Enhanced efficacy of glycoengineered rice cell-produced trastuzumab.

For several decades, a plant-based expression system has been proposed as an alternative platform for the production of biopharmaceuticals including therapeutic monoclonal antibodies (mAbs), but the immunogenicity concerns associated with plant-specific N-glycans attached in plant-based biopharmaceuticals has not been completely solved. To eliminate all plant-specific N-glycan structure, eight genes involved in plant-specific N-glycosylation were mutated in rice (Oryza sativa) using the CRISPR/Cas9 system. The glycoengineered cell lines, PhytoRice®, contained a predominant GnGn (G0) glycoform. The gene for codon-optimized trastuzumab (TMab) was then introduced into PhytoRice® through Agrobacterium co-cultivation. Selected cell lines were suspension cultured, and TMab secreted from cells was purified from the cultured media. The amino acid sequence of the TMab produced by PhytoRice® (P-TMab) was identical to that of TMab. The inhibitory effect of P-TMab on the proliferation of the BT-474 cancer cell line was significantly enhanced at concentrations above 1 µg/mL (****P < 0.0001). P-TMab bound to a FcγRIIIa variant, FcγRIIIa-F158, more than 2.7 times more effectively than TMab. The ADCC efficacy of P-TMab against Jurkat cells was 2.6 times higher than that of TMab in an in vitro ADCC assay. Furthermore, P-TMab demonstrated efficient tumour uptake with less liver uptake compared to TMab in a xenograft assay using the BT-474 mouse model. These results suggest that the glycoengineered PhytoRice® could be an alternative platform for mAb production compared to current CHO cells, and P-TMab has a novel and enhanced efficacy compared to TMab.

Inactivation of the ß (1, 2)-xylosyltransferase and the α (1, 3)-fucosyltransferase gene in rice (Oryza sativa) by multiplex CRISPR/Cas9 strategy.

KEY MESSAGE: CRISPR/Cas9-mediated OsXylT and OsFucT mutation caused the elimination of plant-specific ß1,2-xylose and α1,3-fucose residues on glycoproteins in rice, which is the first report of OsXylT/OsFucT double KO mutation in rice. N-glycosylation pathway is the one of post-translational mechanism and is known as highly conserved in eukaryotes. However, the process for complex-N-glycan modification is different between mammals and plants. In plant-specific manner, ß1,2-xylose and α1,3-fucose residues are transferred to N-glycan core structure on glycoproteins by ß1,2-xylosyltransferase (ß1,2-XylT) and α1,3-fucosyltransferase (α1,3-FucT), respectively. As an effort to use plants as a platform to produce biopharmaceuticals, the plant-specific N-glycan genes of rice (Oryza sativa), ß1,2-xylT (OsXylT) and α1,3-FucT (OsFucT), were knocked out using multiplex CRISPR/Cas9 technology. The double knock-out lines were found to have frameshift mutations by INDELs. Both ß1,2-xylose and α1,3-fucose residues in the lines were not detected in Western blot analysis. Consistently, there was no peak corresponding to the N-glycans in MALDI-TOF/MS analysis. Although α1,3-fucose and ß1,2-xylose residues were not detected in the line, other plant-specific residues of ß1,3-galactose and α1,4-fucose were detected. Thus, we suggest that each enzymes working on the process for complex N-glycan biosynthesis might independently act in rice, hence the double knock-out of both OsXylT and OsFucT might be not enough to humanize N-glycan structure in rice.

Profiling Dynamic Patterns of Single-Cell Motility.

Cell motility plays an essential role in many biological processes as cells move and interact within their local microenvironments. Current methods for quantifying cell motility typically involve tracking individual cells over time, but the results are often presented as averaged values across cell populations. While informative, these ensemble approaches have limitations in assessing cellular heterogeneity and identifying generalizable patterns of single-cell behaviors, at baseline and in response to perturbations. In this study, CaMI is introduced, a computational framework designed to leverage the single-cell nature of motility data. CaMI identifies and classifies distinct spatio-temporal behaviors of individual cells, enabling robust classification of single-cell motility patterns in a large dataset (n = 74 253 cells). This framework allows quantification of spatial and temporal heterogeneities, determination of single-cell motility behaviors across various biological conditions and provides a visualization scheme for direct interpretation of dynamic cell behaviors. Importantly, CaMI reveals insights that conventional cell motility analyses may overlook, showcasing its utility in uncovering robust biological insights. Together, a multivariate framework is presented to classify emergent patterns of single-cell motility, emphasizing the critical role of cellular heterogeneity in shaping cell behaviors across populations.

Single-cell morphology encodes functional subtypes of senescence in aging human dermal fibroblasts.

Cellular senescence is an established driver of aging, exhibiting context-dependent phenotypes across multiple biological length-scales. Despite its mechanistic importance, profiling senescence within cell populations is challenging. This is in part due to the limitations of current biomarkers to robustly identify senescent cells across biological settings, and the heterogeneous, non-binary phenotypes exhibited by senescent cells. Using a panel of primary dermal fibroblasts, we combined live single-cell imaging, machine learning, multiple senescence induction conditions, and multiple protein-based senescence biomarkers to show the emergence of functional subtypes of senescence. Leveraging single-cell morphologies, we defined eleven distinct morphology clusters, with the abundance of cells in each cluster being dependent on the mode of senescence induction, the time post-induction, and the age of the donor. Of these eleven clusters, we identified three bona-fide senescence subtypes (C7, C10, C11), with C10 showing the strongest age-dependence across a cohort of fifty aging individuals. To determine the functional significance of these senescence subtypes, we profiled their responses to senotherapies, specifically focusing on Dasatinib + Quercetin (D+Q). Results indicated subtype-dependent responses, with senescent cells in C7 being most responsive to D+Q. Altogether, we provide a robust single-cell framework to identify and classify functional senescence subtypes with applications for next-generation senotherapy screens, and the potential to explain heterogeneous senescence phenotypes across biological settings based on the presence and abundance of distinct senescence subtypes.

Comparison of Intermediate-Term Clinical Outcomes Between Medial and Lateral Osteochondral Lesions of the Talus Treated With Autologous Osteochondral Transplantation.

BACKGROUND: The conventional operative method to treat an osteochondral lesion of the talus (OLT) is through bone marrow stimulation (BMS). Autologous osteochondral transplantation (AOT) is being used as an alternative option in cases with a large OLT, accompanying subchondral cyst, and/or failed BMS. We aimed to compare the intermediate-term clinical and radiologic results between medial and lateral OLTs after an AOT procedure. METHODS: Among the patients who underwent AOT, 45 cases with at least 3 years' follow-up were included in this retrospective study. We had 15 cases of lateral lesions and selected 30 cases of medial lesions matched for age and gender. Lateral lesions were resurfaced without an osteotomy; medial lesion resurfacing was combined with a medial malleolar osteotomy. Clinical assessment was performed using the Foot and Ankle Outcome Score (FAOS) and Foot and Ankle Ability Measure (FAAM). Radiographic assessment included the irregularity of articular surface (subchondral plate), the progression of degenerative arthritis, and the change of the talar tilt. RESULTS: The mean FAOS and FAAM scores significantly improved after surgery in both groups. Up to 1 year postoperatively, there was significant difference in FAAM scores between the both groups (mean 75.3 points in medial group and 87.2 points in lateral group, P < .001). Delayed union or malunion of the malleolar osteotomy was found in 4 cases (13%) in the medial group. In addition, the progression of joint degeneration was observed in 3 cases (10%) in the medial group. There were no significant differences in the irregularity of articular surface and the change of talar tilt between both groups. CONCLUSION: A comparison between medial and lateral OLTs treated with AOT demonstrated comparable intermediate-term clinical outcomes. However, patients with medial OLT required a longer period to restore ability for daily and sport activities. In addition, we found more complications and higher rate of progression in the radiologic arthritis grade after medial malleolar osteotomy. LEVEL OF EVIDENCE: Level IV, retrospective comparative study.

Neurologic outcomes of prehospital mechanical chest compression device use during transportation of out-of-hospital cardiac arrest patients: a multicenter observational study.

OBJECTIVE: High-quality cardiopulmonary resuscitation with chest compression is important for good neurologic outcomes during out-of-hospital cardiac arrest (OHCA). Several types of mechanical chest compression devices have recently been implemented in Korean emergency medical services. This study aimed to identify the effect of prehospital mechanical chest compression device use on the outcomes of OHCA patients. METHODS: We retrospectively analyzed data drawn from the regional cardiac arrest registry in Daegu, Korea. This registry prospectively collected data from January 2017 to December 2020. Patients aged 18 years or older who experienced cardiac arrest presumed to have a medical etiology were included. The exposure variable was the use of a prehospital mechanical device during transportation by emergency medical technicians. The outcomes measured were neurologic outcomes and survival to discharge. Logistic regression analysis was used. RESULTS: Among 3,230 OHCA patients, 1,111 (34.4%) and 2,119 (65.6%) were managed with manual chest compression and with a mechanical chest compression device, respectively. The mechanical chest compression group showed poorer neurologic outcomes than the manual chest compression group (adjusted odds ratio, 0.12; 95% confidence interval, 0.04-0.33) and decreased survival to discharge (adjusted odds ratio, 0.39; 95% confidence interval, 0.19-0.82) after adjustment for confounding variables. CONCLUSION: Prehospital mechanical chest compression device use in OHCA was associated with poorer neurologic outcomes and survival to discharge compared to manual chest compression.