Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction.

Whereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene, SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carrying SATB1 variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression, and a severe phenotype. In contrast, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability.

EFFECT OF THE C-TERMINUS OF TSHR ON T4 AND TRAB EXPRESSION IN BALB/C MICE.

Context: The expression of TSHR-C on the serum tetraiodothyronine (T4) and TSH receptor antibody (TRAb) levels are rarely studied. Objective: The effect of TSHR-c on T4 and TRAb levels and concomitant thyroid histological changes in mice was investigated. Design: Animal experimental study. Subjects and methods: Female BALB/c mice at 6-8 weeks of age were immunized with the thyroid stimulating hormone receptor antigen C-terminus (TSHR-C), and randomly divided into control group (treated with the corresponding concentrations of normal saline) and four experimental subgroups: TSHR-c1 subgroup (4 µg), TSHR-c2 subgroup (6 µg), TSHR-c3 subgroup (8 µg) and TSHR-c4 subgroup (10 µg). Serum T4 and TRAb levels were determined. Results: The serum T4 level decreased significantly in the experimental mice as the concentration increased. All the experimental mice were positive for serum TRAb (experimental groups: 40 positive/40, 100% vs. control group: 3 positive/10, 30%) compared to the control group (P =0.000). HE staining showed that the follicles in the control mice were composed of small to medium-sized round follicles, whereas the follicles in the experimental mice were irregularly enlarged under light microscope. Conclusions: TSHR-c immunization resulted in thyroid hormone changes like those observed in hypothyroidism, probably due to the induction of TRAb generation.

De novo variants in CELF2 that disrupt the nuclear localization signal cause developmental and epileptic encephalopathy.

We report heterozygous CELF2 (NM_006561.3) variants in five unrelated individuals: Individuals 1-4 exhibited developmental and epileptic encephalopathy (DEE) and Individual 5 had intellectual disability and autistic features. CELF2 encodes a nucleocytoplasmic shuttling RNA-binding protein that has multiple roles in RNA processing and is involved in the embryonic development of the central nervous system and heart. Whole-exome sequencing identified the following CELF2 variants: two missense variants [c.1558C>T:p.(Pro520Ser) in unrelated Individuals 1 and 2, and c.1516C>G:p.(Arg506Gly) in Individual 3], one frameshift variant in Individual 4 that removed the last amino acid of CELF2 c.1562dup:p.(Tyr521Ter), possibly resulting in escape from nonsense-mediated mRNA decay (NMD), and one canonical splice site variant, c.272-1G>C in Individual 5, also probably leading to NMD. The identified variants in Individuals 1, 2, 4, and 5 were de novo, while the variant in Individual 3 was inherited from her mosaic mother. Notably, all identified variants, except for c.272-1G>C, were clustered within 20 amino acid residues of the C-terminus, which might be a nuclear localization signal. We demonstrated the extranuclear mislocalization of mutant CELF2 protein in cells transfected with mutant CELF2 complementary DNA plasmids. Our findings indicate that CELF2 variants that disrupt its nuclear localization are associated with DEE.

Procyanidins Mitigate Osteoarthritis Pathogenesis by, at Least in Part, Suppressing Vascular Endothelial Growth Factor Signaling.

Procyanidins are a family of plant metabolites that have been suggested to mitigate osteoarthritis pathogenesis in mice. However, the underlying mechanism is largely unknown. This study aimed to determine whether procyanidins mitigate traumatic injury-induced osteoarthritis (OA) disease progression, and whether procyanidins exert a chondroprotective effect by, at least in part, suppressing vascular endothelial growth factor signaling. Procyanidins (extracts from pine bark), orally administered to mice subjected to surgery for destabilization of the medial meniscus, significantly slowed OA disease progression. Real-time polymerase chain reaction revealed that procyanidin treatment reduced expression of vascular endothelial growth factor and effectors in OA pathogenesis that are regulated by vascular endothelial growth factor. Procyanidin-suppressed vascular endothelial growth factor expression was correlated with reduced phosphorylation of vascular endothelial growth factor receptor 2 in human OA primary chondrocytes. Moreover, components of procyanidins, procyanidin B2 and procyanidin B3 exerted effects similar to those of total procyanidins in mitigating the OA-related gene expression profile in the primary culture of human OA chondrocytes in the presence of vascular endothelial growth factor. Together, these findings suggest procyanidins mitigate OA pathogenesis, which is mediated, at least in part, by suppressing vascular endothelial growth factor signaling.

Oral and anal sex are key to sustaining gonorrhoea at endemic levels in MSM populations: a mathematical model.

OBJECTIVES: Despite early treatment of urethral infection, gonorrhoea is endemic in urban populations of men who have sex with men (MSM) in Australia. By contrast, gonorrhoea is not common in urban heterosexual populations. Sexual activities among MSM usually involve anal or oral sex, and as these behaviours are becoming increasingly common among heterosexuals, there is a need to investigate their roles in transmission of gonorrhoea. METHODS: We developed individual-based models of transmission of gonorrhoea in MSM and heterosexuals that incorporate anatomical site-specific transmission of gonorrhoea. We estimated the probabilities of transmission for anal sex and oral sex by calibrating an MSM model against prevalence of gonorrhoea and sexual activity data. These probabilities were then applied to a heterosexual model in order to examine whether gonorrhoea can persist in a heterosexual population through the addition of anal sex and oral sex. RESULTS: In the MSM model, gonorrhoea can persist despite prompt treatment of urethral infections. The probability of gonorrhoea persisting is reduced if use of condom for oral sex is increased to more than 15% of acts. Assuming that treatment of symptomatic infections is prompt, gonorrhoea is unlikely to persist in a heterosexual population even with the addition of anal and oral sex. CONCLUSIONS: Our models suggest that oral sex has an important role in sustaining gonorrhoea in a population of MSM by providing a pool of untreated asymptomatic infection. The importance of anal sex or oral sex in sustaining gonorrhoea in a heterosexual population remains uncertain due to the lack of information linking different types of sex acts and transmissibility.

Nutraceuticals: potential for chondroprotection and molecular targeting of osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease and a leading cause of adult disability. There is no cure for OA, and no effective treatments which arrest or slow its progression. Current pharmacologic treatments such as analgesics may improve pain relief but do not alter OA disease progression. Prolonged consumption of these drugs can result in severe adverse effects. Given the nature of OA, life-long treatment will likely be required to arrest or slow its progression. Consequently, there is an urgent need for OA disease-modifying therapies which also improve symptoms and are safe for clinical use over long periods of time. Nutraceuticals-food or food products that provide medical or health benefits, including the prevention and/or treatment of a disease-offer not only favorable safety profiles, but may exert disease- and symptom-modification effects in OA. Forty-seven percent of OA patients use alternative medications, including nutraceuticals. This review will overview the efficacy and mechanism of action of commonly used nutraceuticals, discuss recent experimental and clinical data on the effects of select nutraceuticals, such as phytoflavonoids, polyphenols, and bioflavonoids on OA, and highlight their known molecular actions and limitations of their current use. We will conclude with a proposed novel nutraceutical-based molecular targeting strategy for chondroprotection and OA treatment.

Patient motivation and satisfaction from orthognathic surgery-a case series.

OBJECTIVE: This study attempted to understand the specific motivations of patients who undergo orthognathic surgery and determine their satisfaction with the surgery. The study also assessed the prevalence of complications and how they may impact patient satisfaction. STUDY DESIGN: Patients who underwent orthognathic surgery at the University of California between 2016 and 2019 and had completed postoperative orthodontic treatment for ≥9 months were interviewed. They responded to an open-ended telephone interview regarding their motivations, satisfaction, and complications. RESULTS: The patients showed a high level of satisfaction with the surgery, but there were persistent complications that affected satisfaction. The predominant complication was paresthesia over the distribution of the inferior alveolar nerve. The majority of patients who reported prior headaches and temporomandibular joint problems described improvement in those areas. Comparing the patients' motivations before and after surgery showed that before surgery, patients reported functional concerns, whereas postoperatively they were much more likely to recall aesthetic reasons for the surgery. CONCLUSION: This study showed that although patients are generally satisfied after orthognathic surgery, patients need to be realistically informed of their expectations and adequately informed of possible complications.

Physiological loading of joints prevents cartilage degradation through CITED2.

Both overuse and disuse of joints up-regulate matrix metalloproteinases (MMPs) in articular cartilage and cause tissue degradation; however, moderate (physiological) loading maintains cartilage integrity. Here, we test whether CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), a mechanosensitive transcriptional coregulator, mediates this chondroprotective effect of moderate mechanical loading. In vivo, hind-limb immobilization of Sprague-Dawley rats up-regulates MMP-1 and causes rapid, histologically detectable articular cartilage degradation. One hour of daily passive joint motion prevents these changes and up-regulates articular cartilage CITED2. In vitro, moderate (2.5 MPa, 1 Hz) intermittent hydrostatic pressure (IHP) treatment suppresses basal MMP-1 expression and up-regulates CITED2 in human chondrocytes, whereas high IHP (10 MPa) down-regulates CITED2 and increases MMP-1. Competitive binding and transcription assays demonstrate that CITED2 suppresses MMP-1 expression by competing with MMP transactivator, Ets-1 for its coactivator p300. Furthermore, CITED2 up-regulation in vitro requires the p38δ isoform, which is specifically phosphorylated by moderate IHP. Together, these studies identify a novel regulatory pathway involving CITED2 and p38δ, which may be critical for the maintenance of articular cartilage integrity under normal physical activity levels.

Moderate joint loading reduces degenerative actions of matrix metalloproteinases in the articular cartilage of mouse ulnae.

Joint loading is a recently developed loading modality, which can enhance bone formation and accelerate healing of bone fracture. Since mechanical stimulation alters expression of matrix metalloproteinases (MMPs) in chondrocytes, a question addressed herein was, does joint loading alter actions of MMPs in the articular cartilage? We hypothesized that expression and activity of MMPs are regulated in a load-intensity-dependent manner and that moderate load scan downregulates MMPs. To test this hypothesis, a mouse elbow-loading model was employed. In the articular cartilage of an ulna, the mRNA levels of a group of MMPs as well as their degenerative activities were determined. The result revealed that elbow loading altered the expression and activities of MMPs depending on its loading intensity. Collectively, the data in this study indicate that 0.2 and 0.5 N joint loading significantly reduced the expression of multiple MMPs, that is, MMP-1, MMP-3, MMP-8, and MMP-13, and overall activities of collagenases or gelatinases in articular cartilage, while higher loads increased the expression and activity of MMP-1 and MMP-13. Furthermore, moderate loads at 1 N elevated the mRNA level of CBP/p300-interacting transactivator with ED-rich tail 2 (CITED2), but higher loads at 4 N did not induce a detectable amount of CITED2 mRNA. Since CITED2 is known to mediate the downregulation of MMP-1 and MMP-13, the result indicates that joint loading at moderate intensity reduces MMP activities through potential induction of CITED2. MMPs such as MMP-1 and MMP-13 are predominant collagenases in the pathology of osteoarthritis. Therefore, joint loading could offer an interventional regimen for maintenance of joint tissues.

Mechanical loading: bone remodeling and cartilage maintenance.

Bone remodeling and cartilage maintenance are strongly influenced by biomechanical signals generated by mechanical loading. Although moderate loading is required to maintain bone mass and cartilage homeostasis, loading can cause deleterious effects such as bone fracture and cartilage degradation. Because a tight coupling exists between cartilage and bone, alterations in one tissue can affect the other. Bone marrow lesions are often associated with an increased risk of developing cartilage defects, and changes in the articular cartilage integrity are linked to remodeling responses in the underlying bone. Although mechanisms regulating the maintenance of these two tissues are different, compelling evidence indicates that the signal pathways crosstalk, particularly with the Wnt pathway. A better understanding of the complex tempero-spatial interplay between bone remodeling and cartilage degeneration will help develop a therapeutic loading strategy that prevents bone loss and cartilage degeneration.

Events in articular chondrocytes with aging.

It is well accepted that aging is one of the most prominent risk factors for the initiation and progression of osteoarthritis. One of the most pronounced age-related changes in chondrocytes is the exhibition of a senescent phenotype, which is the result of several factors including the accumulation of reactive oxygen species and advanced glycation end products. Compared with a normal chondrocyte, senescent chondrocytes exhibit an impaired ability to respond to many mechanical and inflammatory insults to the articular cartilage. Furthermore, protein secretion is altered in aging chondrocytes, demonstrated by a decrease in anabolic activity and increased production of proinflammatory cytokines and matrix-degrading enzymes. Together, these events may make the articular cartilage matrix more susceptible to damage and lead to the onset of osteoarthritis. A better understanding of the mechanisms underlying age-related chondrocyte pathophysiology may be critical for the development of novel therapeutic interventions for progressive joint diseases.

Advances in the development of gene therapy, noncoding RNA, and exosome-based treatments for tendinopathy.

Tendinopathy is a common musculoskeletal disorder characterized by chronic low-grade inflammation and tissue degeneration. Tendons have poor innate healing ability and there is currently no cure for tendinopathy. Studies elucidating mechanisms underlying the pathogenesis of tendinopathy and mechanisms mediating the genesis of tendons during development have provided novel targets and strategies to enhance tendon healing and repair. This review summarizes the current understanding and treatments for tendinopathy. The review also highlights recent advances in gene therapy, the potential of noncoding RNAs, such as microRNAs, and exosomes, which are nanometer-sized extracellular vesicles secreted from cells, for the treatment of tendinopathy.

Identification of CITED2 as a negative regulator of fracture healing.

The transcription regulator CITED2 (CBP/p300-Interacting-Transactivator-with-ED-rich-tail-2) is known to suppress genes mediating angiogenesis and extracellular matrix (ECM) remodeling. However, it is unclear whether CITED2 has a role in controlling skeletal repair or remodeling. We tested the hypothesis that CITED2 functions in bone fracture healing by suppressing the expression of genes critical to ECM remodeling, angiogenesis and osteogenesis, importantly the matrix metalloproteinases (MMPs). Three hours following mandibular osteotomy or sham surgery of adult rats, osteotomy fronts were harvested and the expression of CITED2 and genes associated with fracture healing was ascertained by quantitative PCR. In parallel, gain-of-function studies examined the effect of overexpressing CITED2 on the expression and activity of several MMPs. In the fractured mandible, CITED2 expression was inversely related to the expression of MMP-2, -3, -9, -13, VEGF, HIF-1alpha, M-CSF, RANK-L, and OPG. Consistent with this, the over-expression of CITED2 in osteoblasts inhibited the expression and activity of MMP-2, -3, -9, and -13. Taken together, the studies suggest that CITED2 is a critical upstream regulator of fracture healing. The suppression of CITED2 early after fracture may allow an optimal initiation of the healing response.

Epigallocatechin-3-gallate suppresses pain-related and proinflammatory mediators in the subacromial bursa in rotator cuff tendinopathy.

Rotator cuff tendinopathy is one of the leading causes of shoulder pain. However, the mechanisms involved in the development of rotator cuff tendinopathy pain are not fully understood. In this study, we first examined the histological features of subacromial bursa from patients with rotator cuff tendinopathy who had symptoms of pain, and investigated the expression of pain mediators, proinflammatory cytokines, metalloproteinases, growth factors, and alarmins in diseased tendon and bursa tissue by real-time PCR, western blot, and/or immunohistochemistry/immunofluorescence staining. Then we investigated whether epigallocatechin-3-gallate (EGCG) could reduce the expression of pain mediators and proinflammatory cytokines in human primary bursa cells and explored the paracrine effect of these EGCG-treated bursa cells on tenocytes in vitro. Neovascularization and infiltration of immune cells including monocytes/macrophages and mast cells were observed in diseased bursa tissue. Bursa from patients with pain had higher mRNA expression of pain mediators and proinflammatory cytokines, compared to the rotator cuff tendon of the same patients, as well as the bursa from asymptomatic patients. EGCG treatment significantly suppressed the interleukin 1 beta (IL-1ß)-induced elevation of mRNA expression of pain mediators, proinflammatory cytokines, and matrix metalloproteinases (MMPs) in bursa cells in vitro; conditioned medium from EGCG-treated bursa cells significantly reduced IL-1ß-induced expression in human primary tenocytes. Our study suggests that the subacromial bursa might serve as a local source of pain mediators and proinflammatory cytokines in rotator cuff tendinopathy. Moreover, EGCG treatment by primarily targeting the subacromial bursa may be a potential strategy to relieve rotator cuff tendinopathy-related pain and symptoms.

Role of pannexin 1 channels in load-induced skeletal response.

The pannexin 1 (Panx1) channel is a mechanosensitive channel that interacts with P2X7 receptors (P2X7R) to form a functional complex that has been shown in vitro to play an essential role in osteocyte mechanosignaling. While the participation of P2X7R in skeletal responses to mechanical loading has been demonstrated, the role of Panx1 and its interplay with P2X7R still remain to be determined. In this study, we use a global Panx1-/- mouse model and in vivo mechanical loading to demonstrate that Panx1 channels play an essential role in load-induced skeletal responses. We found that absence of Panx1 not only disrupts the P2X7R-Panx1 signaling complex, but also alters load-induced regulation of P2X7R expression. Moreover, lack of Panx1 completely abolished load-induced periosteal bone formation. Load-induced regulation of ß-catenin and sclerostin expression was dysregulated in Panx1-/- , compared to wild-type, bone. This finding suggests that Panx1 deficiency disrupts Wnt/ß-catenin signaling by lowering ß-catenin while favoring inhibition of bone formation by increasing load-induced sclerostin expression. This study demonstrates the existence of a Panx1-dependent mechanosensitive mechanism that not only modulates ATP signaling but also coordinates Wnt/ß-catenin signaling that is essential for proper skeletal response to mechanical loading.

A Converging Strategy for the Generation of a Virtually Sequenced cDNA Library from Unreferenced Pacific Oysters.

The access to biological material of reference species, which were used previously in key experiments such as in the development of novel cell lines or genome sequencing projects, are often difficult to provide for further studies or third parties due to the consumptive nature of the samples. Although now widely distributed over the Pacific coasts of Asia, Australia and North America, individual Pacific oyster specimens are genetically quite diverse and are therefore not directly suitable as the starting material for gene libraries. In this article, we demonstrate the use of unreferenced Pacific oyster specimens obtained from regional seafood markets to generate cDNA libraries. These libraries were then compared to the publicly available oyster genome, and the closest related library was selected using the mitochondrial reference genes Cytochrome C Oxidase subunit I (COX1) and NADH Dehydrogenase (ND). The suitability of the generated cDNA library is also demonstrated by cloning and expression of two genes encoding the enzymes UDP-glucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS), which are responsible for the biosynthesis of UDP-xylose from UDP-glucose.

CITED2 mediates the cross-talk between mechanical loading and IL-4 to promote chondroprotection.

Osteoarthritis (OA) pathogenesis is mediated largely through the actions of proteolytic enzymes such as matrix metalloproteinase (MMP) 13. The transcriptional regulator CITED2, which suppresses the expression of MMP13 in chondrocytes, is induced by interleukin (IL)-4 in T cells and macrophages, and by moderate mechanical loading in chondrocytes. We tested the hypothesis that CITED2 mediates cross-talk between IL-4 signaling and mechanical loading-induced pathways that result in chondroprotection, at least in part, by downregulating MMP13. IL-4 induced CITED2 gene expression in human chondrocytes in a dose- and time-dependent manner through JAK/STAT signaling. Mechanical loading combined with IL-4 resulted in additive effects on inducing CITED2 expression and downregulating of MMP13 in human chondrocytes in vitro. In vivo, IL-4 gene knockout (KO) mice exhibited reduced basal levels of CITED2 expression in chondrocytes. While moderate treadmill running induced CITED2 expression and reduced MMP13 expression in wild-type mice, these effects were blunted (for CITED2) or abolished (for MMP13) in chondrocytes of IL-4 gene KO mice. Moreover, intra-articular injections of mouse recombinant IL-4 combined with regular cage activity mitigated post-traumatic OA to a greater degree compared to immobilized mice treated with IL-4 alone. These data suggest that using moderate loading to enhance IL-4 may be a potential therapeutic strategy for chondroprotection in OA.

CITED2 mediates the mechanical loading-induced suppression of adipokines in the infrapatellar fat pad.

Adipokines secreted from the infrapatellar fat pad (IPFP), such as adipsin and adiponectin, have been implicated in osteoarthritis pathogenesis. CITED2, a mechanosensitive transcriptional regulator with chondroprotective activity, may modulate their expression. Cited2 haploinsufficient mice (Cited2+/- ) on a high-fat diet (HFD) exhibited increased body weight and increased IPFP area compared to wild-type (WT) mice on an HFD. While an exercise regimen of moderate treadmill running induced the expression of CITED2, as well as PGC-1α, and reduced the expression of adipsin and adiponectin in the IPFP of WT mice on an HFD, Cited2 haploinsufficiency abolished the loading-induced expression of PGC-1α and loading-induced suppression of adipsin and adiponectin. Furthermore, knocking down or knocking out CITED2 in adipose stem cells (ASCs)/preadipocytes derived from the IPFP in vitro led to the increased expression of adipsin and adiponectin and reduced PGC-1α, and abolished the loading-induced suppression of adipsin and adiponectin and loading-induced expression of PGC-1α. Overexpression of PGC-1α in these ASC/preadipocytes reversed the effects caused by CITED2 deficiency. The current data suggest that CITED2 is a critical regulator in physiologic loading-induced chondroprotection in the context of an HFD and PGC-1α is required for the inhibitory effects of CITED2 on the expression of adipokines such as adipsin and adiponectin in the IPFP.

Correction to: Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model.

Following publication of the original article [1], the authors reported an error in Figs. 2C and 5C.