Specific Sequences Research Articles

Dysregulated expression of miR-140 and miR-122 compromised microglial chemotaxis and led to reduced restriction of AD pathology

BackgroundDeposition of amyloid β, which is produced by amyloidogenic cleavage of APP by β- and γ-secretase, is one of the primary hallmarks of AD pathology. APP can also be processed by α- and γ-secretase sequentially, to generate sAPPα, which has been shown to be neuroprotective by promoting neurite outgrowth and neuronal survival, etc.MethodsThe global expression profiles of miRNA in blood plasma samples taken from 11 AD patients as well as from 14 age and sex matched cognitively normal volunteers were analyzed using miRNA-seq. Then, overexpressed miR-140 and miR-122 both in vivo and in vitro, and knock-down of the endogenous expression of miR-140 and miR-122 in vitro. Used a combination of techniques, including molecular biology, immunohistochemistry, to detect the impact of miRNAs on AD pathology.ResultsIn this study, we identified that two miRNAs, miR-140-3p and miR-122-5p, both targeting ADAM10, the main α-secretase in CNS, were upregulated in the blood plasma of AD patients. Overexpression of these two miRNAs in mouse brains induced cognitive decline in wild type C57BL/6J mice as well as exacerbated dyscognition in APP/PS1 mice. Although significant changes in APP and total Aβ were not detected, significantly downregulated ADAM10 and its non-amyloidogenic product, sAPPα, were observed in the mouse brains overexpressing miR-140/miR-122. Immunohistology analysis revealed increased neurite dystrophy that correlated with the reduced microglial chemotaxis in the hippocampi of these mice, independent of the other two ADAM10 substrates (neuronal CX3CL1 and microglial TREM2) that were involved in regulating the microglial immunoactivity. Further in vitro analysis demonstrated that both the reduced neuritic outgrowth of mouse embryonic neuronal cells overexpressing miR-140/miR-122 and the reduced Aβ phagocytosis in microglia cells co-cultured with HT22 cells overexpressing miR-140/miR-122 could be rescued by overexpressing the specific inhibitory sequence of miR-140/miR-122 TuD as well as by addition of sAPPα, rendering these miRNAs as potential therapeutic targets.ConclusionsOur results suggested that neuroprotective sAPPα was a key player in the neuropathological progression induced by dysregulated expression of miR-140 and miR-122. Targeting these miRNAs might serve as a promising therapeutic strategy in AD treatment.

The Role of Diffusion-weighted Magnetic Resonance as a Potential Biomarker in the Characterization of Ovarian Tumor Lesions and the Assessment of Post-treatment Evolution

This paper focuses on the role of diffusion-weighted MRI of ovarian tumors as a potential biomarker to detect and characterize malignancy, and to improve the accuracy of preoperative diagnosis and postoperative follow-up to optimize therapeutic management. This is an original retrospective study of 76 patients with ovarian masses who underwent surgery between May 2014 and March 2016 at the "Dr Carol Davila" Central Military University Gynaecology Department. Patients underwent preoperative, 6-month, and 12-month postoperative evaluation of ovarian tumors using diffusion-weighted MRI. Due to the accuracy of this method using specific sequences in tissue characterization, magnetic resonance imaging can be used in the diagnostic work-up of complex adnexal masses.

Application of metagenomic next-generation sequencing for rapid molecular identification in spinal infection diagnosis.

This study aimed to determine the sensitivity and specificity of metagenomic next-generation sequencing (mNGS) for detecting pathogens in spinal infections and to identify the differences in the diagnostic performance between mNGS and targeted next-generation sequencing (tNGS). A total of 76 consecutive patients with suspected spinal infections who underwent mNGS, culture, and histopathological examinations were retrospectively studied. The final diagnosis of the patient was determined by combining the clinical treatment results, pathological examinations, imaging changes and laboratory indicators. The sensitivity and specificity of mNGS and culture were determined. The difference between the two detection rates was statistically significant (p < 0.001), with mNGS exhibiting a significantly higher detection rate (77.6% versus 18.4%). The average diagnosis time of mNGS was significantly shorter than that of bacterial culture (p < 0.001, 1.65 versus 3.07 days). The sensitivity and accuracy of mNGS were significantly higher than that of the culture group (p < 0.001, 82.3% versus 17.5%; 75% versus 27.6%), whereas the specificity of mNGS (42.9%) was lower than that of the culture group (p > 0.05, 42.9% versus 76.9%). The sensitivity, specificity, accuracy, and positive predictive value (PPV) of pus were higher than those of tissue samples for mNGS, whereas for culture, the sensitivity, specificity, accuracy, and PPV of tissue samples were higher than those of pus. tNGS demonstrated higher sensitivity and accuracy in diagnosing tuberculosis (TB) than mNGS (80% versus 50%; 87.5% versus 68.8%). mNGS for spinal infection demonstrated better diagnostic value in developing an antibiotic regimen earlier, and it is recommended to prioritize pus samples for testing through mNGS. Moreover, tNGS outperformed other methods for diagnosing spinal TB and identifying antibiotic-resistance genes in drug-resistant TB.

Products and patterns through which adolescents, young adults, and adults initiate co-use of tobacco and cannabis

BackgroundCo-use of tobacco and cannabis is common. However, few studies have examined the temporal sequencing through which individuals initiate co-use, and how these patterns vary across age. This study addresses this gap by examining the specific products and temporal sequencing through which adolescents, young adults, and adults initiate co-use of tobacco and cannabis. Among adolescents, young adults, and adults who co-used tobacco and cannabis in the past 30 days, we examined (a) whether tobacco or cannabis was used first in their lifetime and (b) which specific tobacco or cannabis product (e.g., nicotine e-cigarette, cannabis edible) was the first used. MethodsData were from a cross-sectional national survey (N = 6,131, 13–40 years old) in which participants reported ever use, past-30-day-use, and order of use for 17 different tobacco and cannabis products. Results were analyzed overall and by age group (13–20; 21–24; 25–40). Results38.4 % of participants reported use of both tobacco and cannabis in the past 30 days. Among these participants, 71.4 % used tobacco first in their lifetime (66.6 % < 21; 71.7 % 21–24; 76.6 % > 24). Approximately 60 % of participants who initiated co-use with tobacco reported nicotine e-cigarettes as their first tobacco product (63.3 % < 21; 66.7 % 21–24; 49.6 % > 24). ConclusionsThe majority of participants who used both tobacco and cannabis used tobacco first in their lifetime, and nicotine e-cigarettes were the most common form of tobacco initiation, particularly among adolescents and young adults. Findings emphasize the need for co-use prevention programs to target common products of initiation.

Optimizing hybrid neural networks for precise COVID-19 mRNA vaccine degradation prediction

Conventional hybrid models often miss an essential factor that can lead to less effective performance: intrinsic sequence dependence when combining various neural network (NN) architectures. This study addresses this issue by highlighting the importance of sequence hybridization in NN architecture integration, aiming to improve model effectiveness. It combines NN layers—dense, long short-term memory (LSTM), and gated recurrent unit (GRU)—using the Keras Sequential API for defining the architecture. To provide better context, bidirectional LSTM (BiLSTM) and bidirectional GRU (BiGRU) replace their unidirectional counterparts, enhancing the models through bidirectional structures. Out of 25 NN models tested, 18 four-layer hybrid NN models consist of one-quarter dense layer and the rest BiLSTM and BiGRU layers. These hybrid NN models undergo supervised learning regression analysis, with mean column-wise root mean square error (MCRMSE) as the performance metric. The results show that each hybrid NN model produces unique outcomes based on its specific hybrid sequence. The Hybrid_LGSS model performs better than existing three-layer BiLSTM networks in predictive accuracy and shows lower overfitting (MCRMSEs of 0.0749 and 0.0767 for training and validation, respectively). This indicates that the optimal hybridization sequence is crucial for achieving a balance between performance and simplicity. In summary, this research could help vaccinologists develop better mRNA vaccines and provide data analysts with new insights for improvement.

DNA sensing based on aggregation of Janus particles using dynamic light scattering

BackgroundThe aggregation of isotropic particles through interparticle reactions poses a challenge in control due to the ability of all surfaces to bind to each other, rendering the quantitative detection of such interparticle reactions based on particle size difficult. Here, we proposed a novel detection scheme for DNA utilizing an assembly of Janus particles (JPs) employing dynamic light scattering (DLS). DNA molecules are tethered on one hemisphere of the JP, while the other hemisphere retains its hydrophobic properties. ResultsAggregation of JPs was induced by the sandwich hybridization of target DNA between them. The assembly of JPs was effectively monitored by the changes in hydrodynamic diameter detected by DLS, revealing that aggregation peaks at 2–3 particles and further reaction was hindered due to the inability of one hemisphere of the JP to interact with another JP. The target DNA demonstrated detectability at concentrations as low as several tens of pM to several nM using a digital sensing method. The two types of target DNA, such as simple (14 base pairs) and HIV-2 specific sequences (20 base pairs) were detectable at nM and pM levels, respectively. Moreover, we substantiated the robustness of our detection scheme through stoichiometric calculations based on an equilibrium model. The present detection mechanism was well explained based on the binding affinity of DNA hybridization. SignificanceThis detection method harnesses the anisotropic nature of JPs and represents the first detection approach based on aggregation. By altering the modification molecules on JPs to match target molecules, such as proteins and organic compounds, a wide range of versatile molecules can be detected using this scheme with high sensitivity. This underscores the broad applicability of the present method.

Gene Editing Technologies: CRISPR/Cas9 and Beyond for Genetic Disease Therapy and Research

Gene editing technologies have revolutionized the field of genetic disease therapy and research. Among these, CRISPR/Cas9 stands out as a versatile tool for precisely targeting and modifying specific sequences within the genome. This paper provides an overview of CRISPR/Cas9 and other emerging gene editing technologies, discussing their potential applications in treating genetic diseases and advancing scientific research. Additionally, ethical considerations and challenges associated with gene editing are explored, along with future directions for this rapidly evolving field. Gene editing technologies have emerged as powerful tools in the field of genetic disease therapy and research, offering unprecedented precision and versatility in manipulating the genome. Among these technologies, CRISPR/Cas9 has garnered significant attention for its simplicity, efficiency, and accuracy in targeted gene modification.

Short Oligourea Foldamers as N‐ or C‐Caps for Promoting α‐Helix Formation in Water

While foldamers have been extensively studied as protein mimics and especially as α‐helix mimics, their use as capping motif to enhance α‐helix propensity remains comparatively much limited. In this study, we leverage the structural similarities between urea‐based helical foldamers and α‐helix to investigate the efficacy of oligoureas as N‐ or C‐caps for reinforcing α‐helical structures in water. Short oligoureas, comprising 3 to 4 residues, were strategically introduced at the N‐ or C‐terminus of two peptide sequences (S‐peptide and an Ala‐rich model sequence). The impact of these foldamer insertions on peptide conformation was examined using electronic circular dichroism (ECD) and solution NMR. This research identifies specific foldamer sequences capable of promoting a‐helicity when incorporated at either terminus of the peptides. Not only does this work broaden the application scope of foldamers, but it also provides valuable insights into novel strategies for modulating peptide conformation in aqueous environments. The findings presented in this study may have implications for peptide design and the development of bioactive foldamer‐based peptide mimics.

Peptide nucleic acids (PNAs) control function of SARS-CoV-2 frameshifting stimulatory element trough PNA-RNA-PNA triplex formation

The highly structured nature of the SARS-CoV-2 genome provides many promising antiviral drug targets. One particularly promising target is a cis-acting RNA pseudoknot found within a critical region called the frameshifting stimulatory element (FSE). In this study, peptide nucleic acids (PNAs) binding to stem 2 of FSE RNA inhibited protein translation and frameshifting, as measured by a cell-free dual luciferase assay, more effectively than PNAs binding to stem 1, stem 3, or the slippery site. Surprisingly, simple antisense PNAs were stronger disruptors of frameshifting than PNA tail-clamps, despite higher thermal stability of the PNA-RNA-PNA triplexes formed by the latter. Another unexpected result was a strong and sequence non-specific enhancement of frameshifting inhibition when using a cationic triplex-forming PNA in conjunction with an antisense PNA targeting key regions of the frameshifting element. Our results illustrate both the potential and the challenges of using antisense PNAs to target highly structured RNAs, such as SARS-CoV-2 pseudoknots. While triplex forming PNAs, including PNA tail-clamps, are emerging as promising ligands for RNA recognition, the binding affinity enhancements when using cationic modifications in triplex-forming PNAs must be carefully balanced to avoid loss of sequence specificity in complex biological systems.

MRI radiomics enhances radiologists’ ability for characterizing intestinal fibrosis in patients with Crohn’s disease

ObjectivesWe aimed to develop MRI-based radiomic models (RMs) to improve the diagnostic accuracy of radiologists in characterizing intestinal fibrosis in patients with Crohn’s disease (CD).MethodsThis retrospective study included patients with refractory CD who underwent MR before surgery from November 2013 to September 2021. Resected bowel segments were histologically classified as none-mild or moderate-severe fibrosis. RMs based on different MR sequence combinations (RM1: T2WI and enhanced-T1WI; RM2: T2WI, enhanced-T1WI, diffusion-weighted imaging [DWI], and apparent diffusion coefficient [ADC]); RM3: T2WI, enhanced-T1WI, DWI, ADC, and magnetization transfer MRI [MTI]), were developed and validated in an independent test cohort. The RMs’ diagnostic performance was compared to that of visual interpretation using identical sequences and a clinical model.ResultsThe final population included 123 patients (81 men, 42 women; mean age: 30.26 ± 7.98 years; training cohort, n = 93; test cohort, n = 30). The area under the receiver operating characteristic curve (AUC) of RM1, RM2, and RM3 was 0.86 (p = 0.001), 0.88 (p = 0.001), and 0.93 (p = 0.02), respectively. The decision curve analysis confirmed a progressive improvement in the diagnostic performance of three RMs with the addition of more specific sequences. All RMs performance surpassed the visual interpretation based on the same MR sequences (visual model 1, AUC = 0.65, p = 0.56; visual model 2, AUC = 0.63, p = 0.04; visual model 3, AUC = 0.77, p = 0.002), as well as the clinical model composed of C-reactive protein and erythrocyte sedimentation rate (AUC = 0.60, p = 0.13).ConclusionsThe RMs, utilizing various combinations of conventional, DWI and MTI sequences, significantly enhance radiologists’ ability to accurately characterize intestinal fibrosis in patients with CD.Critical relevance statementThe utilization of MRI-based RMs significantly enhances the diagnostic accuracy of radiologists in characterizing intestinal fibrosis.Key PointsMRI-based RMs can characterize CD intestinal fibrosis using conventional, diffusion, and MTI sequences.The RMs achieved AUCs of 0.86–0.93 for assessing fibrosis grade.MRI-radiomics outperformed visual interpretation for grading CD intestinal fibrosis.Graphical

Reference-free structural variant detection in microbiomes via long-read co-assembly graphs.

The study of bacterial genome dynamics is vital for understanding the mechanisms underlying microbial adaptation, growth, and their impact on host phenotype. Structural variants (SVs), genomic alterations of 50 base pairs or more, play a pivotal role in driving evolutionary processes and maintaining genomic heterogeneity within bacterial populations. While SV detection in isolate genomes is relatively straightforward, metagenomes present broader challenges due to the absence of clear reference genomes and the presence of mixed strains. In response, our proposed method rhea, forgoes reference genomes and metagenome-assembled genomes (MAGs) by encompassing all metagenomic samples in a series (time or other metric) into a single co-assembly graph. The log fold change in graph coverage between successive samples is then calculated to call SVs that are thriving or declining. We show rhea to outperform existing methods for SV and horizontal gene transfer (HGT) detection in two simulated mock metagenomes, particularly as the simulated reads diverge from reference genomes and an increase in strain diversity is incorporated. We additionally demonstrate use cases for rhea on series metagenomic data of environmental and fermented food microbiomes to detect specific sequence alterations between successive time and temperature samples, suggesting host advantage. Our approach leverages previous work in assembly graph structural and coverage patterns to provide versatility in studying SVs across diverse and poorly characterized microbial communities for more comprehensive insights into microbial gene flux. rhea is open source and available at: https://github.com/treangenlab/rhea.

Conserved Noncoding Cis-Elements Associated with Hibernation Modulate Metabolic and Behavioral Adaptations in Mice.

Our study elucidates functional roles for conserved cis-elements associated with the evolution of mammalian hibernation. Genomic analyses found topologically associated domains (TADs) that disproportionately accumulated convergent genomic changes in hibernators, including the TAD for the Fat Mass & Obesity (Fto) locus. Some hibernation-linked cis-elements in this TAD form regulatory contacts with multiple neighboring genes. Knockout mice for these cis-elements exhibit Fto, Irx3, and Irx5 gene expression changes, impacting hundreds of genes downstream. Profiles of pre-torpor, torpor, and post-torpor phenotypes found distinct roles for each cis-element in metabolic control, while a high caloric diet uncovered different obesogenic effects. One cis-element promoting a lean phenotype influences foraging behaviors throughout life, affecting specific behavioral sequences. Thus, convergent evolution in hibernators pinpoints functional genetic mechanisms of mammalian metabolic control.

Sequence-specific dynamic DNA bending explains mitochondrial TFAM’s dual role in DNA packaging and transcription initiation

Mitochondrial transcription factor A (TFAM) employs DNA bending to package mitochondrial DNA (mtDNA) into nucleoids and recruit mitochondrial RNA polymerase (POLRMT) at specific promoter sites, light strand promoter (LSP) and heavy strand promoter (HSP). Herein, we characterize the conformational dynamics of TFAM on promoter and non-promoter sequences using single-molecule fluorescence resonance energy transfer (smFRET) and single-molecule protein-induced fluorescence enhancement (smPIFE) methods. The DNA-TFAM complexes dynamically transition between partially and fully bent DNA conformational states. The bending/unbending transition rates and bending stability are DNA sequence-dependent—LSP forms the most stable fully bent complex and the non-specific sequence the least, which correlates with the lifetimes and affinities of TFAM with these DNA sequences. By quantifying the dynamic nature of the DNA-TFAM complexes, our study provides insights into how TFAM acts as a multifunctional protein through the DNA bending states to achieve sequence specificity and fidelity in mitochondrial transcription while performing mtDNA packaging.

Finding a road less traveled: Combining analysis and intuition to develop novel problem formulations

AbstractResearch SummaryNovel formulations of strategic problems are key to innovation and exploration. Conventional wisdom suggests that intuitive thinking, rather than rational‐analytic thinking, facilitates novel problem formulations. This article proposes that intuitive thinking is insufficient and that novel formulations instead depend on sequencing rational‐analytic and intuitive thinking across two phases of the problem formulation task. Two experiments using samples of strategists in organizations support the importance of analysis followed by intuition when developing novel problem formulations. This article advances the “both‐and” approach to managerial cognition by investigating how harnessing intuition and analysis in combination may lead to a desirable outcome for a managerial task. This approach moves beyond the typical, “either‐or” approach to cognition in past studies, which pit analysis against intuition in achieving desirable outcomes.Managerial SummaryUnique and potentially contrarian theories on problems—novel problem formulations—are key to innovation, exploration, and value creation. Past research indicates that strategists should rely on intuition and gut feeling rather than analysis to boldly develop novel problem formulations. I propose that intuition alone can lead to premature, obvious conclusions. This article instead claims that novel formulations depend on combining analysis and intuition in a specific sequence. Analysis helps identify hidden needs and pain points that allow strategists to think of diverse possibilities for underlying causes. Intuition helps strategists boldly utilize these ambiguous, unstructured symptoms based on subjective beliefs to develop novel problem formulations. Two experiments with real‐world strategists support the arguments.

Competitive Binding of Methylene Blue to Calf Thymus DNA and Carrageenan: Implications for Sensor Development

Carrageenans are sulfated polysaccharides widely used in the food industry due to their excellent thickening, stabilising, and gelling properties. Objective: This study delves into two key aspects: 1) the binding interaction between the cationic dye methylene blue (MB) and carrageenan compared to other polyanions (alginate, starch, and Arabic gum), and 2) the competitive binding behavior of carrageenan and DNA for MB. Methods: Spectroscopy methods were employed to determine the total amount of carrageenan by utilising MB, which creates complexes with carrageenan. The investigation into the competitive binding between polysaccharides and double-stranded DNA modified with screen-printed electrodes (SPEs) (dsDNA/SPE) for methylene blue (MB) involved the addition of varying quantities of polysaccharide to a solution containing 100 µM MB after a 5-minute accumulation period. Subsequently, cyclic voltammograms were directly recorded to analyse the interactions. Results: Strong electrostatic binding between MB and carrageenan was confirmed using various techniques, including UV-vis, fluorescence, and cyclic voltammetry. Moreover, photoluminescence (PL) spectroscopy was more sensitive than Ultraviolet-visible (UV-vis) spectroscopy, detecting carrageenans in a linear range from 0.5-15 ppm, 2-15 ppm, with correlation coefficients 0.998, 0.997, respectively. Notably, the preference for binding increased with increasing sulfate content, following the order kappa &gt; iota &gt; lambda carrageenan, highlighting the crucial role of sulfate groups in complex formation. Additionally, carrageenan exhibited the strongest competitive binding towards MB at low concentrations compared to other tested polyanions, implying its effectiveness in hindering MB-DNA interactions. Conclusions: These findings pave the way for exciting developments in DNA biosensors. The sensitive colorimetric assay holds promise for the rapid and selective determination of carrageenan levels in food products. Moreover, the competitive binding between carrageenan and MB can be harnessed in DNA biosensors by monitoring changes in MB binding due to specific DNA sequences.

The Role of FOXA1 in Human Normal Development and Its Functions in Sex Hormone-Related Cancers.

Transcription factors (TFs) are essential proteins regulating gene expression by binding to specific nucleotide sequences upstream of genes. Among TF families, the forkhead box (FOX) proteins, characterized by a conserved DNA-binding domain, play vital roles in various cellular processes, including cancer. The FOXA subfamily, encompassing FOXA1, FOXA2, and FOXA3, stands out for its pivotal role in mammalian development. FOXA1, initially identified in the liver, exhibits diverse expression across multiple organ tissues and plays a critical role in cell proliferation, differentiation, and tumor development. Its structural composition includes transactivation domains and a DNA-binding domain, facilitating its function as a pioneer factor, which is crucial for chromatin interaction and the recruitment of other transcriptional regulators. The involvement of FOXA1 in sex hormone-related tumors underscores its significance in cancer biology. This review provides an overview of multifaceted roles of FOXA1 in normal development and its implications in the pathogenesis of hormone-related cancers, particularly breast cancer and prostate cancer.

Specific Sequence Motif of Var Gene as Predictor of Malaria Outcome

Abstract: The Var gene family encodes for Plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP1), a protein responsible for malaria pathogenesis. One of the variants, the var D gene, is hypothesized as a predictor of clinical malaria outcomes. The study aimed to investigate the association between the expression of the var D gene and clinical malaria outcomes. Blood spots on filter paper from uncomplicated and severe malaria patients were collected for DNA and RNA extraction. The RNA was reverse-transcribed into cDNA. DNA and cDNA were amplified by Polymerase Chain Reaction (PCR) technique using specific var D primer, and PCR products were electrophorized in 1% agarose. DNA amplification resulted in double bands of approximately 230 bp and 250 bp in uncomplicated and severe malaria samples. However, the cDNA amplification generated a single band of 230 bp from four out of five severe malaria samples. The existence of band solely in severe malaria transcript suggested its involvement in the pathogenesis of severe malaria. In conclusion, the expression of var D gene-specific sequence can be a potential predictor of severe malaria outcomes.Abstrak: Famili gen var mengkode Plasmodium falciparum Erythrocyte Membrane Protein-1 (PfEMP1), suatu protein yang berperan penting dalam proses patogenesis malaria. Salah satu variannya, gen var D, diduga merupakan prediktor gambaran klinis malaria. Penelitian ini bertujuan untuk mengetahui hubungan antara ekspresi gen var D dan gambaran klinis malaria. Sampel berupa tetesan darah pada kertas filter dari pasien malaria tanpa komplikasi dan malaria berat dikumpulkan untuk diekstraksi DNA dan RNAnya. RNA selanjutnya di trankripsi reverse menjadi cDNA. DNA dan cDNA diamplifikasi dengan teknik PCR menggunakan primer spesifik var D dan produk PCR dielektroforesis menggunakan agarosa 1 %. Amplifikasi DNA menghasilkan beragam pita berukuran sekitar 230 bp dan 250 bp pada sampel malaria tanpa komplikasi dan malaria berat, tetapi amplifikasi cDNA hanya menunjukkan satu pita berukuran 230 bp pada 4 dari 5 sampel malaria berat. Keberadaan pita hanya pada sampel malaria berat mengindikasikan peran gen ini dalam patogenesis malaria berat. Disimpulkan bahwa gen var D dapat ditemukan pada sampel malaria tanpa komplikasi dan berat, namun ekpresi gen tersebut dapat menjadi prediktor yang potensial timbulnya manifestasi klinis malaria berat.

Metal ion-complexed DNA probe coupled with CRISPR/Cas12a amplification and AuNPs for sensitive colorimetric assay of metallothionein in fish

The accurate and sensitive detection of metallothionein (MT) is of great significance in the fields of biomedical, toxicological and environmental sciences. In this work, based on the high affinity interaction between MT and the heavy metal ions of Hg2+ and the significant signal amplification capability of Cas12a/crRNA enzyme as well, we report a simple and highly sensitive method for visual detection of MT, a biomarker in fish for heavy metal ion-induced water bio-pollution. The target MT molecules bind Hg2+ in the Hg2+- complexed hairpin DNA probes to unfold the hairpin structure into ssDNAs, which hybridize with the partial dsDNA duplexes via strand displacement to yield specific sequence-containing dsDNAs. Cas12a/crRNA recognizes these specific sequences to activate its enzyme activity to cyclically cleave the ssDNA linkers in the blue colored gold nanoparticle aggregates to transit their color into red to realize visual detection of MT. Owing to the signal amplification by Cas12a/crRNA, as low as 25 nM of MT can be visually detected with naked eye. In addition, our colorimetric detection method has high selectivity for MT against other interference proteins and can detect MT in the livers and kidneys of crucian carps bought from a local supermarket. Moreover, the developed assay overcomes the limitations of conventional MT detection methods in terms of complexity, high cost and low sensitivity and can therefore offer new methods for monitoring water bio-pollutions.

Adaptive attenuation of virulence in hypervirulent carbapenem-resistant Klebsiella pneumoniae.

The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host's ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.