Frontiers in Bioengineering and Biotechnology最新文献

High-throughput PCR screening is vital in synthetic biology and metabolic engineering, enabling rapid and precise analysis of genetic modifications. However, current methods face challenges including inefficient DNA extraction, high variability across sample types, scalability limitations, and the high cost of template DNA extraction. To address these common challenges, we developed a High-Throughput Genome Releaser (HTGR). This innovative device utilizes a squash-based method for rapid, cost-effective, and efficient DNA extraction, optimized for subsequent PCR reactions. After testing various synthetic materials, we selected a plastic that closely mimics the smooth surface and compression properties of microscope slides, ensuring reliable and consistent performance. The device comprises a 96-well plate and a Shear Applicator, designed for both manual and automated operation, and is compatible with standard liquid-handling robotic platforms. This compatibility simplifies integration into high-throughput PCR workflows. Additionally, we developed software to support its automated functions. Our results demonstrated that the specially engineered 96-well plate and HTGR effectively squash fungal spores, releasing sufficient genomic DNA for PCR screening with 100% efficiency. The genome releaser enables the preparation of PCR-ready genomic DNA from 96 samples within minutes, eliminating the need for an extraction buffer. Its adaptability to a wide range of microorganisms and cell types makes it a versatile tool that could significantly advance biomanufacturing processes.

[This corrects the article DOI: 10.3389/fbioe.2021.635509.].

Background: Pedicle screw loosening (PSL) is a frequent complication in osteoporotic patients undergoing spinal fixation, yet effective risk assessment methods are limited. This study explores the impact of craniocaudal cyclic load on pedicle screw fixation strength using computed tomography-based finite element analysis (CT-FEA) and evaluates its predictive value for PSL.

Methods: A total of 23 PSL cases (7 men and 16 women) and 29 matched controls were analyzed using CT-FEA. Both a simple axial pullout load and a pullout load with a preset craniocaudal cyclic load were applied to calculate the pullout force. Hounsfield unit (HU) values and volumetric bone mineral density (vBMD) of the screw trajectory were also assessed for osteoporosis evaluation. The pullout force and osteoporotic assessment value were compared between PSL and controls.

Results: Craniocaudal cyclic loading significantly reduced the pullout force (924.3 ± 195.1 N vs. 745.2 ± 188.7 N, p < 0.0001). The PSL group had a lower pullout force under cyclic load (629.6 ± 188.2 N vs. 836.9 ± 131.6 N, p < 0.0001) and lower HU value of screw trajectories (183.7 ± 42.6 vs. 206.7 ± 29.72, p = 0.026) than controls, while simple axial pullout force and vBMD showed no significant differences. Receiver operating characteristic (ROC) analysis indicated that pullout force under cyclic load (AUC = 0.806) was a better predictor of PSL than HU values (AUC = 0.629).

Conclusion: This study demonstrates the critical role of craniocaudal cyclic loading in pedicle screw fixation strength and its predictive value for PSL. Craniocaudal cyclic load reduces screw fixation strength significantly. Pullout force under cyclic load assessed by CT-FEA enhances the predictive accuracy for PSL risk.

Recent research has focused on issues related to contamination, nutrient availability, and strain selection, but there has been insufficient focus on harvesting research. This study employed an integrated continuous cultivation and harvesting strategy for a Spirulina microalgae biorefinery. The effects of nutrient-deficiency, harvesting ratio, and NaNO₃ addition on biomass concentration and productivity and phycocyanin accumulation of Spirulina were investigated. The lowest biomass productivity of 0.015 g/L/day was observed in Spirulina cultivated in NaNO₃ deficient medium. A harvesting ratio of 10% showed a consistent range of harvested dry biomass weight (0.20-0.22 g). Addition of 2.50 g/L NaNO₃ resulted in a significant increase in C-phycocyanin (C-PC) and allophycocyanin (APC) concentration from 34.37 mg/g to 68.35 and 27.08 to 33.23 mg/g, respectively. Biomass productivity of 1-L and 10-L batch culture was found to be 0.23 g/L/d and 0.21 g/L/d, respectively. Both 1-L and 10-L batch cultures showed a significant increase in phycocyanin accumulation due to the addition of 2.50 g/L of NaNO₃. These findings highlight the feasibility of continuous cultivation and optimized harvesting for scalable biomass and phycocyanin production, offering valuable insights for industrial biorefineries that seek to enhance microalgae-based bioactive compound extraction.

Nonlinear micro finite element (µFE) models have become the gold-standard for accurate numerical modeling of bone-screw systems. However, the detailed representation of bone microstructure, along with the inclusion of nonlinear material and contact, and pre-damage due to pre-drilling and screw-insertion, constitute significant computational demands and restrict model sizes. The goal of this study was to evaluate the agreement of screw pull-out predictions of computationally efficient, materially nonlinear µFE models with experimental measurements, taking both contact interface and pre-damage into account in a simplified way. Screw pull-out force was experimentally measured in ten porcine radius biopsies, and specimen-specific, voxel-based µFE models were created mimicking the experimental setup. µFE models with three levels of modeling details were compared: Fully bonded interface without pre-damage (FB), simplified contact interface without pre-damage (TED-M), and simplified contact interface with pre-damage (TED-M + P). In the TED-M + P models, the influence of pre-damage parameters (damage zone radial thickness and amount of damage) was assessed and optimal parameters were identified. The results revealed that pre-damage parameters highly impact the pull-out force predictions, and that the optimal parameters are ambiguous and dependent on the chosen bone material properties. Although all µFE models demonstrated high correlations with experimental data (R ² > 0.85), they differed in their 1:1 correspondence. The FB and TED-M models overestimated maximum force predictions (mean absolute percentage error (MAPE) > 52%), while the TED-M + P model with optimized pre-damage parameters improved the predictions (MAPE <17%). In conclusion, screw pull-out forces predicted with computationally efficient, materially nonlinear µFE models showed strong correlations with experimental measurements. To achieve quantitatively accurate results, precise coordination of contact modeling, pre-damage representation, and material properties is essential.

Introduction: Leukocyte adhesion deficiency type 1 (LAD1) is a severe inborn error of immunity caused by mutations in the ITGB2 gene, which encodes the beta-2 integrin subunit (CD18). These mutations lead to the absence or deficiency of CD18/CD11a, b, and c heterodimers, crucial for leukocyte adhesion and immune function. CRISPR-Cas9 Gene editing technology represents a promising approach for correcting these genomic defects restore the stable expression of CD18 and reverse the disease. Methods: We developed a CRISPR-Cas9-based gene correction strategy using Jurkat cells and patient-derived lymphoblastoid cell lines as surrogates for hematopoietic progenitor cells. Three candidate gRNAs were first predicted in silico using CRISPOR and experimentally tested in wild-type ITGB2-expressing Jurkat cells to identify the gRNA with the highest genomic DNA cleavage efficiency. The most efficient gRNA was then paired with espCas9 and used alongside five homology-directed repair templates (HDRs) (single-stranded donor oligonucleotides, ssODNs) to repair ITGB2 defects in patient-derived lymphoblastoid cell lines. CD18 expression levels in edited cells were quantified via flow cytometry, and whole-genome sequencing (WGS) was conducted to assess off-target effects and insertion accuracy. Results: Among the three candidate gRNAs, 2-rev gRNA exhibited the highest genomic cleavage rate in Jurkat cells. Using this gRNA with espCas9 and HDR-2, we achieved a 23% restoration of CD18 expression in LAD1 patient-derived cells, a level sufficient to change the disease course from severe to moderate. Whole-genome sequencing confirmed the absence of off-target mutations or undesired DNA insertions, demonstrating high specificity and precision in gene correction. Discussion: This CRISPR-Cas9-based method provides a precise and effective approach for correcting ITGB2 mutations in LAD1 patients. The high-fidelity gene editing process, validated through WGS, supports its potential for future applications in CD34+ hematopoietic stem cell therapies. The approach can be further optimized for clinical translation, offering a path toward a stable and long-term cure for LAD1.

Delivery of DNA into nucleated eukaryotic cells is known as transfection and has been essential in establishing technologies such as recombinant protein production and gene therapy. Considerable research efforts have led to development of a variety of transfection methods for a multitude of applications and cell types. Many methods are efficient in delivering DNA across the plasma membrane but few focus on subsequent delivery into the nucleus, a necessary step in expression of a recombinant transgene, and the cellular processes governing nuclear import of DNA during transfection have proved elusive. Herein, live confocal microscopy was used to track plasmid DNA during transfection of Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells to map key cellular processes central to nuclear import of DNA showing that there is a strong relationship between events of cell division, promotion of DNA dispersal from endosomes and subsequent nuclear import leading to gene expression. Furthermore, cationic lipid-mediated transfection is more dependent on events of the cell cycle than electroporation to deliver DNA into the nucleus. These findings have informed the design of a method where both CHO and HEK cells are synchronised at G2 phase of the cell cycle followed by timely release enabling cell cycle progression to maximise the frequency of division events immediately after transfection. This led to a 1.2-1.5 fold increase in transfection efficiency for polyethylenimine (PEI) mediated and electroporation transfection respectively. This process enhanced production yields of a monoclonal antibody 4.5 fold in HEK and 18 fold in CHO cells in the first 24 h post transfection. Overall, this study elucidated key cellular processes fundamental to transfection of CHO and HEK cells providing knowledge which can be applied to DNA delivery technologies in a plethora of fields.

Mannitol is a valuable sugar alcohol, extensively used across various industries. Cyanobacteria show potential as future platforms for mannitol production, utilizing CO₂ and solar energy directly. The proof-of-concept has been demonstrated by introducing a two-step pathway in cyanobacteria, converting fructose-6-phosphate to mannitol-1-phosphate and sequentially to mannitol. However, recombinant strains generally faced issues of genetic instability or low titers, consequently affecting the long-term mannitol production. In this work, the construction strategy for engineering mannitol production in Synechococcus elongatus PCC 7942, based on commonly adopted pathway comprising mannitol-1-phosphate dehydrogenase (Mtld) and mannitol-1-phosphatase (M1Pase), was optimized. The results demonstrated that the sequential introduction of m1p and mtld was required to obtain mannitol-producing strains. We further manipulated the abundances of Mtld with a theophylline dose-responsive riboswitch approach, and by combining it with the overexpression of m1p, we successfully obtained a recombinant strain producing 1.5 g/L mannitol under optimal conditions, the highest cyanobacterial yield to date. In addition, the controlled expression of mtld was demonstrated to remarkably augment the genetic stability of the mutant under long-term culturing circumstances, which continued to secrete mannitol after more than 2 months of cultivation without the addition of theophylline, and the mannitol biosynthesis operon did not undergo any spontaneous mutation. The findings in this work provided novel insights into the area of cyanobacteria mannitol metabolism engineering, and would inspire researchers to construct strains with different gene regulatory strategies for efficient photosynthetic biosynthesis.

The early diagnosis rate of gastric cancer is low, and most patients are already at an advanced stage by the time they are diagnosed, posing significant challenges for treatment and exhibiting high recurrence rates, which notably diminish patients' survival time and quality of life. Therefore, there is an urgent need to identify methods that can enhance treatment efficacy. Nanomedicine, distinguished by its small size, high targeting specificity, and strong biological compatibility, is particularly well-suited to address the toxic side effects associated with current diagnostic and therapeutic approaches for gastric cancer. Consequently, the application of nanomedicine and delivery systems in the diagnosis and treatment of gastric cancer has garnered increasing interest from researchers. This review provides an overview of recent advancements in the use of nanomaterials as drugs or drug delivery systems in gastric cancer research, encompassing their applications in diagnosis, chemotherapy, radiotherapy, surgery, and phototherapy, and explores the promising prospects of nanomedicine in the treatment of gastric cancer.

Introduction: Medial Opening-wedge High Tibial Osteotomy (HTO) is an effective treatment for medial compartment osteoarthritis and knee varus in relatively young and active patients. While it can effectively correct lower limb alignment in the coronal plane, it may also affect the posterior tibial slope (PTS) in the sagittal plane. However, the factors influencing PTS and methods for maintaining PTS stability remain controversial.

Methods: A lower limb geometric model was constructed based on the CT data from a patient with medial knee osteoarthritis and varus knee. Multiple models were developed to simulate various conditions: seven different medial cortex inclinations of the proximal tibia (-15°-15°), seven coronal plane inclinations of the central osteotomy plane (-15°-15°), seven sagittal plane inclinations of the hinge axis (-15°-15°), seven hinge axis heights (-7 mm-7 mm), and seven hinge axis inclinations in the axial plane (-15°-15°). Changes in the ratio between anterior and posterior opening gap (RAPOG) and PTS were analyzed.

Results: The medial cortex inclination of the proximal tibia, coronal plane inclination of the central osteotomy plane, inclination of the sagittal plane of the hinge axis, and height of the hinge axis did not alter the PTS; however, these factors did affect RAPOG, with increased values leading to decrease in RAPOG. The ranges of RAPOG for these factors were 76.37%-54.83%, 68.91%-60.94%, 68.04%-64.08%, and 70.38%-62.61%, respectively. However, the hinge axis inclination on the axial plane affects PTS, for inclinations of -15°, -10°, -5°, 0°, 5°, 10°, and 15°, the PTS decreased 2.48°, 1.83°, 0.98°, 0°, -0.97°, -1.82°, and -2.53°, respectively. To maintain a constant PTS, RAPOG should be readjusted to 65.13%, 66.01%, 66.27%, 65.76%, 65.03%, 65.15%, and 65.57%, respectively.

Discussion: The inclination of the hinge axis in the axial plane affects PTS, as its value increases, PTS also increases. To maintain a constant PTS, RAPOG should be readjusted. Understanding these relationships is essential for optimizing surgical techniques to minimize unintended changes in PTS.