Innovative RPA-PfAgo system for rapid MTHFR C677T polymorphism identification.

This study introduces an efficient RPA-PfAgo detection system for the MTHFR C677T polymorphism, proposing a potential strategy to simplify the genotyping process. By optimizing recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute (PfAgo) nucleases, we achieved DNA amplification at a constant temperature. The assay was fine-tuned through meticulous primer and guide DNA selection, with optimal conditions established at 2.0 µL of MgAc, a reaction temperature of 42 °C, and a 10-minute reaction time for RPA. Further optimization of the PfAgo cleavage assay revealed the ideal concentrations of MnCl2, guide DNA, molecular beacon probes, the PfAgo enzyme, and the RPA product to maximize sensitivity and specificity. Clinical validation of 20 samples showed 100 % concordance with Sanger sequencing, confirming the method's precision. The RPA-PfAgo system is a promising tool for on-site genotyping, with broad applications in personalized medicine and disease prevention.

Recombinase polymerase amplification combined with Pyrococcus furiosus Argonaute for fast Salmonella spp. testing in food safety.

Foodborne illness caused by Salmonella spp. is one of the most prevalent public health problems globally, which have brought immeasurable economic burden and social impact to countries around the world. Neither current nucleic acid amplification detection method nor standard culture method (2-3 days) are suitable for field detection in areas with a heavy burden of Salmonella spp. Here, we developed a highly sensitive and accurate assay for Salmonella spp. detection in less than 40 min. Specifically, the invA gene of Salmonella spp. was amplified by recombinase polymerase amplification (RPA), followed by Pyrococcus furiosus Argonaute (PfAgo)-based target sequence cleavage, which could be observed by a fluorescence reader or the naked eye. The assay offered the lowest detectable concentration of 1.05 × 101 colony forming units/mL (CFU/mL). This assay had strong specificity and high sensitivity for the detection of Salmonella spp. in field samples, which indicated the feasibility of this assay.

Ononin triggers ferroptosis-mediated disruption in the triple negative breast cancer both in vitro and in vivo.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is difficult to treat due to a lack of targeted therapies. In this study, we aimed to investigate whether a natural flavonoid compound called ononin could be effective in treating TNBC by triggering ferroptosis in MDA-MB-231 and 4 T1 cell lines, and MDA-MB-231-xenograft nude mice model. Ononin inhibited TNBC through ferroptosis, which was determined by MTT assay, flow cytometry, RT-PCR, immunofluorescence, transmission electron microscopy, histological analysis, western blot and bioluminescence assay. Our results showed that treatment with ononin led to increased levels of malondialdehyde and reactive oxygen species and decreased activity of superoxide dismutase, which are indicatives of ferroptosis. We also found that ononin downregulated two key markers of ferroptosis, SLC7A11 and Nrf2, at both the transcriptional and translational level. Additionally, the administration of ononin resulted in a notable decrease in tumor size and weight in the mouse model. Furthermore, it was observed to enhance the rate of apoptosis in TNBC cells. Importantly, ononin did not induce any histological changes in the kidney, liver, and heart. Taken together, our findings suggest that ononin could be a promising therapeutic strategy for TNBC, and that it works by disrupting the Nrf2/SLC7A11 axis through ferroptosis. These results are encouraging and may lead to the development of new treatments for this challenging cancer subtype.

Integrated 4D label-free proteomics and data mining to elucidate the effects of thermal processing on crisp grass carp protein profiles.

The crisp grass carp (CGC; Ctenopharyngodon idellus C. et V.), known for its unique texture and flavour, is a culinary delicacy whose quality is significantly influenced by thermal processing. This study employed 4D label-free proteomics and data mining techniques to investigate the proteomic changes in CGC muscle tissue induced by various heating temperatures. CGC samples were subjected to a series of heat treatments at increasing temperatures from 20 °C to 90 °C. Proteins were extracted, digested, and analysed using high-resolution mass spectrometry. The proteomic data were then subjected to extensive bioinformatics analysis, including GO and KEGG pathway enrichment. We identified a total of 1085 proteins, 516 of which were shared across all the temperature treatments, indicating a core proteome responsible for CGC textural properties. Differential expression analysis revealed temperature-dependent changes, with significant alterations observed at 90 °C, suggesting denaturation or aggregation of proteins at higher temperatures. Functional enrichment analysis indicated that proteins involved in amino acid metabolism, glutathione metabolism, and nucleotide metabolism were particularly affected by heat. Textural analysis correlated these proteomic changes with alterations in CGC quality attributes, pinpointing 70 °C as the optimum temperature for maintaining the desired texture. A strong positive correlation between specific upregulated proteins was identified, such as the tubulin alpha chain and collagen alpha-1(IV) chain, and the improved textural properties of CGC during thermal processing, suggesting their potential as the potential biomarkers. This study offers a comprehensive proteomic view of the thermal stability and functionality of CGC proteins, delivering invaluable insights for both the culinary processing and scientific management of CGC. Our findings not only deepen the understanding of the molecular mechanisms underpinning the textural alterations in CGC during thermal processing but also furnish practical insights for the aquaculture industry. These insights could be leveraged to optimize cooking techniques, thereby enhancing the quality and consumer appeal of CGC products.

miR-34a/TAN1/CREB Axis Engages in Alleviating Oligodendrocyte Trophic Factor-Induced Myelin Repair Function and Astrocyte-Dependent Neuroinflammation in the Early Stages of Alzheimer's Disease: The Anti-Neurodegenerative Effect of Treadmill Exercise.

Reduced myelin stability observed in the early stages of Alzheimer's disease leads to spatial learning and memory impairment. Exercise has been shown to protect nerves, reduce the risk of Alzheimer's disease, and strengthen synaptic connectivity. However, the underlying mechanisms of how exercise can promote myelin repair and coordinate inflammation and proliferation are still uncertain. In this study, we conducted histological and biochemical assays of cortical lysates after behavioral testing to detect pathological changes, myelin sheath thickness, and mRNA and protein levels. It is notable that D-galactose model mice exhibited elevated miRNA-34a levels, overactive astrocytes, decreased myelin staining scores, increased apoptosis, and decreased synaptic plasticity in the brain. Significantly, after eight weeks of exercise, we observed improvements in LFB scores, NeuN( +) neuron counts, and myelin basic protein (MBP) expression. Additionally, exercise promoted the expression of oligodendrocyte markers Olig2 and PDFGR-α associated with brain proliferation, and improved spatial cognitive function. Furthermore, it decreased the inflammation caused by astrocyte secretions (TNF-α, Cox-2, CXCL2). Interestingly, we also observed downregulation of miR-34a and activation of the TAN1/PI3K/CREB signaling pathway. Our data shed light on a previously unsuspected mechanism by which exercise reduces miR-34a levels and protects neuronal function and survival by preventing excessive demyelination and inflammatory infiltration in the CNS.

Low sucrose availability reduces basal spikelet fertility by inducing abscisic acid and jasmonic acid synthesis in wheat.

Within a spike of wheat, the central spikelets usually generate three to four fertile florets, while the basal spikelets generate zero to one fertile floret. The physiological and transcriptional mechanism behind the difference in fertility between the basal and central spikelets is unclear. This study reports a high temporal resolution investigation of transcriptomes, number and morphology of floret primordia, and physiological traits. The W6.5-W7.5 stage was regarded as the boundary to distinguish between fertile and abortive floret primordia; those floret primordia reaching the W6.5-W7.5 stage during the differentiation phase (3-9 d after terminal spikelet stage) usually developed into fertile florets in the next dimorphism phase (12-27 d after terminal spikelet stage), whereas the others aborted. The central spikelets had a greater number of fertile florets than the basal spikelets, which was associated with more floret primordia reaching the W6.5-W7.5 stage. Physiological and transcriptional results demonstrated that the central spikelets had a higher sucrose content and lower abscisic acid (ABA) and jasmonic acid (JA) accumulation than the basal spikelets due to down-regulation of genes involved in ABA and JA synthesis. Collectively, we propose a model in which ABA and JA accumulation is induced under limiting sucrose availability (basal spikelet) through the up-regulation of genes involved in ABA and JA synthesis; this leads to floret primordia in the basal spikelets failing to reach their fertile potential (W6.5-W7.5 stage) during the differentiation phase and then aborting. This fertility repression model may also regulate spikelet fertility in other cereal crops and potentially provides genetic resources to improve spikelet fertility.

DAXX promotes centromeric stability independently of ATRX by preventing the accumulation of R-loop-induced DNA double-stranded breaks.

Maintaining chromatin integrity at the repetitive non-coding DNA sequences underlying centromeres is crucial to prevent replicative stress, DNA breaks and genomic instability. The concerted action of transcriptional repressors, chromatin remodelling complexes and epigenetic factors controls transcription and chromatin structure in these regions. The histone chaperone complex ATRX/DAXX is involved in the establishment and maintenance of centromeric chromatin through the deposition of the histone variant H3.3. ATRX and DAXX have also evolved mutually-independent functions in transcription and chromatin dynamics. Here, using paediatric glioma and pancreatic neuroendocrine tumor cell lines, we identify a novel ATRX-independent function for DAXX in promoting genome stability by preventing transcription-associated R-loop accumulation and DNA double-strand break formation at centromeres. This function of DAXX required its interaction with histone H3.3 but was independent of H3.3 deposition and did not reflect a role in the repression of centromeric transcription. DAXX depletion mobilized BRCA1 at centromeres, in line with BRCA1 role in counteracting centromeric R-loop accumulation. Our results provide novel insights into the mechanisms protecting the human genome from chromosomal instability, as well as potential perspectives in the treatment of cancers with DAXX alterations.

Danggui Buxue Tang improves therapeutic efficacy of doxorubicin in triple negative breast cancer via ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Danggui Buxue Tang (DBT) has been used for over 800 years to enhance Qi and nourish Blood, and it is particularly beneficial for cancer patients. Recent research has shown that combining DBT with chemotherapy agents leads to superior anti-cancer effects, thereby enhancing therapeutic efficacy. AIM OF THE STUDY: The aim of this study was to evaluate the effectiveness of a combination therapy involving doxorubicin (DOX) and Danggui Buxue Tang (DBT) in the treatment of triple-negative breast cancer (TNBC) and to elucidate the underlying mechanisms of action. MATERIALS AND METHODS: In vitro experiments were performed using MDA-MB-231 and 4T1 cells, while in vivo experiments were carried out using MDA-MB-231 xenograft mice. The therapeutic effects of the combination therapy were evaluated using various techniques, including MTT assay, colony formation assay, flow cytometry, transwell assay, immunofluorescence, transmission electron microscopy (TEM), histological analysis, western blotting, and bioluminescence assay. RESULTS: DBT was found to enhance DOX's anti-TNBC activity in vitro by promoting ferroptosis, as evidenced by the observed mitochondrial morphological changes using TEM. The combination therapy was also found to reduce the expression of Nrf2, HO-1, and GPX4, which are all targets for ferroptosis induction, while simultaneously increasing ROS production. Additionally, the combination therapy reduced nuclear accumulation and constitutive activation of Nrf2, which is a significant cause of chemotherapy resistance and promotes cancer growth. In vivo experiments using an MDA-MB-231 xenograft animal model revealed that the combination therapy significantly reduced tumor cell proliferation and accelerated TNBC deaths by modulating the Nrf2/HO-1/GPX4 axis, with no evidence of tissue abnormalities. Moreover, the combination therapy exhibited a liver protective effect, and administration of Fer-1 was able to reduce the ROS formation produced by the DBT + DOX combination therapy. CONCLUSION: This study provides evidence that the combination therapy of DOX and DBT has the potential to treat TNBC by promoting ferroptosis through the Nrf2/HO-1/GPX4 axis.

[A case report of primary extubation by partial cricotracheal resection for severe subglottic stenosis].

This patient suffered from severe subglottic stenosis(grade Ⅳb). During partial cricotracheal resection, we cut through the cricothyroid membrane and the cricoid arch along the line from the lower edge of the thyroid cartilage to 5 mm of the inferior thyroid cartilage corner anteromedially. This can protect the cricothyroid joint, effectively protect the recurrent laryngeal nerve, and also support the airway. Strictly adhere to airway separation, avoid excessive separation of scars, and combine with reasonable postoperative management to achieve a safe extubation.

Design and development of a rapid meat detection system based on RPA-CRISPR/Cas12a-LFD.

In recent years, meat adulteration safety incidents have occurred frequently, triggering widespread attention and discussion. Although there are a variety of meat quality identification methods, conventional assays require high standards for personnel and experimental conditions and are not suitable for on-site testing. Therefore, there is an urgent need for a rapid, sensitive, high specificity and high sensitivity on-site meat detection method. This study is the first to apply RPA combined with CRISPR/Cas12a technology to the field of multiple meat identification. The system developed by parameter optimization can achieve specific detection of chicken, duck, beef, pork and lamb with a minimum target sequence copy number as low as 1 × 100 copies/µL for 60 min at a constant temperature. LFD test results can be directly observed with the naked eye, with the characteristics of fast, portable and simple operation, which is extremely in line with current needs. In conclusion, the meat identification RPA-CRISPR/Cas12a-LFD system established in this study has shown promising applications in the field of meat detection, with a profound impact on meat quality, and provides a model for other food safety control programs.

A novel FCTF evaluation and prediction model for food efficacy based on association rule mining.

Introduction: Food-components-target-function (FCTF) is an evaluation and prediction model based on association rule mining (ARM) and network interaction analysis, which is an innovative exploration of interdisciplinary integration in the food field. Methods: Using the components as the basis, the targets and functions are comprehensively explored in various databases and platforms under the guidance of the ARM concept. The focused active components, key targets and preferred efficacy are then analyzed by different interaction calculations. The FCTF model is particularly suitable for preliminary studies of medicinal plants in remote and poor areas. Results: The FCTF model of the local medicinal food Laoxianghuang focuses on the efficacy of digestive system cancers and neurological diseases, with key targets ACE, PTGS2, CYP2C19 and corresponding active components citronellal, trans-nerolidol, linalool, geraniol, α-terpineol, cadinene and α-pinene. Discussion: Centuries of traditional experience point to the efficacy of Laoxianghuang in alleviating digestive disorders, and our established FCTF model of Laoxianghuang not only demonstrates this but also extends to its possible adjunctive efficacy in neurological diseases, which deserves later exploration. The FCTF model is based on the main line of components to target and efficacy and optimizes the research level from different dimensions and aspects of interaction analysis, hoping to make some contribution to the future development of the food discipline.

Development of a POCT detection platform based on a locked nucleic acid-enhanced ARMS-RPA-GoldMag lateral flow assay.

In this study, a novel genotyping point-of-care testing (POCT) rapid detection device, the locked nucleic acid (LNA)-amplification refractory mutation system (ARMS)-recombinase polymerase amplification (RPA)-GoldMag lateral flow assay (LFA) platform, was provided by mining and synthesis based on prior technology. Research methods based on system-integrated innovation and knowledge-integrated generation have become a new trend in technology development. Here, we exploit the combination of LNA-coupled ARMS-RPA and gold nanoparticle probe technology for detection signal amplification, thus pioneering a new tool for accurate, rapid, and cost-effective genotyping. We also performed SNP typing detection and clinical validation of this new assay platform using common glucose-6-phosphate dehydrogenase (G6PD) gene single nucleotide polymorphism (SNP) loci, and the results demonstrated the high sensitivity, specificity, stability, accuracy and feasibility of the LNA-ARMS-RPA-GoldMag lateral flow assay platform. It is hoped that this new technology will make a significant contribution to the field of POCT rapid diagnosis and aim to expand the application space, reflecting its clinical application value and development prospects.

Rapid and Ultrasensitive Detection of Plasmodium spp. Parasites via the RPA-CRISPR/Cas12a Platform.

Microscopic examination of thick and thin blood smears stained with Giemsa dye is considered the primary diagnostic tool for the confirmation and management of suspected clinical malaria. However, detecting gametocytes is relatively insensitive, particularly in asymptomatic individuals with low-density Plasmodium infections. To complement existing diagnostic methods, a rapid and ultrasensitive point-of-care testing (POCT) platform for malaria detection is urgently needed and necessary. A platform based on recombinase polymerase amplification (RPA) followed by CRISPR/Cas12a (referred to as RPA-CRISPR/Cas12a) was developed and optimized for the determination of Plasmodium spp. parasites, particularly Plasmodium falciparum, using a fluorescence-based assay (FBDA), lateral flow test strips (LFTS), or naked eye observation (NEO). Then, the established platform was assessed with clinical malaria isolates. Under optimal conditions, the detection threshold was 1 copy/µL for the plasmid, and the limit of detection was 3.11-7.27 parasites/µL for dried blood spots. There was no cross-reactivity against blood-borne pathogens. For the accuracies of RPA-CRISPR/Cas12a, Plasmodium spp. and P. falciparum testing were 98.68 and 94.74%, respectively. The method was consistent with nested PCR results and superior to the qPCR results. RPA-CRISPR/Cas12a is a rapid, ultrasensitive, and reliable platform for malaria diagnosis. The platform requires no or minimal instrumentation for nucleic acid amplification reactions and can be read with the naked eye. Compared with similar diagnostic methods, this platform improves the reaction speed while reducing detection requirements. Therefore, this platform has the potential to become a true POCT for malaria parasites.

Dynamic gene regulatory networks improving spike fertility through regulation of floret primordia fate in wheat.

The developmental process of spike is critical for spike fertility through affecting floret primordia fate in wheat; however, the genetic regulation of this dynamic and complex developmental process remains unclear. Here, we conducted a high temporal-resolution analysis of spike transcriptomes and monitored the number and morphology of floret primordia within spike. The development of all floret primordia in a spike was clearly separated into three distinct phases: differentiation, pre-dimorphism and dimorphism. Notably, we identified that floret primordia with meiosis ability at the pre-dimorphism phase usually develop into fertile floret primordia in the next dimorphism phase. Compared to control, increasing plant space treatment achieved the maximum increasement range (i.e., 50%) in number of fertile florets by accelerating spike development. The process of spike fertility improvement was directed by a continuous and dynamic regulatory network involved in transcription factor and genes interaction. This was based on the coordination of genes related to heat shock protein and jasmonic acid biosynthesis during differentiation phase, and genes related to lignin, anthocyanin and chlorophyll biosynthesis during dimorphism phase. The multi-dimensional association with high temporal-resolution approach reported here allows rapid identification of genetic resource for future breeding studies to realise the maximum spike fertility potential in more cereal crops.

Danggui Buxue Tang: A Review of its Major Components.

Context: Danggui Buxue Tang (DBT) is a classical Chinese medicine that practitioners have used for thousands of years. Historically, those practitioners have used 16 prescriptions of DBT but currently are using only three prescriptions. Objective: The review intended to summarize pharmacological profiles of DBT and also clarify the major active chemicals found within it to provide a better understanding of the significance of DBT clinically. Design: The research team performed a narrative review by searching Pubmed databases. The search used the keywords Danggui Buxue Tang, bioactive chemcials, pharmacological functions. Setting: The databases setting were done by Gong Guowei and Zhou Xuan in the Zunyi Medical University, Zhuhai campus. Results: There are multiple results related to the crude fractions isolated from Danggui Buxue Tang, and also included the clinical trails. Conclusions: Thousands of years of clinical experience have ensured the efficacy of TCM treatments, which can determine the direction of basic research. That research can modify formulas at the molecular level to improve targeting and specificity in the treatment of specific diseases. As a result, the discovery and identification of new compounds within the herbal complex can provide useful research ideas and ensure the viability of new drug development.

A Rapid Antimicrobial Resistance Diagnostic Platform for Staphylococcus aureus Using Recombinase Polymerase Amplification.

Antimicrobial resistance (AMR) has posed a global threat to public health. The Staphylococcus aureus strains have especially developed AMR to practically all antimicrobial medications. There is an unmet need for rapid and accurate detection of the S. aureus AMR. In this study, we developed two versions of recombinase polymerase amplification (RPA), the fluorescent signal monitoring and lateral flow dipstick, for detecting the clinically relevant AMR genes retained by S. aureus isolates and simultaneously identifying such isolates at the species level. The sensitivity and specificity were validated with clinical samples. Our results showed that this RPA tool was able to detect antibiotic resistance for all the 54 collected S. aureus isolates with high sensitivity, specificity, and accuracy (all higher than 92%). Moreover, results of the RPA tool are 100% consistent with that of PCR. In sum, we successfully developed a rapid and accurate AMR diagnostic platform for S. aureus. The RPA might be used as an effective diagnostic test in clinical microbiology laboratories to improve the design and application of antibiotic therapy. IMPORTANCE Staphylococcus aureus is a species of Staphylococcus and belongs to Gram-positive. Meanwhile, S. aureus remains one of the most common nosocomial and community-acquired infections, causing blood flow, skin, soft tissue, and lower respiratory tract infections. The identification of the particular nuc gene and the other eight genes of drug-resistant S. aureus can reliably and quickly diagnose the illness, allowing doctors to prescribe treatment regimens sooner. The detection target in this work is a particular gene of S. aureus, and a POCT is built to simultaneously recognize S. aureus and analyze genes representing four common antibiotic families. We developed and assessed a rapid and on-site diagnostic platform for the specific and sensitive detection of S. aureus. This method allows the determination of S. aureus infection and 10 different AMR genes representing four different families of antibiotics within 40 min. It was easily adaptable in low-resource circumstances and professional-lacking circumstances. It should be supported in overcoming the continuous difficulty of drug-resistant S. aureus infections, which is a shortage of diagnostic tools that can swiftly detect infectious bacteria and numerous antibiotic resistance indicators.

OKseqHMM: a genome-wide replication fork directionality analysis toolkit.

During each cell division, tens of thousands of DNA replication origins are co-ordinately activated to ensure the complete duplication of the human genome. However, replication fork progression can be challenged by many factors, including co-directional and head-on transcription-replication conflicts (TRC). Head-on TRCs are more dangerous for genome integrity. To study the direction of replication fork movement and TRCs, we developed a bioinformatics toolkit called OKseqHMM (https://github.com/CL-CHEN-Lab/OK-Seq, https://doi.org/10.5281/zenodo.7428883). Then, we used OKseqHMM to analyse a large number of datasets obtained by Okazaki fragment sequencing to directly measure the genome-wide replication fork directionality (RFD) and to accurately predict replication initiation and termination at a fine resolution in organisms including yeast, mouse and human. We also successfully applied our analysis to other genome-wide sequencing techniques that also contain RFD information (e.g. eSPAN, TrAEL-seq). Our toolkit can be used to predict replication initiation and fork progression direction genome-wide in a wide range of cell models and growth conditions. Comparing the replication and transcription directions allows identifying loci at risk of TRCs, particularly head-on TRCs, and investigating their role in genome instability by checking DNA damage data, which is of prime importance for human health.

Genome-wide measurement of DNA replication fork directionality and quantification of DNA replication initiation and termination with Okazaki fragment sequencing.

Studying the dynamics of genome replication in mammalian cells has been historically challenging. To reveal the location of replication initiation and termination in the human genome, we developed Okazaki fragment sequencing (OK-seq), a quantitative approach based on the isolation and strand-specific sequencing of Okazaki fragments, the lagging strand replication intermediates. OK-seq quantitates the proportion of leftward- and rightward-oriented forks at every genomic locus and reveals the location and efficiency of replication initiation and termination events. Here we provide the detailed experimental procedures for performing OK-seq in unperturbed cultured human cells and budding yeast and the bioinformatics pipelines for data processing and computation of replication fork directionality. Furthermore, we present the analytical approach based on a hidden Markov model, which allows automated detection of ascending, descending and flat replication fork directionality segments revealing the zones of replication initiation, termination and unidirectional fork movement across the entire genome. These tools are essential for the accurate interpretation of human and yeast replication programs. The experiments and the data processing can be accomplished within six days. Besides revealing the genome replication program in fine detail, OK-seq has been instrumental in numerous studies unravelling mechanisms of genome stability, epigenome maintenance and genome evolution.

Molecular Surveillance of Artemisinin-Based Combination Therapies Resistance in Plasmodium falciparum Parasites from Bioko Island, Equatorial Guinea.

Artemisinin-based combination therapies (ACTs) resistance has emerged and could be diffusing in Africa. As an offshore island on the African continent, the island of Bioko in Equatorial Guinea is considered severely affected and resistant to drug-resistant Plasmodium falciparum malaria. However, the spatial and temporal distribution remain unclear. Molecular monitoring targeting the Pfcrt, Pfk13, Pfpm2, and Pfmdr1 genes was conducted to provide insight into the impact of current antimalarial drug resistance on the island. Furthermore, polymorphic characteristics, haplotype network, and the effect of natural selection of the Pfk13 gene were evaluated. A total of 152 Plasmodium falciparum samples (collected from 2017 to 2019) were analyzed for copy number variation of the Pfpm2 gene and Pfk13, Pfcrt, and Pfmdr1 mutations. Statistical analysis of Pfk13 sequences was performed following different evolutionary models using 96 Bioko sequences and 1322 global sequences. The results showed that the prevalence of Pfk13, Pfcrt, and Pfmdr1 mutations was 73.68%, 78.29%, and 75.66%, respectively. Large proportions of isolates with multiple copies of Pfpm2 were observed (67.86%). In Bioko parasites, the genetic diversity of Pfk13 was low, and purifying selection was suggested by Tajima's D test (-1.644, P > 0.05) and the dN/dS test (-0.0004438, P > 0.05). The extended haplotype homozygosity analysis revealed that Pfk13_K189T, although most frequent in Africa, has not yet conferred a selective advantage for parasitic survival. The results suggested that the implementation of continuous drug monitoring on Bioko Island is an essential measure. IMPORTANCE Malaria, one of the tropical parasitic diseases with a high transmission rate in Bioko Island, Equatorial Guinea, especially caused by P. falciparum is highly prevalent in this region and is commonly treated locally with ACTs. The declining antimalarial susceptibility of artemisinin-based drugs suggested that resistance to artemisinin and its derivatives is developing in P. falciparum. Copy number variants in Pfpm2 and genetic polymorphisms in Pfk13, Pfcrt, and Pfmdr1 can be used as risk assessment indicators to track the development and spread of drug resistance. This study reported for the first time the molecular surveillance of Pfpm2, Pfcrt, Pfk13, and Pfmdr1 genes in Bioko Island from 2017 to 2019 to assess the possible risk of local drug-resistant P. falciparum.

Correlation Between Detection Results of Pepsin in Vocal Fold Polyp Tissues and the Postoperative Efficacy.

PURPOSE: To explore the correlation between detection results of pepsin in vocal fold polyp tissues and the postoperative efficacy. METHODS: The clinical data of 112 patients with vocal fold polyp who received surgical procedures in our hospital from June 2019 to March 2021 were retrospectively analyzed. The vocal fold morphology and vocal acoustic function were assessed at postoperative week 12. Using binary logistic regression, we explored whether the factors, ie, detection result of pepsin in vocal fold polyp tissue, microscopic suturing, the use of CO2 laser, and the history of smoking, affected the postoperative morphological repair of vocal fold polyps. Then, to observe and compare the influence of the detection results of pepsin on the recovery of vocal acoustic function, we divided the enrolled patients into the pepsin group and the pepsin-free group based on the postoperative detection results of pepsin in the polyp tissues by immunohistochemistry, RESULTS: In the 112 patients with vocal fold polyps, positive staining of pepsin in the postoperative samples was found in 76 patients (67.86%) and negative in 36 (32.14%). Totally 80 patients returned to normal in vocal fold morphology, among whom 32 (88.89%) were in pepsin-free group and 48 (63.16%) in pepsin group. Binary logistic regression showed that pepsin was a clinically significant indicator that affected the postoperative morphological recovery of the vocal fold (P = 0.003). Although hoarse voice was improved in all patients at postoperative week 12, the differences were statistically significant in the proportion of patients with grade, roughness, breathiness, asthenia, strain class G, voice handicap index, maximum phonation time, Jitter, Shimmer and noise-to-harmonic ratio between the pepsin group and the pepsin-free group (P < 0.05), with the pepsin-free group being superior to the pepsin group in the improvement of vocal acoustic function. CONCLUSION: Pepsin in vocal fold polyps is a clinically significant indicator affecting the postoperative morphological recovery and acoustic efficacy, and patients with negative pepsin are superior to those with positive pepsin in the postoperative recovery of vocal fold morphology and function.