Journal of biological methods最新文献

Background: For the purification of monoclonal antibodies, we employed a wash step in the cation exchange (CEX) chromatography to reduce acidic charge variants. Although optimal wash conditions, determined under a specific loading density, ensured both effective reduction of charge variants and good process yield, applying the same wash conditions to runs where the loading density moderately deviated from the optimal value could result in insufficient reduction of charge variants or low step yield. This issue is particularly problematic with large-scale manufacturing, where the same wash condition (including buffer and volume) is applied across all runs, despite variations in loading density due to fluctuations in harvest titer.

Objective: To address this problem, we intended to demonstrate that multi-column continuous chromatography could offer an effective solution.

Methods: A multi-column setup was implemented, in which all runs except the final one were performed under optimal conditions to ensure both high product quality and yield.

Results: The multi-column approach allowed for both effective charge variant reduction and achievement of good step yield. Although the final run might be conducted under suboptimal conditions, potentially compromising quality or yield, its impact is minimal, as it contributed only a small fraction to the total product, thereby exerting a limited effect on overall quality and yield.

Conclusion: The current study successfully demonstrated the proof-of-concept using CEX chromatography. In fact, the multi-column strategy proposed here may provide a universal solution to the load-dependence issue in wash steps that is aimed at removing or reducing weakly bound impurities or charge variants in any type of bind-elute mode chromatography.

Background: The biotechnology industry commonly utilizes synthetic media to grow biological control agents (BCAs); however, these media are often considered impractical, particularly in developing countries.

Objective: This study aimed to identify the most suitable locally available organic substrates for the cultivation of BCAs used against cashew fusarium wilt disease.

Materials and methods: Experiments were conducted in 2022 and 2023 on five substrates, namely rice husk (RH), rice bran (RB), hulled millet, parboiled rice, and their combinations, as growth media for combined Bacillus strains and Trichoderma asperellum. The carbohydrate and protein content of the substrates were quantified colorimetrically.

Results: Protein and carbohydrate contents ranged from 13.4 - 19.8% to 76.3 - 82.7%, respectively. The BCAs exhibited high colonization rates on all tested substrates, with combined substrates supporting the highest colonization, followed by RH and RB. Endospore formation and conidiation levels varied significantly over time across substrates and incubation temperatures (p ≤ 0.05). Population densities decreased over time under refrigerated, room temperature, and incubator conditions for most substrates in both seasons, except for combined substrates, RH, and RB. Final population counts were as follows: 2.1 × 10⁷ ± 4.9 × 10⁵ and 1.0 × 10⁷ ± 0.3 × 10⁵ colony-forming units (CFUs)/mL (combined substrates), 2.3 × 10⁷ ± 5.4 × 10⁵ and 5.7 × 10⁷ ± 1.1 × 10⁶ CFUs/mL (RH), 3.6 × 10⁵ ± 2.1 × 10⁴ and 3.3 × 10⁵ ± 1.6 × 10⁴ CFUs/mL (RB), while under refrigerated conditions, population densities remained relatively unchanged throughout the study period.

Conclusion: Based on the findings, it is recommended to use a mixture of substrates, RH alone, or RB alone as appropriate media for the large-scale production of the studied biofungicides.

Background: Despite advancements in surgical treatments, impairments persist after peripheral nerve injuries, prompting a shift in research toward the microenvironment of injured axons. Platelet-rich plasma (PRP), rich in growth factors and derived from autologous blood, emerges as a potential candidate to accelerate nerve healing.

Objective: This study investigated the role of PRP in enhancing peripheral nerve regeneration using a rat sciatic nerve model (n = 8) in female Wistar rats.

Methods: A transected sciatic nerve model was created, with both hindlimbs repaired through end-to-end neurorrhaphy. PRP, prepared from the blood of a healthy Wistar rat, was applied to one hindlimb. Functional recovery was assessed using sciatic indices. At the 20-week time point, histological evaluations were performed to compare PRP-treated hindlimbs with control ones. Statistical analysis was conducted to compare the results between the two groups using three different calculations for specific parameters.

Results: Walking track-based sciatic functional index (SFI) showed an improvement of 66.0%, 47.8%, and 71.6% (p < 0.05). Video analysis-based SFI revealed a 36.7% and 27.3% improvement (p < 0.05). Static sciatic index calculations indicated an improvement of 19.4% for vertical standing and 26.7% for standing on all four limbs (p < 0.001). Histopathological analysis showed a reduction in inflammation, a decrease in fibrosis, and the absence of macrophages in the sciatic nerves of the experimental group. Muscle specimens from the PRP-treated group exhibited fewer macrophages and significantly less fibrosis (p < 0.05). Overall, PRP treatment significantly improved all functional indices.

Conclusion: This study demonstrated PRP's utility in promoting peripheral nerve regeneration, highlighting its potential for both fundamental research and clinical applications.

Background: Colorectal cancer represents one of the most common neoplastic diseases worldwide, making it a frequent focus in routine pathological analyses. Visualizing complex three-dimensional (3D) structures, such as nerves within tumors, requires thick tissue sections, which necessitates the use of optical tissue-clearing methods to achieve transparency. However, following tissue clearing, samples typically require advanced imaging techniques such as light-sheet and two-photon confocal microscopy, which are usually unavailable in standard histological laboratories.

Objective: We aimed to demonstrate how a well-established tissue-clearing approach can be adapted for use in a routine histological laboratory, enabling a robust 3D visualization of nerve fibers in samples of both normal human colon and colon cancer tissues.

Methods: We modified the "clear unobstructed brain/body imaging cocktails" method, originally developed for whole-brain imaging in mice, and applied it to human colon tissue samples measuring approximately 10 mm³, a standard size typically processed in pathological laboratories.

Results: Our protocol, which integrates a tissue-clearing technique, enabled reliable immunofluorescent visualization of colonic nerve fibers labeled with anti-β₃-tubulin antibodies. The labeled nerve fibers could be observed using a standard epifluorescence microscope, and high-quality 3D reconstructions were generated through a simple image analysis approach using the open-source software ilastik, which eliminates the need for confocal microscopy.

Conclusion: The proposed steps provide a valuable method for researchers to visualize complex 3D structures, such as neural cells and processes, in both normal and tumor-transformed tissue settings.

Background: Hepatitis B virus (HBV) genotype B (HBV/B) is the predominant strain in Taiwan and several East Asian countries.

Objective: The aim of this study is to use comprehensive phylogenetic analysis tools to monitor the long-term molecular evolution dynamic of HBV genotype B population in East Asia.

Methods: In this study, full genome sequences of HBV with temporal information were extracted from GenBank and analyzed using the Bayesian Markov chain Monte Carlo method to identify best-fitting coalescent models.

Results: Bayesian Skygrid analysis revealed a viral effective population (phylodynamic) bottleneck for HBV/B in 2003, a pattern similar to the previously described HBV genotype C (HBV/C). Despite these similarities, the viral dynamics for HBV/B and HBV/C diverged after 2005. HBV/C exhibited a marked decrease in genetic diversity across East Asia, whereas HBV/B maintained stable genetic diversity after 2005. Phylogeographic analysis using Neighbor-Joining and Bayesian maximum clade credibility trees indicated that Taiwan was likely the geographic origin of the most recent common ancestor of HBV/B in East Asia. An early clade spread to Japan and subsequently to the West Coast of the United States of America. Another clade dispersed to China, spread widely across the region, and was reintroduced to Taiwan multiple times. In contrast, HBV/C likely originated in China and spread to Japan, Korea, and Taiwan over several decades.

Conclusion: This study highlights the similarities and differences between the viral dynamics and geographical evolutionary pathways between HBV/B and HBV/C.

Background: Lipids are crucial signaling molecules or cellular membrane components orchestrating biological processes. To gain insights into lipid functions and the communication between organelles, it is essential to understand the subcellular localization of individual lipids. Advancements in lipid quantification techniques, improvements in chemical and spatial resolution for detecting various lipid species, and enhancements in organelle isolation speed have allowed for profiling of the organelle lipidome, capturing its temporal-spatial distribution.

Objective: This review examined approaches used to develop organelle lipidome and aimed to gain insights into cellular lipid homeostasis from an organelle perspective. In addition, this review discussed the advancements in lipid-mediated inter-organelle communication within complex physiological and pathological processes.

Conclusion: With the advancement of lipidomic technologies, more detailed explorations of organelle structures and the specific lipid-mediating functions they perform are feasible.

Background: HA14-1 is a small-molecule, stable B-cell lymphoma 2 (Bcl-2) antagonist that promotes apoptosis in malignant cells through an incompletely-defined mechanism of action. Bcl-2 and related anti-apoptotic proteins, such as B-cell lymphoma-extra-large [Bcl-XL]), are predominantly localized to the outer mitochondrial membrane, where they regulate cell death pathways. However, the notably short half-life of HA14-1 in vitro limits its potential therapeutic application. To address this limitation, a more stable analog, ethyl-2-amino-6-phenyl-4-(2-ethoxy-2-oxoethyl)-4H-chromene-3-carboxylate (sHA14-1), was developed.

Objective: This study investigated the relationship between sHA14-1 and Bcl-2/Bcl-XL. The sHA14-1 molecule acts as a hormetic substance. Therefore, it is crucial to determine whether the hormetic zone corresponds to a putative therapeutic window, that is, the optimal concentration at which sHA14-1 selectively kills cancer cells overexpressing Bcl-2 or Bcl-XL while causing minimal damage to normal cells.

Methods: Using classical cell biology and flow cytometry, we examined the main signaling pathways involving Bcl-2 or Bcl-XL, and their modification in the presence of sHA14-1.

Results: We showed that sHA14-1 exerted a dual effect on mitochondria: (i) it sensitized cells to increased permeability, and (ii) it inhibited adenosine diphosphate-stimulated respiration and uncoupled respiration. At relatively low concentrations, sHA14-1 induced mitochondrial swelling, reminiscent of "pore opening" but with distinct characteristics. Over 30 μM, sHA14-1 caused mitochondrial transition depolarization independent of permeability transition and cell death that resembled secondary necrosis (i.e., occurring after maximal mitochondrial permeability) rather than apoptosis. The balance between apoptotic and necrotic cell death induced by sHA14-1 was also evaluated.

Conclusion: Our results suggested that sHA14-1 plays a multifunctional role, involving both mitochondria and the endoplasmic reticulum. Its actions are more complex than its originally intended role in targeting anti-apoptotic Bcl-2 family members, which may complicate its potential application as an anticancer therapy.

Background: The systemic inflammatory response triggered by the carcinogenic process induces significant changes in a wide range of hematological biomarkers, impacting their levels, functions, and overall roles in the body's physiological and pathological processes.

Objective: To evaluate the value of pre-treatment hematological parameters in the prediction of clinical and radiological responses of locally advanced vulvar cancer to definitive chemoradiation.

Methods: We retrospectively reviewed the medical records of patients treated at the King Hussein Cancer Center receiving definitive chemoradiation for pathologically confirmed locally advanced vulvar carcinoma. Response of the primary disease to treatment was classified as complete response (CR) if there was no clinically- or radiologically-confirmed residual disease at 12 weeks after completion of chemoradiation. Univariate analyses on complete response, progression-free survival (PFS), and overall survival (OS) were performed using clinical factors and pre-treatment hematological parameters.

Results: A total of 30 patients were included, with a median age of 57.5 years and a median follow-up of 21 months. Of these, 24 patients (80%) achieved CR. Disease progression occurred in 11 patients (36.7%) during the follow-up period, and 9 (30%) died. Kaplan-Meier analysis demonstrated that only the neutrophil-to-lymphocyte ratio (NLR) (p = 0.007) and basophil-to-lymphocyte ratio (BLR) (p = 0.05) were predictive of OS. Conversely, PFS was significantly associated with white blood cell count (p = 0.042) and BLR (p = 0.004). Receiver operating characteristic (ROC) analysis indicated that NLR and BLR had significant predictive power for survival at the following cutoffs: 3.4 and 0.035, respectively. When categorized by ROC values, BLR was significantly associated with response to treatment (p = 0.026). Moreover, both NLR and BLR were significantly associated with OS and PFS.

Conclusion: Pre-treatment NLR and BLR may be useful predictive markers for clinical and radiological response, as well as for oncological outcomes in locally advanced vulvar cancer treated with definitive chemoradiation.

Background: Protein L-conjugated resins are affinity media that bind to the variable region of the kappa light chain (LC) and have been used for initial product capture in the downstream processing of full-length antibodies and antibody fragments. Previous studies, including ours, have demonstrated that Protein L chromatography effectively separated various byproducts generated during the production of bispecific antibodies (bsAbs), including half-antibody, homodimer, LC-missing species, and aggregates. Cytiva recently launched its second-generation Protein L resin, MabSelect VL, which offers significantly improved binding capacity compared to its predecessor, Capto L.

Objective: This study aimed to explore the feasibility of developing a two-column process, which includes MabSelect VL capture step and a polishing step, for purification of complex antibody molecules.

Methods: We employed two bsAb cases to demonstrate that MabSelect VL's enhanced byproduct removal capability allows for a potential two-column purification process.

Results: For both bsAbs, the developed two-column process yielded a product with quality attributes comparable to those obtained using the traditional three-column process.

Conclusion: The MabSelect VL-based two-column process can be successfully applied to bsAb purification. In addition, it should also be feasible with regular monoclonal antibodies, whose purification is generally less challenging than that of bsAbs. By reducing the downstream process from three columns to two columns, significant savings in terms of time, labor, and materials can be achieved.

Background: Established in vivo radiobiological models are commonly used to assess anti-tumor effects and normal tissue toxicity. However, these models have notable limitations, and additional models are necessary to gain a deeper insights into drug-radiation interactions.

Objective: This study aimed to develop an organotypic ex vivo model by using precision-cut lung slices (PCLSs) to evaluate radiation-induced residual deoxyribonucleic acid (DNA) damage, both alone and in combination with a pharmacological inhibitor of DNA double-strand break (DSB) repair.

Methods: Left lungs from female C57BL/6 mice were dissected, perfused with 4% low-gelling-temperature agarose, and sliced into 250 μm sections. Lung slices were then incubated ex vivo for up to 7 days. The slices were irradiated using ¹³⁷Cs, either with or without a DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441). Tissue sections were subsequently fixed and stained for γH2AX and 53BP1, which serve as histological markers of DNA DSBs.

Results: The established conditions preserved tissue viability for up to 7 days and maintained structural integrity for 2 days. DNA damage, detected through γH2AX and 53BP1 staining, was consistent between lungs irradiated ex vivo and their counterparts irradiated in vivo. In the organotypic model, radiation alone in DNA-PK-deficient SCID mice and radiation combined with DNA-PK inhibition in C57BL/6 mice led to increased residual γH2AX and 53BP1 staining.

Conclusion: This study demonstrates that residual DNA damage levels following ionizing radiation in lung tissue are comparable between in vivo and ex vivo tissue slices, suggesting that PCLSs serve as a valuable organotypic model for investigating the effects of drug-radiation combinations.