Biparatopic antibodies and biparatopic alternative scaffolds are proteins that can simultaneously bind two distinct epitopes, and as such are promising formats for the development of next-generation antibody therapeutics. By design, biparatopic proteins have high avidity and can bridge between epitopes in an intra- or intermolecular manner to form diverse immunocomplexes. Some biparatopic proteins can be efficiently internalized into cells, which may be useful for the targeted delivery of therapeutic molecules into cells. In addition, biparatopic proteins can also lock the conformation of membrane proteins to exert an agonist or antagonist effect. Here, we review the development and characteristics of biparatopic proteins and examine how they differ in terms of antigen-binding efficiency, immunocomplex formation and biological activity compared with conventional monoparatopic proteins.

Core clock proteins play a significant role in maintaining physiological functions, including the metabolism in organisms in a circadian pattern. Metabolism is critical for drug efficiency and pharmacokinetics, which depend on circadian rhythm in animals and humans. Although alternative results are expected in animal experiments, there are limited reports on the influence of circadian rhythm on drug metabolism in culture. We observed the circadian rhythm in mouse embryonic stem (ES) cell-derived cardiomyocytes, as well as in the animal, after forskolin stimulation. The clock-synchronized mouse ES cell-derived cardiomyocytes exhibited time-dependent drug responses. This synchronized circadian rhythm could be maintained for up to three days by forskolin stimulation after every 24 hr. The beating rates of mouse ES cell-derived cardiomyocytes followed a circadian pattern. In conclusion, we established a mouse ES cell-derived cardiomyocyte culture model that exhibited a circadian beating pattern and time-dependent drug responses for up to three days. This model would serve as a valuable tool for chronotherapeutic research in culture.

A next generation fetal electrocardiogram (ECG) that can assess fetal well-being accurately is required in clinical settings. We developed a fetal ECG monitoring system that acts via the maternal abdominal wall and measured the ECG in our clinical studies. To assess the accuracy of the clinical fetal ECG monitoring records, it was necessary to confirm that the waveforms obtained by the indirect lead had the same accuracy as those by the direct lead. This study is translational research in which a murine fetal ECG system with the direct lead that we had already developed was applied and pregnant rabbits that had enough space to place electrodes on the maternal abdominal surface were used. In this study, fetal ECG was measured using the direct and indirect lead simultaneously using pregnant rabbits. The R-squared value between the RR-intervals obtained by the direct lead and those by the indirect lead of the same fetus was used to determine the reliability of the clinically developed fetal ECG monitoring system. The fetal ECG waveform, both with the direct and indirect lead, was obtained from three out of five pregnant rabbits. The average R-squared value was 0.99. Although one of the three pregnant rabbits presented with an atrioventricular block during the measurement, the fetal ECG waveform was successfully extracted with both the direct and indirect lead. The results of this study demonstrate that the fetal ECG monitoring system that acts via the maternal abdominal wall has the same accuracy as that of the direct lead.

C1q/TNF-related protein 3 (CTRP3), a member of the CTRP family, is a soluble protein that is expressed during chondrogenic differentiation and promotes proliferation in chondrogenic cells. However, functional receptor(s) for CTRP3 has not been identified yet. In this study, we reveal progestin and adipoQ receptor family member 2 (PAQR2) [adiponectin receptor 2 (AdipoR2)] as a functional receptor of CTRP3 in chondrogenic cell line (ATDC5). Using RNA interference, we found that PAQR2 inhibition, and not PAQR1, PAQR3, or PAQR4, suppresses CTRP3-induced chondrogenic cell proliferation. In addition, a peptide blocker against PAQR2, and not against PAQR1, was observed to neutralize the growth promoting effect of CTRP3 on chondrogenic cells. Thus, our results suggested that PAQR2 is a functional receptor of CTRP3 in promoting proliferation in chondrogenic cells.

Osteoporosis is associated with low bone density and pathological fractures induced by an imbalance in bone remodeling. It the most common bone disease and a social problem of an aging society. Norzoanthamine (NZ), a newly discovered marine alkaloid, has a suppressive effect on bone loss in osteoporosis animal models and is considered a candidate for anti-osteoporosis drug development. Truncated-norzoanthamine (TZ) is artificially synthesized, and possesses a structure similar to that of NZ. TZ and NZ have been shown to possess collagen protective activity. Here, we hypothesized that TZ also inhibits the bone loss observed in osteoporosis. We explored the effects of TZ treatment in an ovariectomized (OVX) osteoporosis mouse model. We observed that TZ suppressed the decrease in bone volume fraction, trabecular number, and bone mineral density in the femur of OVX mice as analyzed using micro-computed tomography, indicating an improvement in the bone mechanical characters. These findings indicate that TZ suppresses bone loss in OVX mice and suggest that TZ may offer therapeutic effects as a new anti-osteoporosis drug.

Human unilateral renal agenesis (URA) is a urinary malformation characterized by congenital absence of one kidney. The inbred rat strain ACI is a known animal model for studying URA. Recently, a single locus responsible for URA, designated renal agenesis 1 (Renag1), has been mapped to a 379-kb interval that contains a single gene, Kit, which encodes c-kit that plays important roles in stem cell survival, migration, and differentiation. Within the Renag1 interval, an insertion of a long terminal repeat (LTR) sequence has been found as a major variation specific for the ACI genome, and the LTR has been strongly suggested to be a causative factor of URA. Here, we removed the LTR from the ACI Kit gene by the CRISPR/Cas9 system and examined whether these gene-modified rats exhibited URA. Three gene-modified ACI strains were developed that lacked the LTR and flanking sequences of different lengths. Out of a total of 125 gene-modified rats observed, none of the rats exhibited URA. Besides URA, abdominal white spotting, Irish, has also been mapped to the Renag1 locus. We also observed that the gene-modified ACI rats did not exhibit Irish spotting. Thus, we concluded that the LTR was causative of both URA and Irish spotting in ACI rats. This study suggests that the Kit signaling pathway plays an important role in kidney development and that ACI rats would be a promising animal model for regenerative medicine therapy of kidney diseases.