Journal of biological methods最新文献

Background: MicroRNAs (miRNAs) have gained significant attention as potential biomarkers in childhood leukemia, offering insights into diagnosis, prognosis, and therapeutic targeting. However, the clinical translation of miRNA biomarkers faces several challenges, including inconsistencies in results caused by methodological differences, sample processing variability, and a lack of standardized normalization techniques. In addition, miRNA profiles are highly cell-specific, with unique signatures for different blood cell types and leukemic blasts, reflecting their distinct biological roles and disease states. Pre-analytical factors are critical in ensuring the accuracy and reproducibility of miRNA quantification. The selectively enriched and highly stable exosomal miRNAs have shown great promise for studying intercellular communication and disease-specific miRNAs. The selection of the analytical matrix should align with the specific objectives of the research or diagnostic application. Addressing technical challenges and recording potential confounding variables (e.g., age, gender, ethnicity, body mass index, menstrual cycle, fasting, circadian rhythm, comorbidities, medications, smoking, and physical activity) are essential to enhancing the reproducibility and reliability of miRNA biomarkers.

Objective: This review aims to highlight the challenges facing miRNA quantification in childhood leukemias, scrutinizing all relevant pre-analytical issues, including the preferred specimen source, leukemic blast type and count, the selection of suitable blood components, the impact of time and freeze-thaw cycles, collection, processing, and storage variables.

Conclusion: Further research is needed to standardize methodologies and expand our understanding of miRNA interaction networks, ultimately advancing the application of miRNAs in childhood leukemia research.

Background: Leber hereditary optic neuropathy (LHON) is a rare inherited mitochondrial disease that leads to mitochondrial dysfunction, resulting in optic nerve damage and vision loss. Systemic involvement has been reported in several LHON cases, referred to as LHON+ disorders. However, the causes and presentations of such conditions have been poorly studied. It is suggested that 90% of mitochondrial dysfunction is caused by one of three primary point mutations in mitochondrial DNA that affect respiratory complex I (referred to as mtDNA LHON), with unresolved cases of LHON being caused by other variants, known as autosomal recessive LHON. The cardiac, musculoskeletal, neurological, and auditory systems are commonly affected in LHON. For example, hypertrophic cardiomyopathy and sudden cardiac death have been linked to specific mutations. Neurological effects - such as dystonia, epilepsy, polyneuropathy, and ataxia - as well as hearing loss, have also been observed in patients with specific mitochondrial mutations. These findings highlight the need for a more comprehensive evaluation beyond standard ophthalmic assessments. LHON is typically diagnosed based on a combination of ophthalmic imaging, patient age and gender, clinical course (bilateral, rapidly progressive, and sequential visual loss), family history, maternal inheritance, and fundus appearance. However, the advent of genetic testing has significantly expanded the recognized phenotype. In terms of treatment, idebenone is the only FDA-approved therapy for LHON; however, intravitreal gene therapy yields promising improvement, especially for the most common m.11778G>A mutation, which accounts for 70% of causative mutations. At present, these therapies are confined to ocular treatment.

Objective: This review highlights the importance of recognizing systemic manifestations of LHON, which are frequently overlooked in clinical practice.

Conclusion: Early detection of these systemic manifestations, especially in cardiac and neurological systems, could help with prompt intervention and improve patient outcomes. Further research into gene therapy and mitochondrial replacement techniques holds promising potential for developing more effective treatment strategies.

Background: Adult neurogenesis is a regenerative mechanism of the brain that contributes to neuroplasticity and memory consolidation. Aberrant neurogenesis is considered a key pathogenic hallmark of a wide array of neurocognitive disorders. While the functional significance of adult neurogenesis is well established in most experimental and wild animals, its occurrence in the aging human brain remains uncertain.

Objective: Most studies on adult neurogenesis in humans rely on post-mortem analysis, as there is currently no method to accurately evaluate the neurogenic process in the intact brain. Theta rhythm, a neural oscillatory pattern, is believed to originate from hippocampal place cells that play a crucial role in creating cognitive maps. Theta rhythm is positively modulated by various factors, such as physical activities and enriched environment, which also promote adult neurogenesis. The strength and stability of theta rhythm are closely linked to mental well-being and cognitive functions, while its disruptions serve as indicators of neuropathogenic events that directly intersect with the regulation of adult neurogenesis.

Conclusion: Modulation of the theta rhythm may reciprocally reflect the degree of neurogenesis in the adult brain, as newborn neurons can directly integrate with place cells, especially in the hippocampus. Given their electrophysical properties, newborn neurons may hold an intrinsic potential to generate theta rhythm upon motor sensory inputs and different neural activities. Biomedical tools such as electroencephalography, which measures theta rhythm, could thus be utilized to non-invasively monitor ongoing neurogenic processes in intact brains. Consequently, theta rhythm may function as a potential real-time, quantitative marker of adult neurogenesis.

Background: Myasthenia gravis (MG) is an acquired autoimmune disorder characterized by autoantibodies targeting the neuromuscular junction of skeletal muscles. In contrast, congenital myasthenic syndromes (CMSs) represent a clinically diverse group of genetic disorders affecting the neuromuscular junctions, with early onset and autosomal recessive inheritance. CMSs are particularly prevalent in Devon Rex and Sphynx cats. The gold standard for diagnosing MG in cats involves detecting neuromuscular junction autoantibodies by measuring acetylcholine receptor autoantibodies using radioimmunoassay. For CMS, a definitive diagnosis requires the identification of a causative genetic mutation in addition to clinical signs of skeletal muscle weakness and fatigue. With the Sphynx and Devon Rex breeds, data collected have identified a candidate disease region on the feline C2 chromosome, discovered by employing a genome-wide single-nucleotide polymorphism-based homozygosity mapping strategy. Given that MG is an autoimmune condition, it is treated with steroids, immunosuppressive drugs, and sometimes a thymectomy (surgical removal of the thymus gland). CMS is a set of genetic conditions that do not respond to these treatments. Hence, accurate differential diagnosis is critical.

Case presentation: Presented in this case study was a British Shorthair feline which was anamnetically, clinically, paraclinically, and pharmacologically assessed. Genetic testing revealed a positive result for the COLQ gene mutation.

Conclusion: This case study clarified and added criteria to the differential diagnosis between MC and CMS, allowing for more accurate prognostic evaluations and appropriate treatment planning. It also underscores the importance of genetic testing in British Shorthair cats to differentiate between these conditions.

Background: Gastric adenocarcinoma, the malignant proliferation of glandular cells in stomach epithelium, is a type of gastric cancer with a statistical disease burden of the fifth most common cancer globally and the 17^th most common malignancy in the United Kingdom. Prognosis varies with stage (I-IV), with stages I-III showing promising 5-year survival rates up to 71.8%, warranting timely diagnosis and treatment. Surgery is the gold-standard treatment; however, due to complex tumor pathophysiology, there is growing interest in the use of multimodal therapies. Specifically, the combination of surgery and adjuvant chemotherapy has become a key focus of the treatment for stages I-III gastric adenocarcinoma.

Objective: The study reviewed patients with stages I-III (non-advanced) gastric adenocarcinoma to assess whether adjuvant chemotherapy combined with surgery provides better disease-free/disease-specific/cause-specific survival, overall survival, and reduced recurrence rates/improved recurrence-free survival, compared to surgery alone. Analyzed were 17 English-language, full-text, levels I-III peer-reviewed studies from MEDLINE and Embase from the past 10 years were analyzed. No age/sex/ethnicity/country restrictions were applied, and the dimension of interest was limited to stages I-III gastric adenocarcinoma patients who underwent tumor resection through surgery and received chemotherapy as the only adjuvant therapy. Seven (41.2%) studies have more than one statistically significant outcome measure supporting the benefit of adjuvant chemotherapy in combination with surgery over surgery alone. Ten (58.8%) studies showed no statistically significant benefit of adjuvant chemotherapy.

Conclusion: The findings contrasted with previous large-scale meta-analyses, which were limited by sample size and biases in individual studies reviewed. Continued research, incorporating advances in surgical techniques and new chemotherapeutic combinations, is necessary to ascertain best-tailored treatments for gastric adenocarcinoma.

Background: Protein A chromatography is widely used for antibody purification. With conventional packed-bed columns, mass transfer within resin beads is diffusion-limited, entailing long residence time to achieve high binding capacities. Recently, several vendors have introduced Protein A membranes as alternatives to traditional Protein A resins/columns. These membranes feature open pore structures that facilitate the convective transport of protein molecules, enabling high binding capacities within significantly shorter residence time. The use of Protein A membranes can improve throughput, eliminate the need for column packing, and reduce cost. These advantages notwithstanding, Protein A membranes present certain drawbacks. A major limitation is their high dead volume-to-stationary phase ratio, which leads to larger elution volumes compared to their conventional counterparts. This results in significant eluate dilution and increased buffer consumption.

Objective: In the current study, we aimed to demonstrate that ultrafiltration (UF), when used in combination with Protein A membrane chromatography, can address these limitations by allowing eluate concentration and buffer reuse.

Methods: A laboratory model of UF integrated Protein A membrane was set up to test the feasibility and effectiveness of the proposed strategy.

Results: Integrated UF effectively concentrated Protein A membrane eluate to a concentration comparable to that of Protein A column eluate. In addition, reuse of pH-adjusted UF filtrate as elution buffer reduces buffer consumption by 50%.

Conclusion: UF integration is an effective solution for addressing the problem of increased elution volume and buffer consumption associated with Protein A membrane.

Background: Breast cancer is the most frequently diagnosed female malignancy worldwide and one of the primary causes of cancer-related mortality in women. According to the latest the World Health Organization data, it was the most common cancer in 157 out of 185 countries and was culpable for an estimated 670,000 deaths in 2023. With breast cancer incidence continuing to increase, there is a mounting interest in early detection and prevention, with a focus particularly directed on genetic factors, modifiable risk factors, and screening methods.

Objective: This review aimed to examine the genetic landscape of breast cancer, the role of risk factors in disease development, and the importance of advancing diagnostic modalities for early detection. A comprehensive search and analysis of peer-reviewed articles and clinical studies from major medical databases were conducted to assess the most recent advancements and discoveries in the field. The literature review identified several modifiable and non-modifiable risk factors, including genetic predispositions (e.g., BRCA mutations), hormonal influences, lifestyle factors, and reproductive patterns.

Conclusion: By synthesizing current knowledge, this review enhances the understanding of breast cancer's multifactorial nature and provides insights to guide future research on screening strategies and preventive measures.

Background: Early pathogen identification in the bloodstream has long been a key focus for microbiologists and clinicians, given its crucial role in patient management. Matrix-assisted laser desorption ionization-time of flight mass spectrometry has emerged as a valuable tool for the direct and rapid identification of organisms from positive blood cultures.

Objective: This study aimed to evaluate the accuracy, productivity, and feasibility of two methods for the rapid detection of bloodstream infections.

Methods: Two methods were employed in this study: One based on differential centrifugation and the other using a lysis buffer.

Results: The addition of a lysis buffer, sodium dodecyl sulfate (SDS), to the blood culture broth resulted in the identification of a greater number of microorganisms (Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumonia).

Conclusion: The application of SDS into culture broths is user-friendly and can be easily integrated into routine blood culture processing, allowing for species-level identification within hours of a positive BacT/ALERT signal.

Background: Prostate biopsy is a crucial diagnostic tool for detecting clinically significant prostate cancer (csPCa). Traditional transrectal ultrasound (TRUS)-guided biopsy methods are often associated with an increased infection risk of infection and limited accuracy, particularly when diagnosing anterior lesions of the prostate gland.

Objective: This article presented a structured protocol for performing transperineal fusion magnetic resonance imaging/ultrasound (MRI/US) prostate biopsy, highlighting its advantages over the TRUS approach. Our study included biopsy-naïve patients with elevated prostate-specific antigen levels or abnormal digital rectal examination findings, all of whom underwent pre-biopsy multiparametric MRI to guide targeted biopsies. The key objectives of this protocol were to improve the detection rates of csPCa, minimize infection risk, and standardize a transperineal technique that combines both systematic and targeted biopsies. In addition, we provided details on patient preparation, equipment requirements, procedural steps, and follow-up protocols to ensure the safety and effectiveness of the procedure. This protocol aims to serve as a guideline for institutions to adopt MRI/US fusion-guided transperineal biopsy, thereby enhancing diagnostic accuracy and patient safety.

Conclusion: The transperineal fusion MRI/US biopsy protocol enhances diagnostic accuracy, particularly for anterior lesions, while reducing infections risks. Combining targeted and systematic biopsies improves detection rates of csPCa and offers a standardized, safe approach for clinical implementation.

Background: On-line sensing technologies for expanding cell cultures are becoming essential tools for understanding metabolic activity during critical stages of expansion. These tools generate data that indicate when user interventions are required, such as harvesting cells or cell products, inducing cell differentiation, or altering growth medium inputs. The platform must reliably measure the biochemical and physiochemical properties of interest in a dependable, aseptic, and non-invasive manner to benefit users.

Objective: In this proof-of-concept study, we used the Nova Biomedical BioProfile FLEX2 as a platform for metabolic and cellular measurements due to its ability to detect a range of metabolically relevant compounds, measure cell counts and viability, and acquire samples automatically.

Methods: Here, we demonstrated the straightforward integration of the analyzer with the Quantum Flex™ Cell Expansion System (Quantum Flex) using an available sampling port. While this approach can accommodate both suspension and adherent cell types, this study focused on suspension cells only. In addition, we developed a simple method to integrate the sampling adapter into a Quantum cell expansion set, allowing sample collection at precise intervals.

Results: In both cases, the samples were acquired automatically using the analyzer's timing function, facilitating an automated expansion process with increased data collection frequency - previously impractical with manual sampling.

Conclusion: This model provides Quantum Flex users with an option for online sensing to monitor cell expansion at scheduled intervals without requiring additional user input.