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Abstract Many strategies have been developed to produce high levels of biologically active recombinant proteins in plants for biopharmaceutical purposes. However, the production of an active form of human iduronate-2-sulfatase (hIDS) for the treatment of Hunter syndrome by enzyme replacement therapy (ERT) is challenging due to the requirement for cotranslational modification by a formylglycine-producing enzyme encoded by sulfatase modifying factor 1 (hSUMF1) at the Cys84 residue, which converts it to C(alpha)-formylglycine. In this study, we have shown that hIDS can be highly expressed in N. benthamiana by using different constructs. Among them, BiP-GB1-L-dCBD1-2L-8xHis-L-6xHis-3L-EK-hIDS-HDEL (GB1-CBD1-hIDS) showed a high expression level when transiently co-expressed with the turnip crinkle virus gene silencing suppressor P38 and GB1-fused human calreticulin (GB1-CRT1) as a folding enhancer. The hSUMF1 was co-expressed with hIDS for cotranslational modification. The full-length recombinant proteins were purified using Ni2+-NTA affinity resin followed by enterokinase treatment to obtain tag-free hIDS. The N-terminal fragment was removed using microcrystalline cellulose (MCC) beads. The purified active form of hIDS can successfully cleave the sulfate group from an artificial substrate, 4-nitrocatechol sulfate, at a similar level to commercial hIDS expressed in animal cells. These results suggest that plants could be a promising platform for the production of recombinant hIDS.

Abstract Background The formation of shoots plays a pivotal role in plant organogenesis and productivity. Despite its significance, the underlying molecular mechanism of de novo regeneration has not been extensively elucidated in Capsicum annuum ‘Dempsey’, a bell pepper cultivar. To address this, we performed a comparative transcriptome analysis focusing on the differential expression in C. annuum ‘Dempsey’ shoot, callus, and leaf tissue. We further investigated phytohormone-related biological processes and their interacting genes in the C. annuum ‘Dempsey’ transcriptome based on comparative transcriptomic analysis across five species. Results We provided a comprehensive view of the gene networks regulating shoot formation on the callus, revealing a strong involvement of hypoxia responses and oxidative stress. Our comparative transcriptome analysis revealed a significant conservation in the increase of gene expression patterns related to auxin and defense mechanisms in both callus and shoot tissues. Consequently, hypoxia response and defense mechanism emerged as critical regulators in callus and shoot formation in C. annuum ‘Dempsey’. Current transcriptome data also indicated a substantial decline in gene expression linked to photosynthesis within regenerative tissues, implying a deactivation of the regulatory system governing photosynthesis in C. annuum ‘Dempsey’. Conclusion Coupled with defense mechanisms, we thus considered spatial redistribution of auxin to play a critical role in the shoot morphogenesis via primordia outgrowth. Our findings shed light on shoot formation mechanisms in C. annuum ‘Dempsey’ explants, important information for regeneration programs, and have broader implications for precise molecular breeding in recalcitrant crops.

Reducing atmospheric CO2 levels to combat global warming is a pressing concern today. Numerous methods have been employed to capture CO2 from flue gases. One particularly promising approach is the use of carbonic anhydrases (CAs) as biocatalysts for the rapid conversion of CO2 to H2CO3. However, the widespread application of CAs for CO2 capture has been hampered by their inherent instability under real-world conditions. In this study, we have successfully engineered a chimeric carbonic anhydrase with vastly improved physicochemical properties, particularly with respect to its resilience to high temperatures, alkaline pH, and saline environments. Using computational design, we created various hybrid CAs with enhanced resistance to elevated temperatures. Among them, a chimeric CA known as SPS, generated by domain exchange between SazCA and PmCA, exhibited superior heat stability compared to its parent CAs. SPS showed 10 % higher enzymatic activity and retained 80–13 % of its activity during a period of 3 h to 24 h of incubation at 100℃. SPS's apparent kcat and Km values were 4.84 × 108 s−1 and 13.7 mM, respectively. Structural analysis revealed that SPS forms dimers, which contributes to its robustness. Furthermore, we introduced modifications in the form of SPS_1 and SPS_2 variants by incorporating one or two loop sequences from the halotolerant dCAII into SPS. These modifications significantly improved the stability of the CA in alkaline and saline conditions. In particular, SPS showed remarkable efficiency in hydrating CO2 in seawater. Given these compelling results, we propose that hybrid CAs such as SPS, SPS_1, and SPS_2 hold great promise for facilitating CO2 hydration in a wide range of applications. Synopsis: Greenhouse gas sequestration is an immediate need. This study reports an engineered and highly stable carbonic anhydrase for CO2 sequestration and greenhouse gas reduction.

The increasing demand for laparoscopic surgery due to its cosmetic benefits and rapid post-surgery recovery is juxtaposed with a shortage of surgical support staff. This juxtaposition highlights the necessity for improved camera management in laparoscopic procedures, encompassing positioning, zooming, and focusing. Our feasibility study introduces the information and communications technology (ICT) laparoscopy system designed to aid solo laparoscopic surgery. This system tracks a surgeon’s body motion using a controller, manipulating an embedded camera to focus on specific surgical areas. It comprises a camera module, a camera movement controller, and a motor within the main body, operating connected wires according to controller commands for camera movement. Surgeon movements are detected by an inertial measurement unit (IMU) sensor, facilitating precise camera control. Additional features include a foot pedal switch for motion tracking, a dedicated trocar for main body stability, and a display module. The system’s effectiveness was evaluated using an abdomen phantom model and animal experimentation with a porcine model. The camera responded to human movement within 100 ms, a delay that does not significantly affect procedural performance. The ICT laparoscopy system with advanced motion-tracking technology is a promising tool for solo laparoscopic surgery, potentially improving surgical outcomes and overcoming staff shortages.

Plants and their use as bioreactors for the generation of recombinant proteins have become one of the hottest topics in the field of Plant Biotechnology and Plant Synthetic Biology. Plant bioreactors offer superior engineering potential compared to other types, particularly in the realm of subcellular accumulation strategies for increasing production yield and quality. This review explores established and emerging strategies for subcellular accumulation of recombinant proteins in tobacco bioreactors, highlighting recent advancements in the field. Additionally, the review provides reference to the crucial initial step of selecting an optimal subcellular localization for the target protein, a design that substantially impacts production outcomes.