Over-expressed wild-type UBQLN2 formed protein inclusions attracting the autophagy substrate sequestosome-1 and the proteasome component 26S proteasome regulatory subunit 7. These findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that the enhancement of UBQLN2 functions is involved in UBQLN2 pathogenesis. Pathogenic mutation in ubiquilin 2 (UBQLN2) causes neurodegeneration in ALS and FTLD. Studies in rodent models suggest a gain of toxic function in mutant UBQLN2. We created new transgenic rats as a relevant model and examined whether enhancing wild-type UBQLN2 expression is implicated in the pathogenesis of mutant UBQLN2. We observed that over-expression of human or rat wild-type Ubqln2 caused protein aggregation and neuronal death in transgenic rats. Our findings suggest that excess UBQLN2 is toxic rather than protective to neurons and that uncontrolled enhancement of UBQLN2 function is involved in UBQLN2 pathogenesis. Read the Editorial Highlight for this article on page 159.

Pathogenic Ubqln2 gains toxic properties to induce neuron death.

Mutations in ubiquilin 2 (Ubqln2) is linked to amyotrophic lateral sclerosis and frontotemporal lobar degeneration. A foremost question regarding Ubqln2 pathogenesis is whether pathogenically mutated Ubqln2 causes neuron death via a gain or loss of functions. To better understand Ubqln2 pathobiology, we created Ubqln2 transgenic and knockout rats and compared phenotypic expression in these novel rat models. Overexpression of Ubqln2 with a pathogenic mutation (P497H substitution) caused cognitive deficits and neuronal loss in transgenic rats at the age of 130 days. In the transgenic rats, neuronal loss was preceded by the progressive formation of Ubqln2 aggregates and was accompanied by the progressive accumulation of the autophagy substrates p62 and LC3-II and the impairment of endosome pathways. In contrast, none of these pathologies observed in mutant Ubqln2 transgenic rats was detected in Ubqln2 knockout rats at the age of 300 days. Together, our findings in Ubqln2 transgenic and knockout rats collectively suggest that pathogenic Ubqln2 causes neuron death mainly through a gain of unrevealed functions rather than a loss of physiological functions.

Expression of ALS-linked TDP-43 mutant in astrocytes causes non-cell-autonomous motor neuron death in rats.

Mutation of Tar DNA-binding protein 43 (TDP-43) is linked to amyotrophic lateral sclerosis. Although astrocytes have important roles in neuron function and survival, their potential contribution to TDP-43 pathogenesis is unclear. Here, we created novel lines of transgenic rats that express a mutant form of human TDP-43 (M337V substitution) restricted to astrocytes. Selective expression of mutant TDP-43 in astrocytes caused a progressive loss of motor neurons and the denervation atrophy of skeletal muscles, resulting in progressive paralysis. The spinal cord of transgenic rats also exhibited a progressive depletion of the astroglial glutamate transporters GLT-1 and GLAST. Astrocytic expression of mutant TDP-43 led to activation of astrocytes and microglia, with an induction of the neurotoxic factor Lcn2 in reactive astrocytes that was independent of TDP-43 expression. These results indicate that mutant TDP-43 in astrocytes is sufficient to cause non-cell-autonomous death of motor neurons. This motor neuron death likely involves deficiency in neuroprotective genes and induction of neurotoxic genes in astrocytes.

Reactive astrocytes secrete lcn2 to promote neuron death.

Glial reaction is a common feature of neurodegenerative diseases. Recent studies have suggested that reactive astrocytes gain neurotoxic properties, but exactly how reactive astrocytes contribute to neurotoxicity remains to be determined. Here, we identify lipocalin 2 (lcn2) as an inducible factor that is secreted by reactive astrocytes and that is selectively toxic to neurons. We show that lcn2 is induced in reactive astrocytes in transgenic rats with neuronal expression of mutant human TAR DNA-binding protein 43 (TDP-43) or RNA-binding protein fused in sarcoma (FUS). Therefore, lcn2 is induced in activated astrocytes in response to neurodegeneration, but its induction is independent of TDP-43 or FUS expression in astrocytes. We found that synthetic lcn2 is cytotoxic to primary neurons in a dose-dependent manner, but is innocuous to astrocytes, microglia, and oligodendrocytes. Lcn2 toxicity is increased in neurons that express a disease gene, such as mutant FUS or TDP-43. Conditioned medium from rat brain slice cultures with neuronal expression of mutant TDP-43 contains abundant lcn2 and is toxic to primary neurons as well as neurons in cultured brain slice from WT rats. Partial depletion of lcn2 by immunoprecipitation reduced conditioned medium-mediated neurotoxicity. Our data indicate that reactive astrocytes secrete lcn2, which is a potent neurotoxic mediator.

XBP1 depletion precedes ubiquitin aggregation and Golgi fragmentation in TDP-43 transgenic rats.

Protein inclusion is a prominent feature of neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) that is characterized by the presence of ubiquitinated TDP-43 inclusion. Presence of protein inclusions indicates an interruption to protein degradation machinery or the overload of misfolded proteins. In response to the increase in misfolded proteins, cells usually initiate a mechanism called unfolded protein response (UPR) to reduce misfolded proteins in the lumen of endoplasmic reticules. Here, we examined the effects of mutant TDP-43 on the UPR in transgenic rats that express mutant human TDP-43 restrictedly in the neurons of the forebrain. Over-expression of mutant TDP-43 in rats caused prominent aggregation of ubiquitin and remarkable fragmentation of Golgi complexes prior to neuronal loss. While ubiquitin aggregates and Golgi fragments were accumulating, neurons expressing mutant TDP-43 failed to up-regulate chaperones residing in the endoplasmic reticules and failed to initiate the UPR. Prior to ubiquitin aggregation and Golgi fragmentation, neurons were depleted of X-box-binding protein 1 (XBP1), a key player of UPR machinery. Although it remains to determine how mutation of TDP-43 leads to the failure of the UPR, our data demonstrate that failure of the UPR is implicated in TDP-43 pathogenesis.

The first case of cutaneous infection with Mycobacterium parascrofulaceum.

The authors present the first, to the best of their knowledge, reported case of cutaneous infection caused by Mycobacterium parascrofulaceum. A 42-year-old woman presented with asymptomatic reddish papules, nodules, plaques, and patches on the right side of her face and on her forehead that had persisted for 5 years, with the lesions gradually increasing in size over that time. No previous intervening medical treatment had been applied. No history or evidence of immunosuppression was found. A skin biopsy was performed for routine histological examination. Samples of lesioned skin were inoculated on Löwenstein-Jensen medium to determine the presence of acid-fast bacilli. Ziehl-Neelsen staining was used to confirm the presence of the organism. In vitro drug susceptibility testing was conducted using the microtiter plate method. Mycobacterium was identified by polymerase chain reaction-restriction fragment length polymorphism analysis and sequencing of the hsp65 and 16S rDNA genes. Cultures for aerobic and anaerobic bacteria, as well as fungus, were also conducted. Routine histopathology revealed granulomatous changes without caseation. Ziehl-Neelsen staining showed that the organisms in both the lesions and the cultures were acid-fast bacilli. The cultured colonies were grown in Löwenstein-Jensen medium and incubated at two different temperatures (32°C and 37°C) for 2-3 weeks, developing pigmentation both in the dark and in the light. In vitro drug susceptibility tests showed that the organism was sensitive to clarithromycin and moxifloxacin. Polymerase chain reaction-restriction fragment length polymorphism analysis and sequencing of the hsp65 and 16S rDNA genes confirmed that the isolated organisms were M. parascrofulaceum. Fungal and other standard bacterial cultures were negative. In conclusion, identification and diagnosis of nontuberculous mycobacteria should be performed promptly to obtain better prognoses. Empirical treatments may be feasible, and drug susceptibility tests are important.

Entorhinal cortical neurons are the primary targets of FUS mislocalization and ubiquitin aggregation in FUS transgenic rats.

Ubiquitin-positive inclusion containing Fused in Sarcoma (FUS) defines a new subtype of frontotemporal lobar degeneration (FTLD). FTLD is characterized by progressive alteration in cognitions and it preferentially affects the superficial layers of frontotemporal cortex. Mutation of FUS is linked to amyotrophic lateral sclerosis and to motor neuron disease with FTLD. To examine FUS pathology in FTLD, we developed the first mammalian animal model expressing human FUS with pathogenic mutation and developing progressive loss of memory. In FUS transgenic rats, ubiquitin aggregation and FUS mislocalization were developed primarily in the entorhinal cortex of temporal lobe, particularly in the superficial layers of affected cortex. Overexpression of mutant FUS led to Golgi fragmentation and mitochondrion aggregation. Intriguingly, aggregated ubiquitin was not colocalized with either fragmented Golgi or aggregated mitochondria, and neurons with ubiquitin aggregates were deprived of endogenous TDP-43. Agonists of peroxisome proliferator-activated receptor gamma (PPAR-γ) possess anti-glial inflammation effects and are also shown to preserve the dendrite and dendritic spines of cortical neurons in culture. Here we show that rosiglitazone, a PPAR-γ agonist, rescued the dendrites and dendritic spines of neurons from FUS toxicity and preserved rats' spatial memory. Our FUS transgenic rats would be useful to the mechanistic study of cortical dementia in FTLD. As rosiglitazone is clinically used to treat diabetes, our results would encourage immediate application of PPAR-γ agonists in treating patients with cortical dementia.