Efficient and accurate BmNPV bacmid editing system by two-step golden gate assembly.

The silkworm-baculovirus expression vector system (silkworm-BEVS), using Bombyx mori nucleopolyhedrovirus (BmNPV) and silkworm larvae or pupae, has been used as a cost-effective expression system for the production of various recombinant proteins. Recently, several gene knockouts in baculoviruses have been shown to improve the productivity of recombinant proteins. However, the gene editing of the baculovirus genome (approximately 130 kb) remains challenging and time-consuming. In this study, we sought to further enhance the productivity of the silkworm-BEVS by synthesizing and gene editing the BmNPV bacmid from plasmids containing fragments of BmNPV genomic DNA using a two-step Golden Gate Assembly (GGA). The BmNPV genome, divided into 19 fragments, was amplified by PCR and cloned into the plasmids. From these initial plasmids, four intermediate plasmids containing the BmNPV genomic DNA were constructed by GGA with the type IIS restriction enzyme BsaI. Subsequently, the full-length bacmid was successfully synthesized from the four intermediate plasmids by GGA with another type IIS restriction enzyme PaqCI with a high efficiency of 97.2 %. Furthermore, this methodology enabled the rapid and straightforward generation of the BmNPV bacmid lacking six genes, resulting in the suppression of degradation of recombinant proteins expressed in silkworm pupae. These results indicate that the BmNPV bacmid can be quickly and efficiently edited using only simple cloning techniques and enzymatic reactions, marking a significant advancement in the improvement of the silkworm-BEVS.

Associations between increased renal resistive index and cardiovascular events.

BACKGROUND AND PURPOSE: Chronic kidney disease is a risk factor for cardiovascular disease (CVD). Renal resistive index (RI) measured by Doppler ultrasonography is associated with renal impairment. We investigated the relationship between RI and cardiac function, and evaluated the utility of RI for predicting cardiac events in patients with CVD. METHODS AND RESULTS: Renal Doppler ultrasonography and echocardiography were performed in a total of 452 patients with CVD. Correlations of RI with serum creatinine and estimated glomerular filtration rate (eGFR) were significant but not strong (r = 0.37, p < 0.001; r = -0.42, p < 0.001, respectively). RI correlated positively with age, left atrial volume index, left ventricular mass index, and early transmitral velocity to mitral annular early diastolic velocity (e') ratio (E/e'), and showed significant negative correlations with e' and diastolic blood pressure. Between two subgroups-112 patients hospitalized with cardiovascular events (Group A) and 200 age- and eGFR-matched controls (Group B)-RI was significantly higher in Group A than in Group B, although age and eGFR were similar. CONCLUSIONS: RI reflects the impairment of intrarenal hemodynamics that cannot be adequately elucidated by eGFR alone. Assessment of renal RI may be useful in conjunction with prognostic estimates for patients with CVD.