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International Journal of Molecular... Jun 2024This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of... (Review)
Review
This review covers the analytical applications of protein partitioning in aqueous two-phase systems (ATPSs). We review the advancements in the analytical application of protein partitioning in ATPSs that have been achieved over the last two decades. Multiple examples of different applications, such as the quality control of recombinant proteins, analysis of protein misfolding, characterization of structural changes as small as a single-point mutation, conformational changes upon binding of different ligands, detection of protein-protein interactions, and analysis of structurally different isoforms of a protein are presented. The new approach to discovering new drugs for a known target (e.g., a receptor) is described when one or more previous drugs are already available with well-characterized biological efficacy profiles.
Topics: Water; Proteins; Protein Folding; Humans; Protein Binding; Protein Conformation; Ligands; Recombinant Proteins
PubMed: 38928046
DOI: 10.3390/ijms25126339 -
International Journal of Molecular... Jun 2024We analyzed the thermal stability of the HPr protein through the site-directed point mutation Lys62 replaced by Ala residue using molecular dynamics simulations at five...
We analyzed the thermal stability of the HPr protein through the site-directed point mutation Lys62 replaced by Ala residue using molecular dynamics simulations at five different temperatures: 298, 333, 362, 400, and 450 K, for periods of 1 μs and in triplicate. The results from the mutant thermophilic HPrm protein were compared with those of the wild-type thermophilic HPr protein and the mesophilic HPr protein. Structural and molecular interaction analyses show that proteins lose stability as temperature increases. Mutant and wild-type proteins behave similarly up to 362 K. However, at 400 K the mutant protein shows greater structural instability, losing more buried hydrogen bonds and exposing more of its non-polar residues to the solvent. Therefore, in this study, we confirmed that the salt bridge network of the Glu3-Lys62-Glu36 triad, made up of the Glu3-Lys62 and Glu36-Lys62 ion pairs, provides thermal stability to the thermophilic HPr protein.
Topics: Molecular Dynamics Simulation; Protein Stability; Hydrogen Bonding; Temperature; Mutation; Bacterial Proteins; Amino Acid Substitution; Protein Conformation; Mutagenesis, Site-Directed
PubMed: 38928023
DOI: 10.3390/ijms25126316 -
Cancers Jun 2024The PI3K/AKT/mTOR signalling pathway is one of the most frequently activated pathways in breast cancer and also plays a central role in the regulation of several... (Review)
Review
The PI3K/AKT/mTOR signalling pathway is one of the most frequently activated pathways in breast cancer and also plays a central role in the regulation of several physiologic functions. There are major efforts ongoing to exploit precision medicine by developing inhibitors that target the three kinases (PI3K, AKT, and mTOR). Although multiple compounds have been developed, at present, there are just three inhibitors approved to target this pathway in patients with advanced ER-positive, HER2-negative breast cancer: everolimus (mTOR inhibitor), alpelisib (PIK3CA inhibitor), and capivasertib (AKT inhibitor). Like most targeted cancer drugs, resistance poses a major problem in the clinical setting and is a factor that has frequently limited the overall efficacy of these agents. Drug resistance can be categorised into intrinsic or acquired resistance depending on the timeframe it has developed within. Whereas intrinsic resistance exists prior to a specific treatment, acquired resistance is induced by a therapy. The majority of patients with ER-positive, HER2-negative advanced breast cancer will likely be offered an inhibitor of the PI3K/AKT/mTOR pathway at some point in their cancer journey, with the options available depending on the approval criteria in place and the cancer's mutation status. Within this large cohort of patients, it is likely that most will develop resistance at some point, which makes this an area of interest and an unmet need at present. Herein, we review the common mechanisms of resistance to agents that target the PI3K/AKT/mTOR signalling pathway, elaborate on current management approaches, and discuss ongoing clinical trials attempting to mitigate this significant issue. We highlight the need for additional studies into AKT1 inhibitor resistance in particular.
PubMed: 38927964
DOI: 10.3390/cancers16122259 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To analyze thalassemia genotypes and distribution of children in Wuzhou Guangxi, and evaluate the diagnostic value of HbA2 in children's thalassemia screening, so as to...
OBJECTIVE
To analyze thalassemia genotypes and distribution of children in Wuzhou Guangxi, and evaluate the diagnostic value of HbA2 in children's thalassemia screening, so as to provide scientific evidence for the prevention and control strategies of thalassemia.
METHODS
Four hundred and fifty-eight children suspected with thalassemia in Wuzhou were enrolled from March 2017 to June 2022. The level of HbA2 was detected using Bio-Rad VARIANT II Hb analysis system. The deletion of α-thalassemia was measured with gap-PCR assay, and the point mutation of α- and β-thalassemia was tested with DNA reverse dot blot hybridization assay. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic value of HbA2 for children's thalassemia.
RESULTS
A total of 304 thalassemia carriers were detected in 458 children, accounting for 66.38%. One hundred and seventy-five cases were defined to be α-thalassemia, with the main type of --/αα (54.86%). Thirty-six cases were defined to be intermediate α-thalassemia, with the main type of -α/-- (9.72%). In 108 cases with β-thalassemia, / was the main type, accounting for 49.07%, followed by / (14.81%). Seven cases were moderate/severe β-thalassemia (predominantly / and //). Twenty-one genotypes of α- and β-thalassemia were found in the children. There was significant difference of HbA2 level between the children with different types of thalassemia and healthy controls (all < 0.001). ROC curve analysis showed that the sensitivities of HbA2 for α-thalassemia, β-thalassemia and αβ-thalassemia were 74.3%, 82.4% and 85.7%, with the optimal cut-off values of 2.60%, 3.60% and 3.70%, respectively, the specificities were 64.3%, 96.1% and 96.8%, and the area under the curve were 0.690, 0.887 and 0.916, respectively.
CONCLUSION
The thalassemia genotypes of children in Wuzhou are diverse. It is necessary to further strengthen the prevention and control measure of thalassemia to reduce birth defects and improve birth quality.
Topics: Humans; Genotype; China; Child; alpha-Thalassemia; beta-Thalassemia; Hemoglobin A2; Point Mutation; Male
PubMed: 38926975
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.027 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To investigate gene mutation characteristics of primary central nervous system lymphoma (PCNSL) through whole exome sequencing (WES) to 18 patients with PCNSL.
OBJECTIVE
To investigate gene mutation characteristics of primary central nervous system lymphoma (PCNSL) through whole exome sequencing (WES) to 18 patients with PCNSL.
METHODS
Tumor tissues from 18 patients with diffuse large B-cell lymphoma who were diagnosed with PCNSL in Department of Hematology, Lanzhou University Second Hospital from September 2018 to December 2020 and had normal immune function, no history of HIV or immunosuppressant therapy were collected. High-throughput-based WES was performed on the tumor tissues, with an average sequencing depth of >100×. After data processing and bioinformatics analysis of sequencing results, the mutation maps and mutation characteristics of 18 PCNSL patients were obtained.
RESULTS
Obvious somatic mutations were detected in all 18 patients. The median number of somatic mutations was 321. Missense mutations were most prominent (accounting for about 90%), and the mutation type was dominated by C>T (50.2%), reflecting the age-related mutation pattern. Among the top 15 frequently mutated genes, , and were the driver genes. The enrichment results of driver gene pathways showed that RTK-RAS, Wnt, NOTCH, Hippo and Cell-Cycle pathways were significantly enriched. The tumor mutation burden was between 3.558 48/Mb and 8.780 89/Mb, and the average was 4.953 32/Mb, which was significantly higher than other cancer research cohorts in the TCGA database.
CONCLUSIONS
PCNSL occurs somatic missense mutations frequently, mainly point mutations, and the mutation type is mainly C>T. The driver genes are mainly involved in RTK-RAS, Wnt, NOTCH and Hippo pathways, indicating that the above pathways may be related to the pathogenesis of PCNSL. PCNSL has a significantly high tumor mutation burden, which may explain the efficacy of PD-1 inhibitors in PCNSL.
Topics: Humans; Central Nervous System Neoplasms; Exome Sequencing; Mutation; Lymphoma, Large B-Cell, Diffuse; Mutation, Missense
PubMed: 38926963
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.015 -
Anticancer Research Jul 2024Genomic examination of tumor tissue has been clinically accepted, and the identification of actionable mutations for molecular-targeted therapy may provide substantial...
BACKGROUND/AIM
Genomic examination of tumor tissue has been clinically accepted, and the identification of actionable mutations for molecular-targeted therapy may provide substantial survival benefit for patients with advanced malignancies.
CASE REPORT
A female patient in her 60s showed a stenosis of the afferent loop of the small intestine because of circumferential metastatic tumor 14 months after curative surgery for hilar cholangiocarcinoma. Chemotherapy with gemcitabine plus cisplatin was administered for 18 months. An oncopanel examination was performed during chemotherapy, and a high tumor mutation burden was revealed. At 38 months after surgery, a new recurrent tumor, 2.7 cm in size, was observed in the abdominal wall, which was histologically proven to be metastatic adenocarcinoma. Atezolizumab was administered. After three cycles of treatment, treatment was switched to pembrolizumab because of its acceptance by healthcare insurance. The recurrent tumors in the abdominal wall and small intestine disappeared 6 months after the administration of immune checkpoint inhibitor, and the patient has continued pembrolizumab, surviving for 76 months after surgery without any clinical evidence of tumor.
CONCLUSION
Immune checkpoint blockade successfully prolonged the survival of a patient with advanced hilar cholangiocarcinoma with high tumor mutation burden, although the optimal number of mutations for such a successful response needs to be clarified.
Topics: Humans; Female; Mutation; Immune Checkpoint Inhibitors; Bile Duct Neoplasms; Cholangiocarcinoma; Middle Aged; Antibodies, Monoclonal, Humanized; Antineoplastic Combined Chemotherapy Protocols; Treatment Outcome
PubMed: 38925819
DOI: 10.21873/anticanres.17135 -
ACS Nano Jun 2024CRISPR/Cas systems have been widely employed for nucleic acid biosensing and have been further advanced for mutation detection by virtue of the sequence specificity of...
CRISPR/Cas systems have been widely employed for nucleic acid biosensing and have been further advanced for mutation detection by virtue of the sequence specificity of crRNA. However, existing CRISPR-based genotyping methods are limited by the mismatch tolerance of Cas effectors, necessitating a comprehensive screening of crRNAs to effectively distinguish between wild-type and point-mutated sequences. To circumvent the limitation of conventional CRISPR-based genotyping, here, we introduce Single-Molecule kinetic Analysis via a Real-Time digital CRISPR/Cas12a-assisted assay (SMART-dCRISPR). SMART-dCRISPR leverages the differential kinetics of the signal increase in CRISPR/Cas systems, which is modulated by the complementarity between crRNA and the target sequence. It employs single-molecule digital measurements to discern mutations based on kinetic profiles that could otherwise be obscured by variations in the target concentrations. We applied SMART-dCRISPR to genotype notable mutations in SARS-CoV-2, point mutation (K417N) and deletion (69/70DEL), successfully distinguishing wild-type, Omicron BA.1, and Omicron BA.2 SARS-CoV-2 strains from clinical nasopharyngeal/nasal swab samples. Additionally, we introduced a portable digital real-time sensing device to streamline SMART-dCRISPR and enhance its practicality for point-of-care settings. The combination of a rapid and sensitive isothermal CRISPR-based assay with single-molecule kinetic analysis in a portable format significantly enhances the versatility of CRISPR-based nucleic acid biosensing and genotyping.
PubMed: 38922290
DOI: 10.1021/acsnano.4c05312 -
Analytical Chemistry Jun 2024Detection of circulating tumor DNA (ctDNA) in liquid biopsy is of great importance for tumor diagnosis but difficult due to its low amount in bodily fluids. Herein, a...
Detection of circulating tumor DNA (ctDNA) in liquid biopsy is of great importance for tumor diagnosis but difficult due to its low amount in bodily fluids. Herein, a novel ctDNA detection platform is established by quantifying DNA amplification by-product pyrophosphate (PPi) using a newly designed bivariable lanthanide metal-organic framework (Ln-MOF), namely, Ce/Eu-DPA MOF (CE-24, DPA = pyridine-2,6-dicarboxylic acid). CE-24 MOF exhibits ultrafast dual-response (fluorescence enhancement and enzyme-activity inhibition) to PPi stimuli by virtue of host-guest interaction. The platform is applied to detecting colon carcinoma-related ctDNA (KARS G12D mutation) combined with the isothermal nucleic acid exponential amplification reaction (EXPAR). ctDNA triggers the generation of a large amount of PPi, and the ctDNA quantification is achieved through the ratio fluorescence/colorimetric dual-mode assay of PPi. The combination of the EXPAR and the dual-mode PPi sensing allows the ctDNA assay method to be low-cost, convenient, bioreaction-compatible (freedom from the interference of bioreaction systems), sensitive (limit of detection down to 10 fM), and suitable for on-site detection. To the best of our knowledge, this work is the first application of Ln-MOF for ctDNA detection, and it provides a novel universal strategy for the rapid detection of nucleic acid biomarkers in point-of-care scenarios.
PubMed: 38922180
DOI: 10.1021/acs.analchem.4c01207 -
The Journal of General Virology Jun 2024Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family , genus and is transmitted by biting midges. Infection of naïve ruminants in a critical...
Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family , genus and is transmitted by biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts . It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.
Topics: Animals; Orthobunyavirus; Bunyaviridae Infections; Ceratopogonidae; Genome, Viral; Cricetinae; Cell Line; Virus Replication; Point Mutation; Cattle; Sheep; Phylogeny; RNA, Viral
PubMed: 38921821
DOI: 10.1099/jgv.0.002005 -
Current Oncology (Toronto, Ont.) Jun 2024Small cell bladder cancer (SCBC) is a rare and aggressive disease, often treated with platinum/etoposide-based chemotherapy. Key molecular drivers include the...
Small cell bladder cancer (SCBC) is a rare and aggressive disease, often treated with platinum/etoposide-based chemotherapy. Key molecular drivers include the inactivation of onco-suppressor genes (, ) and amplifications in proto-oncogenes (). We report a patient with SCBC who achieved an objective and prolonged response to lurbinectedin, which has been approved for metastatic small cell lung cancer, after developing disease progression on cisplatin/etoposide and nivolumab/ipilimumab. A genomic analysis of a metastatic biopsy prior to lurbinectedin initiation revealed a mutation and amplification of the cell cycle regulators and . A repeat biopsy following the development of lurbinectedin resistance showed a new actionable ERBB2 alteration without significant change in the tumor mutation burden (six mutations/Mb). The present report suggests that lurbinectedin may be active and should be further explored in SCBC harboring mutations and amplifications in E2F3 and MYC family complexes.
Topics: Humans; Carbolines; Urinary Bladder Neoplasms; Heterocyclic Compounds, 4 or More Rings; Mutation; Tumor Suppressor Protein p53; Male; Carcinoma, Small Cell; Heterocyclic Compounds, 3-Ring; Antineoplastic Agents; Middle Aged
PubMed: 38920737
DOI: 10.3390/curroncol31060254