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Biology Jun 2024The proliferation of large green macroalgae in marine environments has led to the occurrence of green tides, particularly in the South Yellow Sea region of China, where... (Review)
Review
The proliferation of large green macroalgae in marine environments has led to the occurrence of green tides, particularly in the South Yellow Sea region of China, where has been identified as the primary species responsible for the world's largest green tide events. Allelopathy among plants is a critical factor influencing the dynamics of green tides. This review synthesizes previous research on allelopathic interactions within green tides, categorizing four extensively studied allelochemicals: fatty acids, aldehydes, phenols, and terpenes. The mechanisms by which these compounds regulate the physiological processes of green tide algae are examined in depth. Additionally, recent advancements in the rapid detection of allelochemicals are summarized, and their potential applications in monitoring green tide events are discussed. The integration of advanced monitoring technologies, such as satellite observation and environmental DNA (eDNA) analysis, with allelopathic substance detection is also explored. This combined approach addresses gaps in understanding the dynamic processes of green tide formation and provides a more comprehensive insight into the mechanisms driving these phenomena. The findings and new perspectives presented in this review aim to offer valuable insights and inspiration for researchers and policymakers.
PubMed: 38927336
DOI: 10.3390/biology13060456 -
Biomolecules Jun 2024Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography...
Lung cancer is the leading cause of cancer deaths globally, necessitating effective early detection methods. Traditional diagnostics like low-dose computed tomography (LDCT) often yield high false positive rates. gene methylation has emerged as a promising biomarker. This study aimed to develop and validate a novel semi-nested real-time PCR assay enhancing sensitivity and specificity for detecting methylation using extendable blocking probes (ExBPs). The assay integrates a semi-nested PCR approach with ExBPs, enhancing the detection of low-abundance methylated DNA amidst unmethylated sequences. It was tested on spiked samples with varied methylation levels and on clinical samples from lung cancer patients and individuals with benign lung conditions. The assay detected methylated DNA down to 0.01%. Clinical evaluations confirmed its ability to effectively differentiate between lung cancer patients and those with benign conditions, demonstrating enhanced sensitivity and specificity. The use of ExBPs minimized non-target sequence amplification, crucial for reducing false positives. The novel semi-nested real-time PCR assay offers a cost-effective, highly sensitive, and specific method for detecting methylation, enhancing early lung cancer detection and monitoring, particularly valuable in resource-limited settings.
Topics: Humans; Lung Neoplasms; DNA Methylation; Homeodomain Proteins; Real-Time Polymerase Chain Reaction; Biomarkers, Tumor; Sensitivity and Specificity
PubMed: 38927132
DOI: 10.3390/biom14060729 -
BMC Biotechnology Jun 2024Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with...
BACKGROUND
Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase.
METHODS
In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration.
RESULTS
The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion.
CONCLUSIONS
This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.
Topics: Animals; CHO Cells; Integrases; Cricetulus; Nuclear Localization Signals; Cell Nucleus; Serine; Green Fluorescent Proteins; Cricetinae; Xenopus laevis
PubMed: 38926833
DOI: 10.1186/s12896-024-00871-4 -
Scientific Reports Jun 2024Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones...
Excess amounts of histones in the cell induce mitotic chromosome loss and genomic instability, and are therefore detrimental to cell survival. In yeast, excess histones are degraded by the proteasome mediated via the DNA damage response factor Rad53. Histone expression, therefore, is tightly regulated at the protein level. Our understanding of the transcriptional regulation of histone genes is far from complete. In this study, we found that calcineurin inhibitor treatment increased histone protein levels, and that the transcription factor NFATc1 (nuclear factor of activated T cells 1) repressed histone transcription and acts downstream of the calcineurin. We further revealed that NFATc1 binds to the promoter regions of many histone genes and that histone transcription is downregulated in a manner dependent on intracellular calcium levels. Indeed, overexpression of histone H3 markedly inhibited cell proliferation. Taken together, these findings suggest that NFATc1 prevents the detrimental effects of histone H3 accumulation by inhibiting expression of histone at the transcriptional level.
Topics: NFATC Transcription Factors; Histones; Calcineurin; Humans; Cell Proliferation; Gene Expression Regulation; Promoter Regions, Genetic; Signal Transduction; Transcription, Genetic; Calcium
PubMed: 38926604
DOI: 10.1038/s41598-024-65769-9 -
Scientific Reports Jun 2024Detecting aberrant cell-free DNA (cfDNA) methylation is a promising strategy for lung cancer diagnosis. In this study, our aim is to identify methylation markers to...
Detecting aberrant cell-free DNA (cfDNA) methylation is a promising strategy for lung cancer diagnosis. In this study, our aim is to identify methylation markers to distinguish patients with lung cancer from healthy individuals. Additionally, we sought to develop a deep learning model incorporating cfDNA methylation and fragment size profiles. To achieve this, we utilized methylation data collected from The Cancer Genome Atlas and Gene Expression Omnibus databases. Then we generated methylated DNA immunoprecipitation sequencing and genome-wide Enzymatic Methyl-seq (EM-seq) form lung cancer tissue and plasma. Using these data, we selected 366 methylation markers. A targeted EM-seq panel was designed using the selected markers, and 142 lung cancer and 56 healthy samples were produced with the panel. Additionally, cfDNA samples from healthy individuals and lung cancer patients were diluted to evaluate sensitivity. Its lung cancer detection performance reached an accuracy of 81.5% and an area under the receiver operating characteristic curve of 0.87. In the serial dilution experiment, we achieved tumor fraction detection of 1% at 98% specificity and 0.1% at 80% specificity. In conclusion, we successfully developed and validated a combination of methylation panel and a deep learning model that can distinguish between patients with lung cancer and healthy individuals.
Topics: Humans; Lung Neoplasms; DNA Methylation; Deep Learning; Biomarkers, Tumor; Female; Male; Middle Aged; Aged; Cell-Free Nucleic Acids; ROC Curve
PubMed: 38926407
DOI: 10.1038/s41598-024-63411-2 -
Cell Genomics Jun 2024Single-cell RNA sequencing (scRNA-seq) datasets contain true single cells, or singlets, in addition to cells that coalesce during the protocol, or doublets. Identifying...
Single-cell RNA sequencing (scRNA-seq) datasets contain true single cells, or singlets, in addition to cells that coalesce during the protocol, or doublets. Identifying singlets with high fidelity in scRNA-seq is necessary to avoid false negative and false positive discoveries. Although several methodologies have been proposed, they are typically tested on highly heterogeneous datasets and lack a priori knowledge of true singlets. Here, we leveraged datasets with synthetically introduced DNA barcodes for a hitherto unexplored application: to extract ground-truth singlets. We demonstrated the feasibility of our framework, "singletCode," to evaluate existing doublet detection methods across a range of contexts. We also leveraged our ground-truth singlets to train a proof-of-concept machine learning classifier, which outperformed other doublet detection algorithms. Our integrative framework can identify ground-truth singlets and enable robust doublet detection in non-barcoded datasets.
PubMed: 38925122
DOI: 10.1016/j.xgen.2024.100592 -
Microbial Biotechnology Jun 2024Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the...
Beauveria bassiana is an entomopathognic fungus, which is widely employed in the biological control of pests. Gene disruption is a common method for studying the functions of genes involved in fungal development or its interactions with hosts. However, generating gene deletion mutants was a time-consuming work. The transcriptional factor OpS3 has been identified as a positive regulator of a red secondary metabolite oosporein in B. bassiana. In this study, we have designed a new screening system by integrating a constitutive OpS3 expression cassette outside one of the homologous arms of target gene. Ectopic transformants predominantly exhibit a red colour with oosporein production, while knockout mutants appear as white colonies due to the loss of the OpS3 expression cassette caused by recombinant events. This screening strategy was used to obtain the deletion mutants of both tenS and NRPS genes. Correct mutants were obtained by screening fewer than 10 mutants with a positive efficiency ranging from 50% to 75%. This system significantly reduces the workload associated with DNA extraction and PCR amplification, thereby enhancing the efficiency of obtaining correct transformants in B. bassiana.
Topics: Beauveria; Gene Knockout Techniques; Animals; Gene Deletion; Fungal Proteins; Insecta; Genetics, Microbial
PubMed: 38923821
DOI: 10.1111/1751-7915.14512 -
The Kaohsiung Journal of Medical... Jun 2024Thyroid dyshormonogenesis (TDH) is responsible for 15%-25% of congenital hypothyroidism (CH) cases. Pathogenetic variants of this common inherited endocrine disorders...
Thyroid dyshormonogenesis (TDH) is responsible for 15%-25% of congenital hypothyroidism (CH) cases. Pathogenetic variants of this common inherited endocrine disorders vary geographically. Unraveling the genetic underpinnings of TDH is essential for genetic counseling and precise therapeutic strategies. This study aims to identify genetic variants associated with TDH in Southern Taiwan using whole exome sequencing (WES). We included CH patients diagnosed through newborn screening at a tertiary medical center from 2011 to 2022. Permanent TDH was determined based on imaging evidence of bilateral thyroid structure and the requirement for continuous medication beyond 3 years of age. Genomic DNA extracted from blood was used for exome library construction, and pathogenic variants were detected using an in-house algorithm. Of the 876 CH patients reviewed, 121 were classified as permanent, with 47 (40%) confirmed as TDH. WES was conducted for 45 patients, and causative variants were identified in 32 patients (71.1%), including DUOX2 (15 cases), TG (8 cases), TSHR (7 cases), TPO (5 cases), and DUOXA2 (1 case). Recurrent variants included DUOX2 c.3329G>A, TSHR c.1349G>A, TG c.1348delT, and TPO c.2268dupT. We identified four novel variants based on genotype, including TSHR c.1135C>T, TSHR c.1349G>C, TG c.2461delA, and TG c.2459T>A. This study underscores the efficacy of WES in providing definitive molecular diagnoses for TDH. Molecular diagnoses are instrumental in genetic counseling, formulating treatment, and developing management strategies. Future research integrating larger population cohorts is vital to further elucidate the genetic landscape of TDH.
PubMed: 38923290
DOI: 10.1002/kjm2.12871 -
Methods and Protocols Jun 2024Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production,...
Timely and accurate detection and characterization of microbial threats is crucial for effective infection and outbreak management. Additionally, in food production, rapid microbe identification is indispensable for maintaining quality control and hygiene standards. Current methods for typing microbial strains often rely on labor-intensive, time-consuming, and expensive DNA- and sera-serotyping techniques, limiting their applicability in rapid-response scenarios. In this context, the IR Biotyper, utilizing Fourier-transform infrared (FTIR) spectroscopy, offers a novel approach, providing specific spectra for fast strain typing within 3 h. This methodology article serves as a comprehensive resource for researchers and technicians aiming to utilize FTIR spectroscopy for microbial strain typing. It encompasses detailed guidelines on sample preparation, data acquisition, and analysis techniques, ensuring the generation of reliable and reproducible results. We highlight the IR Biotyper's rapid and accurate discrimination capabilities, showcasing its potential for real-time pathogen monitoring and source-tracking to enhance public health and food safety. We propose its integration as an early screening method, followed by more detailed analysis with whole-genome sequencing, to optimize detection accuracy and response efficiency in microbial surveillance systems.
PubMed: 38921827
DOI: 10.3390/mps7030048 -
Pediatric Reports May 2024Leukocytosis in neonates can occur because of infectious, inflammatory, malignant, or physiological processes. Hyperleukocytosis is defined as a total leukocyte count...
Leukocytosis in neonates can occur because of infectious, inflammatory, malignant, or physiological processes. Hyperleukocytosis is defined as a total leukocyte count (TLC) exceeding 100,000 per mm, warranting immediate evaluation. Neonates with hyperleukocytosis are at risk of leukostasis and the associated severe complications, including respiratory distress, myocardial ischemia, hyperuricemia, acute renal failure, infarction, and hemorrhage. Differentiating leukemia and leukemoid reactions in neonates presenting with elevated TLC is challenging but critical. We present a unique case of a preterm male neonate with hyperleukocytosis, initially suspected to have an underlying malignancy. The neonate's clinical course was complicated by respiratory distress syndrome and anemia of prematurity, necessitating neonatal intensive care unit management. Further investigation revealed high human herpesvirus 6 (HHV-6) DNA levels in the whole blood, leading to a chromosomally integrated HHV-6 (ciHHV-6) diagnosis. CiHHV-6 is characterized by HHV-6 DNA integration into the host genome. Accurate diagnosis relies on whole-blood quantitative PCR, distinguishing ciHHV-6 from an active infection. The neonate remained asymptomatic, and antiviral treatment was deemed unnecessary. This case underscores the importance of recognizing ciHHV-6 as a potential cause of hyperleukocytosis in neonates and highlights the value of whole-blood PCR for differentiation. Understanding the spectrum of HHV-6 infection in neonates is vital for appropriate management and prognostication.
PubMed: 38921702
DOI: 10.3390/pediatric16020037