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Cobalt-catalyzed hydroalkynylation of alkynes, alkenes, and imines affords internal alkynes with various functional groups adjacent to the carbon–carbon triple bond moiety in an atom-economical manner. In addition, cross-coupling of in situ generated alkynylcobalt species from terminal alkynes, haloalkynes, and metal acetylides with (hetero)aromatic compounds and organic halides selectively provides various internal aryl- and alkylalkynes.1 Introduction2 Hydroalkynylation of Alkynes for 1,3-Enyne Synthesis3 Hydroalkynylation of Polar and Nonpolar Double Bonds4 Dehydrogenative Cross-Coupling Reaction Using Terminal Alkynes with Aromatic Compounds5 Cross-Coupling Reactions Using Haloalkynes as the Coupling Partners6 Cross-Coupling Reactions Using Metal Acetylides7 Conclusion

Immune checkpoint inhibitors (ICIs) frequently cause immune-related adverse events (irAEs) that often involve endocrine organs. Pembrolizumab and atezolizumab are currently administered in combination with chemotherapy for several malignancies. Although transient thyrotoxicosis within 6 weeks after the first ICI dose is the typical course of thyroid irAEs with ICI monotherapy, we encountered a unique course of a thyroid irAE in a patient who received combination therapy consisting of pembrolizumab plus pemetrexed and carboplatin. Delayed onset of thyrotoxicosis occurred at 22 weeks after the first dose of pembrolizumab. To understand more about this curious event, we conducted a retrospective cohort study of the following groups: pembrolizumab monotherapy (Pem-mono), pembrolizumab plus chemotherapy (Pem-combi), atezolizumab monotherapy (Atezo-mono), and atezolizumab plus chemotherapy (Atezo-combi). There were no differences in the incidence of overt thyroid irAEs: Pem-mono, 12 of 151 patients (7.9%) versus Pem-combi, 4 of 56 patients (7.1%) (p = 0.85) and Atezo-mono, 5 of 27 patients (18.5%) versus Atezo-combi, 5 of 57 patients (8.8%) (p = 0.20). Through detailed analyses of patients with thyrotoxicosis, we found some patients with delayed-onset thyroid irAE, defined as development at 16 weeks or more after the first ICI dose. Delayed-onset thyroid irAEs were only observed in the combination therapy groups: Pem-combi or Atezo-combi, 3 of 8 patients versus Pem-mono or Atezo-mono, 0 of 10 patients. Our observation that thyroid irAE development can be delayed with ICIs when used in combination with chemotherapy suggests longer monitoring of thyroid function is needed to avoid missing irAEs.

OBJECTIVE A unique clinical course was observed in a patient with resistance to thyroid hormone β (RTHβ) caused by a variant of the THRB gene leading to the replacement of glycine with arginine in codon 347 (p.G347R). He presented with the syndrome of inappropriate secretion of thyrotropin (TSH) (free T4 [fT4]: 32.43 pmol/L, TSH: 4.67 mIU/L), but slowly developed progressive hypothyroidism (fT4: 8.37 pmol/L, TSH: 100.90 mIU/L) that resolved after suspending bezafibrate (BZ) treatment (fT4: 32.18 pmol/L, TSH: 7.14 mIU/L). This study clinically and experimentally evaluated this interesting phenomenon. METHODS A retrospective cohort analysis of non-RTHβ patients was performed at Kyoto University Hospital. Data before BZ treatment were compared to the first data after treatment. Using reporter assays of iodothyronine deiodinases (DIO1, DIO2, DIO3) in HEK293T cells, we performed functional analyses of mutant thyroid hormone receptor β with p.G347R (G347R TRβ). Mice with G347R TRβ were generated by hydrodynamic gene delivery. RESULTS In non-RTHβ patients (n = 7), BZ treatment did not change serum free T3 and TSH but significantly increased fT4 (p = .008). BZ administration increased DIO3 reporter activity in the context of G347R TRβ, whereas did not change DIO1 and DIO2 reporter activity. In the livers of mice with G347R TRβ, BZ administration increased reverse T3 content, which corresponded to an increase in Dio3 messenger RNA. CONCLUSIONS While hypothyroidism associated with BZ treatment did not occur in non-RTHβ patients, it was observed in a patient with RTHβ due to the p.G347R variant. Liver DIO3 upregulation might involve this hypothyroidism.

3,3’,5-triiodothyroacetic acid (TRIAC) is a metabolite of endogenous thyroid hormones (THs) that can bind to and activate TH receptors. As TRIAC was previously detected in sewage effluent, we aimed to investigate exogenous TRIAC’s potential for endocrine disruption. We administered either TRIAC or 3,3’,5-triiodo-L-thyronine (LT3) to both euthyroid mice and 6-propyl-2-thiouracil-induced hypothyroid mice. In hypothyroid mice, TRIAC alleviated growth retardation, suppressed the hypothalamus-pituitary-thyroid (HPT) axis, and upregulated TH-responsive genes in the pituitary gland, liver, and heart. We observed that, unlike LT3, TRIAC does not upregulate the expression of TH-responsive genes in the cerebrum. Measurement of organ-specific TRIAC levels suggested that TRIAC was not efficiently trafficked into the cerebrum. Furthermore, by analyzing euthyroid mice, we found that cerebral TRIAC levels did not increase despite TRIAC administration at higher concentrations, whereas serum and cerebral TH levels were substantially decreased. Hence, TH-responsive genes in the cerebrum appear to be downregulated by TRIAC. In summary, TRIAC administration decreases circulating TH levels by suppressing the HPT axis, while the consequent attenuation of TH actions was compensated by TRIAC in peripheral tissues but not in the cerebrum due to the relative impermeability of the blood–brain barrier towards TRIAC. We verified that exogenous TRIAC disrupts TH actions in the cerebrum. This disruption is apparently due to the additive effects of circulating endogenous THs being depleted via a negative feedback loop involving the HPT axis and heterogenous distribution of TRIAC among different organs. Our findings indicate that environmental TRIAC poses a potential neurodevelopmental risk.

Background In peripheral tissues, triiodothyronine (T3) production and consequent thyroid hormone actions are mainly regulated by iodothyronine deiodinases (DIOs) classified into 3 types: D1, D2, and D3. Although thyroid hormone economy in the DIO-deficient mice and patients carrying genetic variants of DIOs was well clarified, a condition of DIOs upregulation was not enough elucidated. We aimed to investigate effects of peripheral DIOs on thyroid hormone economy and consequent thyroid hormone action using novel DIO-overexpressing mice that were not yet generated. Methods We cloned coding sequences of human DIOs with FLAG-tag and HiBiT-tag sequences into a pcDNA3.1 vector. To obtain full-length proteins, we modified these vectors by cloning selenocysteine insertion sequence of each DIO (SECIS vectors). Western blot analyses and HiBiT lytic assay using HEK293T cells revealed that SECIS vectors expressed full-length proteins with substantial activity. Subsequently, in vivo transfections of pLIVE-based SECIS vectors into male C57BL/6J mice were performed by hydrodynamic gene delivery to generate mice overexpressing DIOs (D1, D2, and D3 mice). Results First, we verified that DIOs were successfully overexpressed in the livers using HiBiT lytic assay as well as RT-PCR. Analyses of multiple organs of D3 mice assured that full-length protein was not significantly expressed in the heart, the kidney, and the skeletal muscle. RT-PCR analyses also suggested that the pituitary glands and the thyroid glands were not transfected. We evaluated thyroid hormone economy of DIO-overexpressing mice compared to Empty mice transfected with pLIVE-Empty vector. D1 mice did not have changes in serum thyroid hormone levels, whereas D2 mice had higher serum fT3 levels. Clearer changes were found in D3 mice. They had hypothyroidism with higher serum rT3 levels. Correspondingly, in their livers, rT3 concentration was higher and T3-responsive genes were downregulated. We elucidated by adding the cohort with levothyroxine (LT4) administration because DIO-overexpressing mice had liver T4 insufficiency probably due to consumption by DIOs. Comparisons between the DIO-overexpressing mice with and without LT4 administration revealed that D2 mice presented upregulation of T3-responsive genes by LT4 administration distinct from Empty mice. Conclusions We generated DIO-overexpressing mice that expressed human DIOs predominantly in the liver. They provided physiological evidence to integrate the phenotypes of the knockout mice and specific clinical situations. D2 mice seemed to recruit T3 into the circulation as well as in the liver. D3 mice had an overt phenotype characterized by consumptive hypothyroidism. In addition, D3 mice are expected to be a novel hypothyroidism model that does not involve disruption of thyroidal hormone secretion. DIO-overexpressing mice can be generated by single injections of plasmid vectors of interest and can contribute to progress in the field of thyroid hormone economy and action. Presentation: No date and time listed

Regulatory mechanisms of iodothyronine deiodinases (DIOs) require further elucidation, and conventional methods for evaluating DIOs are unsuitable for high-throughput screening (HTS). Here we explored factors of transcriptional regulation of 3 types of DIOs (DIO1, DIO2, and DIO3) from a chemical library using our designed HTS. We constructed HTS based on a promoter assay and performed a screen of 2480 bioactive compounds. For compounds that were clinically approved, we validated hit compounds through a retrospective cohort study in our department that evaluated changes in thyroid function in patients using the compounds as drug therapy. Furthermore, we verified the involvement of DIOs using mice treated with the compounds. Of the hit compounds, 6 and 7 compounds transcriptionally up- and downregulated DIO1, respectively; 34 transcriptionally upregulated DIO2; and 5 and 2 compounds transcriptionally up- and downregulated DIO3, respectively. The cohort study clarified the clinical effects of some hit compounds: ritodrine increased free triiodothyronine (fT3)/free thyroxine (fT4) ratio and decreased serum thyroid-stimulating hormone (TSH) levels, tadalafil increased serum fT3 levels, and tyrosine kinase inhibitors (TKIs) decreased serum fT3 and fT4 levels and increased serum TSH levels. Following in vivo experiments using treated mice, consistent results were observed in ritodrine, which upregulated DIO2 in the thyroid gland. In conclusion, we completed HTS for DIOs and obtained attractive hit compounds. Our cohort study revealed the clinical significance of ritodrine, sildenafil, and TKIs. We hope our unique method will contribute to analyzing various targets and lists of hit compounds will promote understanding of DIOs.

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide evidence from experiments using mutant mice, indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel–TRPM7 channel–CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

In peripheral tissues, triiodothyronine (T3) production and consequent thyroid hormone actions are mainly regulated by iodothyronine deiodinases (DIOs) classified into 3 types: D1, D2, and D3. We aimed to investigate the effects of peripheral DIOs on thyroid hormone economy independent of the hypothalamus-pituitary-thyroid axis. We cloned coding sequences of human DIOs with FLAG-tag and HiBiT-tag sequences into a pcDNA3.1 vector. To obtain full-length proteins, we modified these vectors by cloning the selenocysteine insertion sequence of each DIO (SECIS vectors). Western blot analyses and HiBiT lytic assay using HEK293T cells revealed that SECIS vectors expressed full-length proteins with substantial activity. Subsequently, in vivo transfections of pLIVE-based SECIS vectors into male C57BL/6J mice were performed by hydrodynamic gene delivery to generate mice overexpressing DIOs predominantly in the liver (D1, D2, and D3 mice). After 7 days from transfections, mice were analyzed to clarify phenotypes. To summarize, serum thyroid hormone levels did not change in D1 mice but D2 mice had higher serum free T3 levels. D3 mice developed hypothyroidism with higher serum reverse T3 (rT3) levels. Transfections with levothyroxine administration suggested that thyroid hormone action was upregulated in D2 mice. Our DIO-overexpressing mice provided insights on the physiological properties of upregulated DIOs: D2 augments local thyroid hormone action and recruits T3 into the circulation: D3 decreases circulating T3 and T4 levels with elevated rT3, leading to consumptive hypothyroidism. As D3 mice are expected to be a novel hypothyroidism model, they can contribute to progress in the field of thyroid hormone economy and action.

Skeletal alterations in the head and neck region, such as midfacial hypoplasia, foramen magnum stenosis and spinal canal stenosis, are commonly observed in patients with mucopolysaccharidosis (MPS). However, enzyme replacement therapy (ERT), one of the major treatment approaches for MPS, shows limited efficacy for skeletal conditions. In this study, we analysed the craniofacial morphology of mice with MPS type VII, and investigated the underlying mechanisms promoting jaw deformities in these animals. Furthermore, we investigated the effects of C-type natriuretic peptide (CNP), a potent endochondral ossification promoter, on growth impairment of the craniofacial region in MPS VII mice when administered alone or in combination with ERT. MPS VII mice exhibited midfacial hypoplasia caused by impaired endochondral ossification, and histological analysis revealed increased number of swelling cells in the resting zone of the spheno-occipital synchondrosis (SOS), an important growth centre for craniomaxillofacial skeletogenesis. We crossed MPS VII mice with transgenic mice in which CNP was expressed in the liver under the control of the human serum amyloid-P component promoter, resulting in elevated levels of circulatory CNP. The maxillofacial morphological abnormalities associated with MPS VII were ameliorated by CNP expression, and further prevented by a combination of CNP and ERT. Histological analysis showed that ERT decreased the swelling cell number, and CNP treatment increased the width of the proliferative and hypertrophic zones of the SOS. Furthermore, the foramen magnum and spinal stenoses observed in MPS VII mice were significantly alleviated by CNP and ERT combination. These results demonstrate the therapeutic potential of CNP, which can be used to enhance ERT outcome for MPS VII-associated head and neck abnormalities.

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca2+ influx in growth plate chondrocytes, leading to activation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) for promoting bone growth. In this report, we provide experimental evidence indicating a functional link between CNP and TRPM7 channels. Our pharmacological data suggest that CNP-evoked NPR2 activation elevates cellular cGMP content and stimulates big-conductance Ca2+-dependent K+ (BK) channels as a substrate for cGMP-dependent protein kinase (PKG). BK channel-induced hyperpolarization likely enhances the driving force of TRPM7-mediated Ca2+ entry and seems to accordingly activate CaMKII. Indeed, ex vivo organ culture analysis indicates that CNP-facilitated bone growth is abolished by chondrocyte-specific Trpm7 gene ablation. The defined CNP signaling pathway, the NPR2-PKG-BK channel-TRPM7 channel-CaMKII axis, likely pinpoints promising target proteins for developing new therapeutic treatments for divergent growth disorders.

Opioids are widely used for treatment of acute and chronic pain. However, opioids have several well-known clinical adverse effects such as constipation, nausea, respiratory depression and drowsiness. Endocrine dysfunctions are also opioid-induced adverse effects but remain under-diagnosed in clinical settings, especially opioid-induced adrenal insufficiency (OIAI). A 46-year-old woman was treated with transdermal fentanyl at a dose of 90-120 mg daily morphine milligram equivalent for non-malignant chronic pain for four years. Fatigue, loss of appetite and decrease in vitality began about two years after starting fentanyl. Subsequently, constipation and abdominal pain appeared and became worse, which led to suspicion of adrenal insufficiency. Clinical diagnosis of OIAI was established based on laboratory findings of secondary adrenal insufficiency, including corticotropin-releasing hormone stimulation test, clinical history of long-term fentanyl use, and exclusion of other hypothalamic-pituitary diseases. Oral corticosteroid replacement therapy was unable to relieve her abdominal pain and constipation; opioid-rotation and dose-reduction of fentanyl were not feasible because of her persistent pain and severe anxiety. While her clinical course clearly suggested that long-term, relatively high-dose transdermal fentanyl treatment may have contributed to the development of secondary adrenal insufficiency, the symptoms associated with OIAI are generally non-specific and complex. Together with under-recognition of OIAI as a clinical entity, the non-specific, wide range of symptoms can impede prompt diagnosis. Thus, vigilance for early symptoms enabling treatments including corticosteroid replacement therapy is necessary for patients taking long-term and/or high dose opioid treatment.

Growth retardation is an important side effect of glucocorticoid (GC)-based drugs, which are widely used in various preparations to treat many pediatric diseases. We investigated the therapeutic effect of exogenous CNP-53, a stable molecular form of intrinsic CNP, on a mouse model of GC-induced growth retardation. We found that CNP-53 successfully restored GC-induced growth retardation when both dexamethasone (DEX) and CNP-53 were injected from 4 to 8 weeks old. Notably, CNP-53 was not effective during the first week. From 4 to 5 weeks old, neither CNP-53 in advance of DEX, nor high-dose CNP-53 improved the effect of CNP. Conversely, when CNP-53 was started at 5 weeks old, final body length at 8 weeks old was comparable to that when CNP-53 was started at 4 weeks old. As for the mechanism of resistance to the CNP effect, DEX did not impair the production of cGMP induced by CNP. CNP reduced Erk phosphorylation even under treatment with DEX, while CNP did not changed that of p38 or GSK3β. Collectively, the effect of CNP-53 on GC-induced growth retardation is dependent on age in a mouse model, suggesting adequate and deliberate use of CNP would be effective for GC-induced growth retardation in clinical settings.

The growth plates are cartilage tissues found at both ends of developing bones, and vital proliferation and differentiation of growth plate chondrocytes are primarily responsible for bone growth. C-type natriuretic peptide (CNP) stimulates bone growth by activating natriuretic peptide receptor 2 (NPR2) which is equipped with guanylate cyclase on the cytoplasmic side, but its signaling pathway is unclear in growth plate chondrocytes. We previously reported that transient receptor potential melastatin-like 7 (TRPM7) channels mediate intermissive Ca

This study aimed to determine the effect of C-type natriuretic peptide (CNP) overexpression on craniofacial growth during the pubertal growth period in mice.Six-week-old C57BL/6 mice were injected with pLIVE-Empty vectors (Control mice) and pLIVE-NPPC vectors (CNP mice) using the hydrodynamic method. Morphological analyses were performed at the age of 12 weeks.Micro-computed tomography (µCT) images showed significant (p 0.05) hyperplasia in the maxilla along the sagittal plane (CNP mice: 13.754 mm, Control mice: 13.215 mm). Further, the images revealed significant bone overgrowth in the sagittal direction in the sphenoid (CNP mice: 6.936 mm, Control mice: 6.411 mm) and occipital (CNP mice: 4.051 mm, Control mice: 3.784 mm) bones in the CNP mice compared with that in the Control mice. Compared with SAP-Nppc-Tg mice in previous studies, although there was no effect on nose length and nasal bone length, the effect was sufficient to improve craniofacial hypogrowth. Furthermore, CNP promoted sagittal cranial growth by increasing the thickness of the spheno-occipital synchondrosis in organ cultures and nasal septal cartilage in micromass cultures, which were derived from 6-week-old mice.We have previously shown that the elevated blood levels of CNP from the neonatal period affect midfacial skeletogenesis by promoting endochondral ossification using mice (SAP-Nppc-Tg mice). The overexpression of CNP, even in 6-weeks-old mice, promoted growth in the sagittal direction within the maxillary region. These findings indicate the therapeutic potential of CNP for the treatment of midfacial hypoplasia during the pubertal growth spurt.

AimsProgrammed cell death-1 (PD-1) blockade therapy frequently results in immune-related adverse events involving the thyroid gland (thyroid irAEs). Although clinical features of thyroid irAEs are known, the mechanisms remain unclear. Here, we conducted a pilot study to investigate mechanisms of thyroid irAE development from the perspective of autoantibodies.MethodsWe performed immunoprecipitation-based assays using sera of 3 patients who developed thyroid irAEs with PD-1 blockade therapy by nivolumab and HEK293T cell lysates, including overexpressed proteins of interest (NKX2-1, PAX8, FOXE1, and HHEX; thyroid-specific transcriptional factors). The pellets were analyzed by western blot to detect the HiBit tag attached to the C-terminus of the proteins.ResultsRelevant changes to NKX2-1 bands were not seen in all 3 patients, but PAX8 bands were augmented in patient 2 with lung cancer and patient 3 with renal cell carcinoma. In addition, FOXE1 bands were augmented in patient 1 with malignant melanoma and patient 3, and a HHEX band was augmented in patient 3. Thus, we revealed novel thyroid-specific autoantibodies, PAX8Ab, FOXE1Ab, and HHEXAb. Expression patterns of the antigens recognized by these antibodies were not identical to the primary sites, so autoimmune responses in thyroid irAE may originate from the thyroid gland, and not the malignancy. Considering that TPOAb rather than TgAb is often negative in patients with thyroid irAEs, other mechanisms such as cytotoxic T cell and antigenicity of thyroglobulin may be involved.ConclusionsAlthough the significance of these novel autoantibodies needs further examination, the present study provides new insights for thyroid autoimmunity.

Objective Programmed cell death-1 (PD-1) blockade therapy, an immune checkpoint treatment, can induce hypophysitis or hypopituitarism as an immune-related adverse event (pituitary irAE). We aimed to clarify the clinical features of pituitary irAEs during PD-1 blockade therapy. Design, patients and measurements This retrospective study investigated consecutive patients treated with nivolumab, an anti-PD-1 antibody, at Kyoto University Hospital between 1 September 2014 and 31 August 2019. We examined patients' baseline characteristics and analysed the clinical data of those who developed pituitary irAEs. Results Of the 374 recruited patients, 7 (1.9%) developed pituitary irAEs, and each presented with isolated secondary adrenal insufficiency. In 4 patients, changes in ACTH were delayed relative to those in cortisol: when serum cortisol levels fell below the reference range, plasma ACTH levels were still normal. Pituitary irAEs were accompanied by elevated serum-free T3 (fT3) levels, which resolved with glucocorticoid replacement. Serum TSH levels were not suppressed despite elevated serum fT3 levels and 1 patient even presented with high fT3 level above the reference range (fT3, 7.1 pmol/L; free T4 (fT4), 13.9 pmol/L; and TSH, 5.1 mIU/L). Conclusions Isolated secondary adrenal insufficiency was a common pituitary irAE during PD-1 blockade therapy. This condition was accompanied by thyroid dysfunction, including elevation of fT3 without TSH suppression.

Hyperledger Fabric is an implementation that enables permissioned blockchains, which provide a general blockchain framework with identifiable participants for a variety of business applications. Although many performance issues of Hyperledger Fabric have been alleviated to some extent, its performance is still limited - e.g. 2.2k transactions per second in our experiment that executes two reads and two writes in a transaction. A major performance bottleneck is incurred by accesses to the databases that store the latest key-value pairs in the ledger data, indexes to transactions, and the update history. In this paper, we characterize the performance of database systems used in Hyperledger Fabric to identify optimization opportunities by running a Hyperledger Fabric GoLevelDB (HLF-GLDB) benchmark. We developed HLF-GLDB as a standalone benchmark to simulate database accesses in Hyperledger Fabric. Results of the performance characterization revealed that: (1) the data compression of GoLevelDB is a major performance bottleneck in Hyperledger Fabric, and disabling the compression improved the performance by 54%; (2) the size of a database affects the performance significantly. For example, when the size increased by four times, the performance degraded by 25%; (3) To reduce the database access overhead in chaincode, it is better to combine small values so that they can be represented by a single key.

We developed a non-toxic cyanation reaction of various aryl halides and triflates in acetonitrile using a catalyst system of [Ni(MeCN)6](BF4)2, 1,10-phenanthroline, and 1,4-bis(trimethylsilyl)-2,3,5,6-tetramethyl-1,4-dihydropyrazine (Si-Me4-DHP). Si-Me4-DHP was found to function as a reductant for generating nickel(0) species and a silylation reagent to achieve the catalytic cyanation via C-CN bond cleavage.

Background: Immune-related adverse events by immune checkpoint inhibitors often involve several endocrine-related organs. PD-1 pathway blockade therapy by anti-PD-1 antibodies including nivolumab frequently causes thyroid dysfunction (thyroid irAE). Thyroid irAE seems to be distinctive compared to conventional painless thyroiditis in terms of a clinical course: transient thyrotoxicosis and subsequent persistent hypothyroidism [1]. Our retrospective cohort study regarding nivolumab provided several suggestions [2]. The thyroid irAE (+) group had a longer median overall survival than the thyroid irAE (−) group in patients with lung cancer, but this observation was not seen in patients with malignant melanoma: In addition, 5 of 17 patients tested at the point of thyroid dysfunction development were double negative for TPOAbs and TgAbs, known thyroid autoantibodies. From these findings, we set a hypothesis that antibodies for unknown antigens mediate prognostic effects of thyroid irAEs if tumor tissues express the same antigens. Methods: We performed co-immunoprecipitation using Protein G beads, sera of three patients with thyroid irAEs, and lysates of HEK293T cells overexpressing candidate proteins tagged with FLAG and HiBit (NKX2-1, PAX8, FOXE1, and HHEX). The pellets were analyzed by western blot. Results: FOXE1 bands were augmented in patient 1 with lung cancer, a PAX8 band in patient 2 with malignant melanoma, and bands of FOXE1, PAX8, and HHEX in patient 3 with renal cell carcinoma, compared to a control sample of a normal subject. We performed subcutaneous injections of purified IgG fraction from the serum of patient 3 to C57BL/6 mice every 2 weeks. The mice were sacrificed after 4 weeks, but no significant changes were observed in their thyroid glands and thyroid function. Conclusions: We identified novel autoantibodies for FOXE1, PAX8, and HHEX, thyroid-specific transcriptional factors. In our experiments, the pathogenicity of antibodies were not suggested. Considering our previous observation that the thyroid gland expresses both PD-L1 and PD-L2, ligands of PD-1 receptor [1], PD-1 pathway blockade may particularly disrupt immune tolerance of the thyroid gland, resulting in autoantibody production. Because thyroid irAEs have been revealed to be a prognostic factor, these novel autoantibodies are candidate biomarkers for PD-1 pathway blockade therapy. References: 1. Yamauchi et al. Clinical Features of Nivolumab-Induced Thyroiditis: A Case Series Study. Thyroid. 2017; 27(7): 894–901. 2. Yamauchi et al. Incidence, features, and prognosis of immune-related adverse events involving the thyroid gland induced by nivolumab. PLoS One. 2019; 14(5): e0216954.

Growth impairment in mucopolysaccharidoses (MPSs) is an unresolved issue as it is resistant to enzyme replacement therapy (ERT) and growth hormone therapy. C-type natriuretic peptide (CNP) is a promising agent that has growth-promoting effects. Here we investigate the effects of CNP on growth impairment of MPSs using Gusbmps-2J mice, a model for MPS type VII, with combination therapy of CNP and ERT by hydrodynamic gene delivery. Although monotherapies were not sufficient to restore short statures of treated mice, combination therapy resulted in successful restoration. The synergistic effects of CNP and ERT were not only observed in skeletal growth but also in growth plates. ERT reduced cell swelling in the resting zone and increased cell number by accelerating proliferation or inhibiting apoptosis. CNP thickened the proliferative and hypertrophic zones. Regarding changes in the bone, ERT restored bone sclerosis through decreased bone formation and increased bone resorption, and CNP did not adversely affect this process. In addition, improvement of joint deformation by ERT was suggested by analyses of joint spaces and articular cartilage. CNP additively provided restoration of the short stature of MPS VII mice in combination with ERT, which improved abnormalities of growth plates and bone metabolism.