NADP+ or CO2 reduction by frhAGB-encoded hydrogenase through interaction with formate dehydrogenase 3 in the hyperthermophilic archaeon Thermococcus onnurineus NA1.

IMPORTANCE: The strategy using structural homology with the help of structure prediction by AlphaFold was very successful in finding potential targets for the frhAGB-encoded hydrogenase of Thermococcus onnurineus NA1. The finding that the hydrogenase can interact with FdhB to reduce the cofactor NAD(P)+ is significant in that the enzyme can function to supply reducing equivalents, just as F420-reducing hydrogenases in methanogens use coenzyme F420 as an electron carrier. Additionally, it was identified that T. onnurineus NA1 could produce formate from H2 and CO2 by the concerted action of frhAGB-encoded hydrogenase and formate dehydrogenase Fdh3.

Bioconversion of CO to formate by artificially designed carbon monoxide:formate oxidoreductase in hyperthermophilic archaea.

Ferredoxin-dependent metabolic engineering of electron transfer circuits has been developed to enhance redox efficiency in the field of synthetic biology, e.g., for hydrogen production and for reduction of flavoproteins or NAD(P)+. Here, we present the bioconversion of carbon monoxide (CO) gas to formate via a synthetic CO:formate oxidoreductase (CFOR), designed as an enzyme complex for direct electron transfer between non-interacting CO dehydrogenase and formate dehydrogenase using an electron-transferring Fe-S fusion protein. The CFOR-introduced Thermococcus onnurineus mutant strains showed CO-dependent formate production in vivo and in vitro. The maximum formate production rate from purified CFOR complex and specific formate productivity from the bioreactor were 2.2 ± 0.2 µmol/mg/min and 73.1 ± 29.0 mmol/g-cells/h, respectively. The CO-dependent CO2 reduction/formate production activity of synthetic CFOR was confirmed, indicating that direct electron transfer between two unrelated dehydrogenases was feasible via mediation of the FeS-FeS fusion protein.

A Novel NADP-Dependent Formate Dehydrogenase From the Hyperthermophilic Archaeon Thermococcus onnurineus NA1.

The genome of the hyperthermophilic archaeon Thermococcus onnurineus NA1 contains three copies of the formate dehydrogenase (FDH) gene, fdh1, fdh2, and fdh3. Previously, we reported that fdh2, clustered with genes encoding the multimeric membrane-bound hydrogenase and cation/proton antiporter, was essential for formate-dependent growth with H2 production. However, the functionality of the other two FDH-coding genes has not yet been elucidated. Herein, we purified and characterized cytoplasmic Fdh3 to understand its functionality. The purified Fdh3 was identified to be composed of a tungsten-containing catalytic subunit (Fdh3A), an NAD(P)-binding protein (Fdh3B), and two Fe-S proteins (Fdh3G1 and Fdh3G2). Fdh3 oxidized formate with specific activities of 241.7 U/mg and 77.4 U/mg using methyl viologen and NADP+ as electron acceptors, respectively. While most FDHs exhibited NAD+-dependent formate oxidation activity, the Fdh3 of T. onnurineus NA1 showed a strong preference for NADP+ over NAD+ as a cofactor. The catalytic efficiency (k cat /K m) of Fdh3 for NADP+ was measured to be 5,281 mM-1 s-1, which is the highest among NADP-dependent FDHs known to date. Structural modeling suggested that Arg204 and Arg205 of Fdh3B may contribute to the stabilization of the 2'-phosphate of NADP(H). Fdh3 could also use ferredoxin as an electron acceptor to oxidize formate with a specific activity of 0.83 U/mg. Furthermore, Fdh3 showed CO2 reduction activity using reduced ferredoxin or NADPH as an electron donor with a specific activity of 0.73 U/mg and 1.0 U/mg, respectively. These results suggest a functional role of Fdh3 in disposing of reducing equivalents by mediating electron transfer between formate and NAD(P)H or ferredoxin.

Use of oral paclitaxel for the treatment of bladder tumors in dogs.

An oral paclitaxel formulation that overcomes the hypersensitivity reaction of paclitaxel has been evaluated for safety and efficacy in humans, but not in dogs. We present the first case report on the use of oral paclitaxel in dogs. In this study, oral paclitaxel was well-tolerated in four dogs with either transitional cell carcinoma or prostate cancer; adverse effects were limited to mild neutropenia. Each of the dogs had progressive disease at the end, but clinical responses, including changes in mass size and improvement of clinical symptoms, were confirmed in some of the animals following oral paclitaxel chemotherapy. Although this study is somewhat limited by a small sample size, it suggests that oral paclitaxel may be a chemotherapeutic option for malignant tumors in dogs.

Preconditioning of canine adipose tissue-derived mesenchymal stem cells with deferoxamine potentiates anti-inflammatory effects by directing/reprogramming M2 macrophage polarization.

Preconditioning with hypoxia or hypoxia-mimetic agents has been tried with mesenchymal stem cells (MSCs) to improve the secretion of anti-inflammatory factors. These preconditioning procedures upregulate hypoxia inducible factor (HIF) 1-alpha leading to the transcription of HIF-dependent tissue protective and anti-inflammatory genes. Due to the limited number of studies exploring the activity of deferoxamine (DFO)-a hypoxia-mimetic agent-in MSCs, we aimed to determine whether DFO can enhance the secretion of anti-inflammatory substances in canine adipose tissue-derived (cAT)-MSCs. Furthermore, we investigated whether this activity of DFO could affect macrophage polarization and activate anti-inflammatory reactions. cAT-MSCs preconditioned with DFO exhibited enhanced secretion of anti-inflammatory factors such as prostaglandin E2 and tumor necrosis factor-α-stimulated gene-6. To evaluate the interaction between DFO preconditioned cAT-MSCs and macrophages, RAW 264.7 cells were co-cultured with cAT-MSCs using the Transwell system, and changes in the expression of factors related to macrophage polarization were analyzed using the quantitative real-time PCR and western blot assays. When RAW 264.7 cells were co-cultured with DFO preconditioned cAT-MSCs, the expression of M1 and M2 markers decreased and increased, respectively, compared to co-culturing with non-preconditioned cAT-MSCs. Thus, cAT-MSCs preconditioned with DFO can more effectively direct and reprogram macrophage polarization into the M2 phase, an anti-inflammatory state.

Antitumour effects of Liporaxel (oral paclitaxel) for canine melanoma in a mouse xenograft model.

Paclitaxel, a member of the taxane family, exhibits antitumour effects by targeting the microtubules in cancer cells. Recently, oral paclitaxel has been developed to overcome the side effects of intravenous paclitaxel administration in human patients. The objective of this study was to investigate the antitumour effects of oral paclitaxel in vitro and in vivo. Three weeks after inoculation, oral paclitaxel (25 and 50 mg/kg) or saline was administered every week for three consecutive weeks. To explore the underlying mechanism, tumour angiogenesis was examined by immunohistochemistry with an anti-CD31 antibody. Tumour cell apoptosis was detected by Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling assay, and cell cycle arrest was confirmed by western blot analysis. Oral paclitaxel treatment of canine melanoma cells exerted mediated antiproliferative effects and mediated cell cycle arrest in vitro. In animal experiments, after oral paclitaxel administration, the average tumour size decreased to approximately 30% of that in the control. Histologically, oral paclitaxel showed anti-angiogenic effects and induced the apoptosis in tumour tissues. Oral paclitaxel also downregulated the intratumoural expression of cyclin D1 and inhibited cell proliferation. The study findings support potential application of oral paclitaxel as a novel chemotherapeutic strategy to treat canine melanoma. This is the first study to investigate the potential of oral paclitaxel as a therapeutic drug against canine tumours.

Enhanced angiogenic activity of dimethyloxalylglycine-treated canine adipose tissue-derived mesenchymal stem cells.

The paracrine function of mesenchymal stem cells (MSCs) during transplantation has been recently studied due to its poor differentiation ratio. Dimethyloxalylglycine (DMOG) has been used to promote angiogenesis in experimental animal models, however, comparable approaches for canine MSCs are not sufficient. In the present study, we assessed whether DMOG improves angiogenesis in canine adipose tissue-derived mesenchymal stem cells (cAT-MSCs). cAT-MSCs were treated with DMOG and their effect on angiogenesis was investigated by cell proliferation assay, western blotting, and tube formation assay. Dimethyloxalylglycine preconditioning enhanced the expression of vascular endothelial growth factor (VEGF) among pro-angiogenic factors in cAT-MSCs via hypoxia-inducible factor-1α stabilization. Dimethyloxalylglycine primed-cAT-MSC-conditioned media increased angiogenesis in human umbilical vein endothelial cells. These results suggest that DMOG conditioning of cAT-MSCs augmented the secretion of VEGF, which acted as a prominent pro-angiogenic factor during angiogenesis. DMOG-primed cAT-MSCs may have the potential to induce beneficial effects in ischemic diseases in clinical trials.

Prostaglandin E2 secreted from feline adipose tissue-derived mesenchymal stem cells alleviate DSS-induced colitis by increasing regulatory T cells in mice.

BACKGROUND: Inflammatory bowel disease (IBD) is an intractable autoimmune disease, relatively common in cats, with chronic vomiting and diarrhea. Previous studies have reported that mesenchymal stem cells (MSCs) alleviate inflammation by modulating immune cells. However, there is a lack of research on cross-talk mechanism between feline adipose tissue-derived mesenchymal stem cells (fAT-MSCs) and immune cells in IBD model. Hence, this study aimed to evaluate the therapeutic effects of fAT-MSC on mice model of colitis and to clarify the therapeutic mechanism of fAT-MSCs. RESULTS: Intraperitoneal infusion of fAT-MSC ameliorated the clinical and histopathologic severity of colitis, including body weight loss, diarrhea, and inflammation in the colon of Dextran sulfate sodium (DSS)-treated mice (C57BL/6). Since regulatory T cells (Tregs) are pivotal in modulating immune responses and maintaining tolerance in colitis, the relation of Tregs with fAT-MSC-secreted factor was investigated in vitro. PGE2 secreted from fAT-MSC was demonstrated to induce elevation of FOXP3 mRNA expression and adjust inflammatory cytokines in Con A-induced feline peripheral blood mononuclear cells (PBMCs). Furthermore, in vivo, FOXP3+ cells of the fAT-MSC group were significantly increased in the inflamed colon, relative to that in the PBS group. CONCLUSION: Our results suggest that PGE2 secreted from fAT-MSC can reduce inflammation by increasing FOXP3+ Tregs in mice model of colitis. Consequently, these results propose the possibility of administration of fAT-MSC to cats with not only IBD but also other immune-mediated inflammatory diseases.