Long-Read Sequencing Resolves a Complex Structural Variant in PRKN Parkinson's Disease.

BACKGROUND: Parkin RBR E3 ubiquitin-protein ligase (PRKN) mutations are the most common cause of young onset and autosomal recessive Parkinson's disease (PD). PRKN is located in FRA6E, which is one of the common fragile sites in the human genome, making this region prone to structural variants. However, complex structural variants such as inversions of PRKN are seldom reported, suggesting that there are potentially unrevealed complex pathogenic PRKN structural variants. OBJECTIVES: To identify complex structural variants in PRKN using long-read sequencing. METHODS: We investigated the genetic cause of monozygotic twins presenting with a young onset dystonia-parkinsonism using targeted sequencing, whole exome sequencing, multiple ligation probe amplification, and long-read sequencing. We assessed the presence and frequency of complex inversions overlapping PRKN using whole-genome sequencing data of Accelerating Medicines Partnership Parkinson's disease (AMP-PD) and United Kingdom (UK)-Biobank datasets. RESULTS: Multiple ligation probe amplification identified a heterozygous exon three deletion in PRKN and long-read sequencing identified a large novel inversion spanning over 7 Mb, including a large part of the coding DNA sequence of PRKN. We could diagnose the affected subjects as compound heterozygous carriers of PRKN. We analyzed whole genome sequencing data of 43,538 participants of the UK-Biobank and 4941 participants of the AMP-PD datasets. Nine inversions in the UK-Biobank and two in AMP PD were identified and were considered potentially damaging and likely to affect PRKN expression. CONCLUSIONS: This is the first report describing a large 7 Mb inversion involving breakpoints outside of PRKN. This study highlights the importance of using long-read sequencing for structural variant analysis in unresolved young-onset PD cases. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Mutations in CHCHD2 cause α-synuclein aggregation.

Mutations in CHCHD2 are linked to a familial, autosomal dominant form of Parkinson's disease (PD). The gene product may regulate mitochondrial respiratory function. However, whether mitochondrial dysfunction induced by CHCHD2 mutations further yields α-synuclein pathology is unclear. Here, we provide compelling genetic evidence that mitochondrial dysfunction induced by PD-linked CHCHD2 T61I mutation promotes α-synuclein aggregation using brain autopsy, induced pluripotent stem cells (iPSCs) and Drosophila genetics. An autopsy of an individual with CHCHD2 T61I revealed widespread Lewy pathology with both amyloid plaques and neurofibrillary tangles that appeared in the brain stem, limbic regions and neocortex. A prominent accumulation of sarkosyl-insoluble α-synuclein aggregates, the extent of which was comparable to that of a case with α-synuclein (SNCA) duplication, was observed in CHCHD2 T61I brain tissue. The prion-like activity and morphology of α-synuclein fibrils from the CHCHD2 T61I brain tissue were similar to those of fibrils from SNCA duplication and sporadic PD brain tissues. α-Synuclein insolubilization was reproduced in dopaminergic neuron cultures from CHCHD2 T61I iPSCs and Drosophila lacking the CHCHD2 ortholog or expressing the human CHCHD2 T61I. Moreover, the combination of ectopic α-synuclein expression and CHCHD2 null or T61I enhanced the toxicity in Drosophila dopaminergic neurons, altering the proteolysis pathways. Furthermore, CHCHD2 T61I lost its mitochondrial localization by α-synuclein in Drosophila. The mislocalization of CHCHD2 T61I was also observed in the patient brain. Our study suggests that CHCHD2 is a significant mitochondrial factor that determines α-synuclein stability in the etiology of PD.

Soluble epoxide hydrolase plays a key role in the pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is characterized as a chronic and progressive neurodegenerative disorder, and the deposition of specific protein aggregates of α-synuclein, termed Lewy bodies, is evident in multiple brain regions of PD patients. Although there are several available medications to treat PD symptoms, these medications do not prevent the progression of the disease. Soluble epoxide hydrolase (sEH) plays a key role in inflammation associated with the pathogenesis of PD. Here we found that MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced neurotoxicity in the mouse striatum was attenuated by subsequent repeated administration of TPPU, a potent sEH inhibitor. Furthermore, deletion of the sEH gene protected against MPTP-induced neurotoxicity, while overexpression of sEH in the striatum significantly enhanced MPTP-induced neurotoxicity. Moreover, the expression of the sEH protein in the striatum from MPTP-treated mice or postmortem brain samples from patients with dementia of Lewy bodies (DLB) was significantly higher compared with control groups. Interestingly, there was a positive correlation between sEH expression and phosphorylation of α-synuclein in the striatum. Oxylipin analysis showed decreased levels of 8,9-epoxy-5Z,11Z,14Z-eicosatrienoic acid in the striatum of MPTP-treated mice, suggesting increased activity of sEH in this region. Interestingly, the expression of sEH mRNA in human PARK2 iPSC-derived neurons was higher than that of healthy control. Treatment with TPPU protected against apoptosis in human PARK2 iPSC-derived dopaminergic neurons. These findings suggest that increased activity of sEH in the striatum plays a key role in the pathogenesis of neurodegenerative disorders such as PD and DLB. Therefore, sEH may represent a promising therapeutic target for α-synuclein-related neurodegenerative disorders.

Increased Risk of Aortic Dissection with Perlecan Deficiency.

Perlecan (HSPG2), a basement membrane-type heparan sulfate proteoglycan, has been implicated in the development of aortic tissue. However, its role in the development and maintenance of the aortic wall remains unknown. Perlecan-deficient mice (Hspg2-/--Tg: Perl KO) have been found to show a high frequency (15-35%) of aortic dissection (AD). Herein, an analysis of the aortic wall of Perl KO mice revealed that perlecan deficiency caused thinner and partially torn elastic lamina. Compared to the control aortic tissue, perlecan-deficient aortic tissue showed a significant decrease in desmosine content and an increase in soluble tropoelastin levels, implying the presence of immature elastic fibers in Perl KO mice. Furthermore, the reduced expression of the smooth muscle cell contractile proteins actin and myosin in perlecan-deficient aortic tissue may explain the risk of AD. This study showed that a deficiency in perlecan, which is localized along the elastic lamina and at the interface between elastin and fibrillin-1, increased the risk of AD, largely due to the immaturity of extracellular matrix in the aortic tissue. Overall, we proposed a new model of AD that considers the deficiency of extracellular molecule perlecan as a risk factor.

NDP52 interacts with mitochondrial RNA poly(A) polymerase to promote mitophagy.

Parkin-mediated mitophagy is a quality control pathway that selectively removes damaged mitochondria via the autophagic machinery. Autophagic receptors, which interact with ubiquitin and Atg8 family proteins, contribute to the recognition of damaged mitochondria by autophagosomes. NDP52, an autophagy receptor, is required for autophagic engulfment of damaged mitochondria during mitochondrial uncoupler treatment. The N-terminal SKICH domain and C-terminal zinc finger motif of NDP52 are both required for its function in mitophagy. While the zinc finger motif contributes to poly-ubiquitin binding, the function of the SKICH domain remains unclear. Here, we show that NDP52 interacts with mitochondrial RNA poly(A) polymerase (MTPAP) via the SKICH domain. During mitophagy, NDP52 invades depolarized mitochondria and interacts with MTPAP dependent on the proteasome but independent of ubiquitin binding. Loss of MTPAP reduces NDP52-mediated mitophagy, and the NDP52-MTPAP complex attracts more LC3 than NDP52 alone. These results indicate that NDP52 and MTPAP form an autophagy receptor complex, which enhances autophagic elimination of damaged mitochondria.

Heparan sulfate alterations in extracellular matrix structures and fibroblast growth factor-2 signaling impairment in the aged neurogenic niche.

Adult neurogenesis in the subventricular zone of the lateral ventricle decreases with age. In the subventricular zone, the specialized extracellular matrix structures, known as fractones, contact neural stem cells and regulate neurogenesis. Fractones are composed of extracellular matrix components, such as heparan sulfate proteoglycans. We previously found that fractones capture and store fibroblast growth factor 2 (FGF-2) via heparan sulfate binding, and may deliver FGF-2 to neural stem cells in a timely manner. The heparan sulfate (HS) chains in the fractones of the aged subventricular zone are modified based on immunohistochemistry. However, how aging affects fractone composition and subsequent FGF-2 signaling and neurogenesis remains unknown. The formation of the FGF-fibroblast growth factor receptor-HS complex is necessary to activate FGF-2 signaling and induce the phosphorylation of extracellular signal-regulated kinase (Erk1/2). In this study, we observed a reduction in HS 6-O-sulfation, which is critical for FGF-2 signal transduction, and failure of the FGF-2-induced phosphorylation of Erk1/2 in the aged subventricular zone. In addition, we observed increased HS 6-O-endo-sulfatase, an enzyme that may be responsible for the HS modifications in aged fractones. In conclusion, the data revealed that heparan sulfate 6-O-sulfation is reduced and FGF-2-dependent Erk1/2 signaling is impaired in the aged subventricular zone. HS modifications in fractones might play a role in the reduced neurogenic activity in aging brains.

Identification of peptides derived from the C-terminal domain of fibulin-7 active for endothelial cell adhesion and tube formation disruption.

Despite the research done on pathological angiogenesis, there is still a need for the development of new therapies against angiogenesis-related diseases. Fibulin-7 (Fbln7) is a member of the extracellular matrix fibulin protein family. The Fbln7 C-terminal fragment, Fbln7-C, binds to endothelial cells and inhibits their tube formation in culture. In this study, we screened 12 synthetic peptides, covering the fibulin-globular domain of Fbln7-C, to identify active sites for endothelial cell adhesion and in vitro antiangiogenic activity. Three peptides, fc10, fc11, and fc12, promoted Human Umbilical Vein Endothelial Cells (HUVECs) adhesion, and the morphology of HUVECs on fc10 was similar to that on Fbln7-C. EDTA and the anti-integrin ß1 function-blocking antibody inhibited HUVECs adhesion to both fc10 and fc12, and heparin inhibited HUVECs adhesion to both fc11 and fc12. fc10 and fc11 inhibited HUVECs tube formation. Our results suggest that three peptides from Fbln7-C are biologically active for endothelial cell adhesion and disrupt the tube formation, suggesting a potential therapeutic use of these peptides for angiogenesis-related diseases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 184-195, 2016.

Laminin α1 regulates age-related mesangial cell proliferation and mesangial matrix accumulation through the TGF-ß pathway.

Laminin α1 (LAMA1), a subunit of the laminin-111 basement membrane component, has been implicated in various biological functions in vivo and in vitro. Although LAMA1 is present in kidney, its roles in the kidney are unknown because of early embryonic lethality. Herein, we used a viable conditional knockout mouse model with a deletion of Lama1 in the epiblast lineage (Lama1(CKO)) to study the role of LAMA1 in kidney development and function. Adult Lama1(CKO) mice developed focal glomerulosclerosis and proteinuria with age. In addition, mesangial cell proliferation was increased, and the mesangial matrix, which normally contains laminin-111, was greatly expanded. In vitro, mesangial cells from Lama1(CKO) mice exhibited significantly increased proliferation compared with those from controls. This increased proliferation was inhibited by the addition of exogenous LAMA1-containing laminin-111, but not by laminin-211 or laminin-511, suggesting a specific role for LAMA1 in regulating mesangial cell behavior. Moreover, the absence of LAMA1 increased transforming growth factor (TGF)-ß1-induced Smad2 phosphorylation, and inhibitors of TGF-ß1 receptor I kinase blocked Smad2 phosphorylation in both control and Lama1(CKO) mesangial cells, indicating that the increased Smad2 phosphorylation occurred in the absence of LAMA1 via the TGF-ß1 receptor. These findings suggest that LAMA1 plays a critical role in kidney function and kidney aging by regulating the mesangial cell population and mesangial matrix deposition through TGF-ß/Smad signaling.

A C-terminal fragment of fibulin-7 interacts with endothelial cells and inhibits their tube formation in culture.

We have previously demonstrated that fibulin-7 (Fbln7) is expressed in teeth by pre-odontoblast and odontoblast cells, localized in the basement membrane and dentin matrices, and is an adhesion molecule for dental mesenchyme cells and odontoblasts. Fbln7 is also expressed in blood vessels by endothelial cells. In this report, we show that a recombinant C-terminal Fbln7 fragment (Fbln7-C) bound to Human Umbilical Vein Endothelial Cells (HUVECs) but did not promote cell spreading and actin stress fiber formation. Fbln7-C binding to HUVECs induced integrin clustering at cell adhesion sites with other focal adhesion molecules, and sustained activation of FAK, p130Cas, and Rac1. In addition, RhoA activation was inhibited, thereby preventing HUVEC spreading. As endothelial cell spreading is an important step for angiogenesis, we examined the effect of Fbln7-C on angiogenesis using in vitro assays for endothelial cell tube formation and vessel sprouting from aortic rings. We found that Fbln7-C inhibited the HUVEC tube formation and the vessel sprouting in aortic ring assays. Our findings suggest potential anti-angiogenic activity of the Fbln7 C-terminal region.

Domain 36 of tropoelastin in elastic fiber formation.

Elastic fiber assembly is a complex stepwise process involving multiple different proteins and enzymes. Domain 36, encoded by the last exon of the elastin gene, is recognized to be an important domain for deposition onto microfibrils, an essential step in elastic fiber assembly. However, the role of domain 36 in elastic fiber assembly has not been clarified. Here, we utilized our established in vitro assembly model to identify the importance of domain 36 for the assembly process. Our results showed that the lack of domain 36 in bovine tropoelastin results in deficient elastic fiber assembly. A similar result was obtained with the point mutation of two cysteine residues and the deletion of the Lysine-Arginine-Lysine-Arginine (RKRK) sequence in domain 36. Double immunofluorescence of tropoelastin and fibrillin-1, a main component of microfibrils, demonstrated reduced localization of these mutant tropoelastin molecules on fibrillin-1 fibers. Moreover, the binding affinity of these mutants to fibrillin-1 and microfibril-associated glycoprotein (MAGP) was significantly decreased. These data indicate that domain 36 of tropoelastin facilitates elastic fiber assembly by interacting with microfibrils via two cysteine residues and the RKRK sequence.

A defined method for differentiating human iPSCs into midbrain dopaminergic progenitors that safely restore motor deficits in Parkinson's disease.

Background: Parkinson's disease (PD) is a progressive neurodegenerative condition that primarily affects motor functions; it is caused by the loss of midbrain dopaminergic (mDA) neurons. The therapeutic effects of transplanting human-induced pluripotent stem cell (iPSC)-derived mDA neural progenitor cells in animal PD models are known and are being evaluated in an ongoing clinical trial. However, However, improvements in the safety and efficiency of differentiation-inducing methods are crucial for providing a larger scale of cell therapy studies. This study aimed to investigate the usefulness of dopaminergic progenitor cells derived from human iPSCs by our previously reported method, which promotes differentiation and neuronal maturation by treating iPSCs with three inhibitors at the start of induction. Methods: Healthy subject-derived iPS cells were induced into mDA progenitor cells by the CTraS-mediated method we previously reported, and their proprieties and dopaminergic differentiation efficiency were examined in vitro. Then, the induced mDA progenitors were transplanted into 6-hydroxydopamine-lesioned PD model mice, and their efficacy in improving motor function, cell viability, and differentiation ability in vivo was evaluated for 16 weeks. Results: Approximately ≥80% of cells induced by this method without sorting expressed mDA progenitor markers and differentiated primarily into A9 dopaminergic neurons in vitro. After transplantation in 6-hydroxydopamine-lesioned PD model mice, more than 90% of the engrafted cells differentiated into the lineage of mDA neurons, and approximately 15% developed into mature mDA neurons without tumour formation. The grafted PD model mice also demonstrated significantly improved motor functions. Conclusion: This study suggests that the differentiation protocol for the preparation of mDA progenitors is a promising option for cell therapy in patients with PD.

Long-read sequencing resolves a complex structural variant in PRKN Parkinson's disease.

Background: PRKN mutations are the most common cause of young onset and autosomal recessive Parkinson's disease (PD). PRKN is located in FRA6E which is one of the common fragile sites in the human genome, making this region prone to structural variants. However, complex structural variants such as inversions of PRKN are seldom reported, suggesting that there are potentially unrevealed complex pathogenic PRKN structural variants. Objectives: To identify complex structural variants in PRKN using long-read sequencing. Methods: We investigated the genetic cause of monozygotic twins presenting with a young onset dystonia-parkinsonism using targeted sequencing, whole exome sequencing, multiple ligation probe amplification, and long-read. We assessed the presence and frequency of complex inversions overlapping PRKN using whole-genome sequencing data of AMP-PD and UK-Biobank datasets. Results: Multiple ligation probe amplification identified a heterozygous exon 3 deletion in PRKN and long-read sequencing identified a large novel inversion spanning over 7Mb, including a large part of the coding DNA sequence of PRKN. We could diagnose the affected subjects as compound heterozygous carriers of PRKN. We analyzed whole genome sequencing data of 43,538 participants of the UK-Biobank and 4,941 participants of the AMP-PD datasets. Nine inversions in the UK-Biobank and two in AMP PD were identified and were considered potentially damaging and likely to affect PRKN isoforms. Conclusions: This is the first report describing a large 7Mb inversion involving breakpoints outside of PRKN. This study highlights the importance of using long-read whole genome sequencing for structural variant analysis in unresolved young-onset PD cases.

Differentiation of Midbrain Dopaminergic Neurons from Human iPS Cells.

Human-induced pluripotent stem (iPS) cells provide a powerful means for analyzing disease mechanisms and drug screening, especially for neurological diseases, considering the difficulty to obtain live pathological tissue. The midbrain dopaminergic neurons of the substantia nigra are mainly affected in Parkinson's disease, but it is impossible to obtain and analyze viable dopaminergic neurons from live patients. This problem can be overcome by the induction of dopaminergic neurons from human iPS cells. Here, we describe an efficient method for differentiating human iPS cells into midbrain dopaminergic neurons. This protocol holds merit for obtaining a deeper understanding of the disease and for developing novel treatments.

Irradiation by a Combination of Different Peak-Wavelength Ultraviolet-Light Emitting Diodes Enhances the Inactivation of Influenza A Viruses.

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock. This study aimed to determine the ideal irradiation by ultraviolet-light emitting diodes (UV-LEDs) for IAV disinfection. We irradiated the IAV H1N1 subtype with 4.8 mJ/cm2 UV using eight UV-LEDs [peak wavelengths (WL) = 365, 310, 300, 290, 280, 270, and 260 nm)] or a mercury low pressure (LP)-UV lamp (Peak WL = 254 nm). Inactivation was evaluated by the infection ratio of Madin-Darby canine kidney (MDCK) cells or chicken embryonated eggs. Irradiation by the 260 nm UV-LED showed the highest inactivation among all treatments. Because the irradiation-induced inactivation effects strongly correlated with damage to viral RNA, we calculated the correlation coefficient (RAE) between the irradiant spectrum and absorption of viral RNA. The RAE scores strongly correlated with the inactivation by the UV-LEDs and LP-UV lamp. To increase the RAE score, we combined three different peak WL UV-LEDs (hybrid UV-LED). The hybrid UV-LED (RAE = 86.3) significantly inactivated both H1N1 and H6N2 subtypes to a greater extent than 260 nm (RAE = 68.6) or 270 nm (RAE = 42.2) UV-LEDs. The RAE score is an important factor for increasing the virucidal effects of UV-LED irradiation.

Distinct steps of cross-linking, self-association, and maturation of tropoelastin are necessary for elastic fiber formation.

Elastic fibers play an important role in the characteristic resilience of many tissues. The assembly of tropoelastin into a fibrillar matrix is a complex stepwise process and the deposition and cross-linking of tropoelastin are believed to be key steps of elastic fiber formation. However, the detailed mechanisms of elastic fiber assembly have not been defined yet. Here, we demonstrate the relationship between deposition and the cross-linking/maturation of tropoelastin. Our data show that a C-terminal half-fragment of tropoelastin encoded by exons 16-36 (BH) is deposited onto microfibrils, yet we detect very limited amounts of the cross-linking amino acid, desmosine, an indicator of maturation, whereas the N-terminal half-fragment encoded by exons 2-15 (FH) was deficient for both deposition and cross-linking, suggesting that elastic fiber formation requires full-length tropoelastin molecules. A series of experiments using mutant BH fragments, lacking either exon 16 or 30, or a deletion of both exons showed that self-association of tropoelastin polypeptides was an early step in elastic fiber assembly. Immunofluorescence and Western blot assay showed that the treatment of cell culture medium or conditioned medium with beta-aminopropionitrile to inhibit cross-linking, prevented both the deposition and polymerization of tropoelastin. In conclusion, our present results support the view that self-association and oxidation by lysyl oxidase precedes tropoelastin deposition onto microfibrils and the entire molecule of tropoelastin is required for this following maturation process.

Characterization of the molecular interaction between tropoelastin and DANCE/fibulin-5.

Fibulin-5 is believed to play an important role in the elastic fiber formation. The present experiments were carried out to characterize the molecular interaction between fibulin-5 and tropoelastin. Our data showed that the divalent cations of Ca(2+), Ba(2+) and Mg(2+) significantly enhanced the binding of fibulin-5 to tropoelastin. In addition, N-linked glycosylation of fibulin-5 does not require for the binding to tropoelastin. To address the fibulin-5 binding site on tropoelastin constructs containing, exons 2-15 and exons 16-36, of tropoelastin were used. Fibulin-5 binding was significantly reduced to either fragment and also to a mixture of the two fragments. These results suggested that the whole molecule of tropoelastin was required for the interaction with fibulin-5. In co-immunoprecipitation experiments, tropoelastin binding to fibulin-5 was enhanced by an increase of temperature and sodium chloride concentration, conditions that enhance the coacervation of tropoelastin. The binding of tropoelastin fragments to fibulin-5 was directly proportional to their propensity to coacervate. Furthermore, the addition of fibulin-5 to tropoelastin facilitated coacervation. Taken together, the present study shows that fibulin-5 enhances elastic fiber formation in part by improving the self-association properties of tropoelastin.

Domains 16 and 17 of tropoelastin in elastic fibre formation.

Naturally occurring mutations are useful in identifying domains that are important for protein function. We studied a mutation in the elastin gene, 800-3G>C, a common disease allele for SVAS (supravalvular aortic stenosis). We showed in primary skin fibroblasts from two different SVAS families that this mutation causes skipping of exons 16-17 and results in a stable mRNA. Tropoelastin lacking domains 16-17 (Delta16-17) was synthesized efficiently and secreted by transfected retinal pigment epithelium cells, but showed the deficient deposition into the extracellular matrix compared with normal as demonstrated by immunofluorescent staining and desmosine assays. Solid-phase binding assays indicated normal molecular interaction of Delta16-17 with fibrillin-1 and fibulin-5. However, self-association of Delta16-17 was diminished as shown by an elevated coacervation temperature. Moreover, negative staining electron microscopy confirmed that Delta16-17 was deficient in forming fibrillar polymers. Domain 16 has high homology with domain 30, which can form a beta-sheet structure facilitating fibre formation. Taken together, we conclude that domains 16-17 are important for self-association of tropoelastin and elastic fibre formation. This study is the first to discover that domains of elastin play an essential role in elastic fibre formation by facilitating homotypic interactions.

Irradiation by ultraviolet light-emitting diodes inactivates influenza a viruses by inhibiting replication and transcription of viral RNA in host cells.

Influenza A viruses (IAVs) pose a serious global threat to humans and their livestock, especially poultry and pigs. This study aimed to investigate how to inactivate IAVs by using different ultraviolet-light-emitting diodes (UV-LEDs). We developed sterilization equipment with light-emitting diodes (LEDs) those peak wavelengths were 365 nm (UVA-LED), 310 nm (UVB-LED), and 280 nm (UVC-LED). These UV-LED irradiations decreased dose fluence-dependent plaque-forming units of IAV H1N1 subtype (A/Puerto Rico/8/1934) infected Madin-Darby canine kidney (MDCK) cells, but the inactivation efficiency of UVA-LED was significantly lower than UVB- and UVC-LED. UV-LED irradiations did not alter hemagglutination titer, but decreased accumulation of intracellular total viral RNA in infected MDCK cells was observed. Additionally, UV-LED irradiations suppressed the accumulation of intracellular mRNA (messenger RNA), vRNA (viral RNA), and cRNA (complementary RNA), as measured by strand-specific RT-PCR. These results suggest that UV-LEDs inhibit host cell replication and transcription of viral RNA. Both UVB- and UVC-LED irradiation decreased focus-forming unit (FFU) of H5N1 subtype (A/Crow/Kyoto/53/2004), a highly pathogenic avian IAV (HPAI), in infected MDCK cells, and the amount of FFU were lower than the H1N1 subtype. From these results, it appears that IAVs may have different sensitivity among the subtypes, and UVB- and UVC-LED may be suitable for HPAI virus inactivation.

Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression.

We previously reported that perlecan, a heparan-sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage-synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2-/- -Tg (Hspg2-/- ;Col2a1-Hspg2Tg/- ) mice, in which the perlecan-knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic-, osteogenic-, and adipogenic-potentials were examined in the Hspg2-/- -Tg SMCs compared to the control SMCs prepared from wild-type Hspg2+/+ -Tg (Hspg2+/+ ;Col2a1-Hspg2Tg/- ) littermates. In a culture condition permitting proliferation, both control and Hspg2-/- -Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2-/- -Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2-/- -Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:837-846, 2017.