Staining patterns of PNA and UEA-I lectins in the postnatal developing male genital excurrent duct epithelium in mice.

Objective: The research was aimed at determining the staining patterns of Peanut agglutinin (PNA) lectin and Ulex europaeus agglutinin I (UEA-I) lectin in postnatal developing (day 1 to 8 weeks) male genital excurrent duct epithelium in mice. Materials and Methods: Lectin staining was performed on testis and epididymis of 1-day-old and 1-, 2-, 3-, 4-, 5-, 6-, and 8-week-old ICR mice. Results: Rete testis epithelia were UEA-I- and PNA-negative at all postnatal ages. PNA lectin unclearly stained the epithelial apical surface in efferent ductules (ED) and ductus epididymis up to 2 weeks, while UEA-I was negative in those epithelia. By contrast, at 3 weeks of age, PNA and/or UEA-I turned to clearly label epithelia in the excurrent duct system. At 5 weeks, efferent ductules epithelium was UEA-I-negative but strongly PNA-positive; segment I was PNA-negative but faintly UEA-I positive; segment II was weakly PNA-positive but strongly UEA-I-positive; segment III was strongly-PNA- and UEA-I-positive; segment IV was weakly UEA-I-positive but strongly PNA-positive; and segment V was faintly UEA-I-positive but strongly PNA-positive. At 5 and 8 weeks (the adult), the staining patterns of PNA and UEA-I lectins were nearly identical. Moreover, an increasing staining intensity was observed in both lectins with the advancement of age until 5 weeks of age. Additionally, it was noted that, with the exception of segment I, the staining intensity of PNA gradually increased while that of UEA-I gradually decreased towards the distal section of the ductus epididymis. Conclusion: Lectin PNA and UEA-I staining, in conclusion, indicated epithelial segments in the male genital duct of mice from the postnatal developing age of 3 weeks, and up until 5 weeks, their staining patterns were nearly identical to those in adults.

Peptide Nucleic Acids: From Origami to Editing.

Peptide nucleic acids (PNAs) combine the programmability of native nucleic acids with the robustness and ease of synthesis of a peptide backbone. These designer biomolecules have demonstrated tremendous utility across a broad range of applications, from the formation of bespoke biosupramolecular architectures to biosensing and gene regulation. Herein, we explore some of the key developments in the application of PNA in chemical biology and biotechnology in the last 5 years and present anticipated key areas of future development.

Engineering of a DNA/γPNA Hybrid Nanoreporter for ctDNA Mutation Detection via γPNA Urinalysis.

Detection of circulating tumor DNA (ctDNA) mutations, which are molecular biomarkers present in bodily fluids of cancer patients, can be applied for tumor diagnosis and prognosis monitoring. However, current profiling of ctDNA mutations relies primarily on polymerase chain reaction (PCR) and DNA sequencing and these techniques require preanalytical processing of blood samples, which are time-consuming, expensive, and tedious procedures that increase the risk of sample contamination. To overcome these limitations, here the engineering of a DNA/γPNA (gamma peptide nucleic acid) hybrid nanoreporter is disclosed for ctDNA biosensing via in situ profiling and recording of tumor-specific DNA mutations. The low tolerance of γPNA to single mismatch in base pairing with DNA allows highly selective recognition and recording of ctDNA mutations in peripheral blood. Owing to their remarkable biostability, the detached γPNA strands triggered by mutant ctDNA will be enriched in kidneys and cleared into urine for urinalysis. It is demonstrated that the nanoreporter has high specificity for ctDNA mutation in peripheral blood, and urinalysis of cleared γPNA can provide valuable information for tumor progression and prognosis evaluation. This work demonstrates the potential of the nanoreporter for urinary monitoring of tumor and patient prognosis through in situ biosensing of ctDNA mutations.

Role of comammox bacteria in granular bioreactor for nitrogen removal via partial nitritation/anammox.

In this study, two bioprocess models were first constructed with the newly-discovered comammox process described as one-step and two-step nitrification and evaluated against relevant experimental data. The validated models were then applied to reveal the potential effect of comammox bacteria on the granular bioreactor particularly suitable for undertaking partial nitritation/anammox (PN/A) under different operating conditions of bulk dissolved oxygen (DO) and influent NH4+. The results showed although comammox bacteria-based PN/A could achieve > 80.0 % total nitrogen (TN) removal over a relatively wider range of bulk DO and influent NH4+ (i.e., 0.25-0.40 g-O2/m3 and 470-870 g-N/m3, respectively) without significant nitrous oxide (N2O) production (< 0.1 %), the bulk DO should be finely controlled based on the influent NH4+ to avoid the undesired full nitrification by comammox bacteria. Comparatively, conventional ammonium-oxidizing bacteria (AOB)-based PN/A not only required higher bulk DO to achieve > 80.0 % TN removal but also suffered from 1.7 %∼2.8 % N2O production.

Preclinical Pharmacokinetics in Tumors and Normal Tissues of the Antigene PNA Oligonucleotide MYCN-Inhibitor BGA002.

Although MYCN has been considered an undruggable target, MYCN alterations confer poor prognosis in many pediatric and adult cancers. The novel MYCN-specific inhibitor BGA002 is an antigene peptide nucleic acid oligonucleotide covalently bound to a nuclear localization signal peptide. In the present study, we characterized the pharmacokinetics (PK) of BGA002 after single and repeated administration to mice using a novel specific enzyme-linked immunosorbent assay. BGA002 concentrations in plasma showed linear PK, with dose proportional increase across the tested dose levels and similar exposure between male and female and between intravenous and subcutaneous route of administration. Repeated dosing resulted in no accumulation in plasma. Biodistribution up to 7 days after single subcutaneous administration of [14C]-radiolabeled BGA002 showed broad tissues and organ distribution (suggesting a potential capability to reach primary tumor and metastasis in several body sites), with high concentrations in kidney, liver, spleen, lymph nodes, adrenals, and bone marrow. Remarkably, we demonstrated that BGA002 concentrates in tumors after repeated systemic administrations in three mouse models with MYCN amplification (neuroblastoma, rhabdomyosarcoma, and small-cell lung cancer), leading to a significant reduction in tumor weight. Taking into account the available safety profile of BGA002, these data support further evaluation of BGA002 in patients with MYCN-positive tumors.

Use of peptide nucleic acid probe to determine telomere dynamics in improving chromosome analysis in genetic toxicology studies.

Genetic toxicology, strategically located at the intersection of genetics and toxicology, aims to demystify the complex interplay between exogenous agents and our genetic blueprint. Telomeres, the protective termini of chromosomes, play instrumental roles in cellular longevity and genetic stability. Traditionally karyotyping and fluorescence in situ hybridisation (FISH), have been indispensable tools for chromosomal analysis following exposure to genotoxic agents. However, their scope in discerning nuanced molecular dynamics is limited. Peptide Nucleic Acids (PNAs) are synthetic entities that embody characteristics of both proteins and nucleic acids and have emerged as potential game-changers. This perspective report comprehensively examines the vast potential of PNAs in genetic toxicology, with a specific emphasis on telomere research. PNAs' superior resolution and precision make them a favourable choice for genetic toxicological assessments. The integration of PNAs in contemporary analytical workflows heralds a promising evolution in genetic toxicology, potentially revolutionizing diagnostics, prognostics, and therapeutic avenues. In this timely review, we attempted to assess the limitations of current PNA-FISH methodology and recommend refinements.

Surface Modification of Polyurethane Sponge with Zeolite and Zero-Valent Iron Promotes Short-Cut Nitrification.

Partial nitrification-Anammox (PN-A) is a cost-effective, environmentally friendly, and efficient method for removing ammonia (NH4+-N) pollutants from water. However, the limited accumulation of nitrite (NO2--N) represents a bottleneck in the development of PN-A processes. To address this issue, this study developed a composite carrier loaded with nano zero-valent iron (nZVI) and zeolite to enhance NO2--N accumulation during short-cut nitrification. The modified composite carrier revealed electropositive, hydrophilicity, and surface roughness. These surface characteristics correlate positively with the carrier's total biomass adsorption capacity; the initial adsorption of microorganisms by the composite carrier was increased by 8.7 times. Zeolite endows the carrier with an NH4+-N adsorption capacity of 4.50 mg/g carrier. The entropy-driven ammonia adsorption process creates an ammonia-rich microenvironment on the surface of the carrier, providing effective inhibition of nitrite-oxidizing bacteria (NOB). In tests conducted with a moving bed biofilm reactor and a sequencing batch reactor, the composite carrier achieved a 95% NH4+-N removal efficiency, a NO2--N accumulation efficiency of 78%, and a doubling in total nitrogen removal efficiency. This composite carrier enhances NO2--N accumulation by preventing biomass washout, inhibiting NOB, and enriching PN-A functional bacteria, suggesting its potential for large-scale, stable PN-A applications.

Detecting the FLJ22447 lncRNA in Ovarian Cancer with Cyclopentane-Modified FIT-PNAs (cpFIT-PNAs).

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.

Development and utility of a SARS-CoV-2 pseudovirus assay for compound screening and antibody neutralization assays.

Background: The highly infectious nature of SARS-CoV-2 necessitates using bio-containment facilities to study viral pathogenesis and identify potent antivirals. However, the lack of high-level bio-containment laboratories across the world has limited research efforts into SARS-CoV-2 pathogenesis and the discovery of drug candidates. Previous research has reported that non-replicating SARS-CoV-2 Spike-pseudotyped viral particles are effective tools to screen for and identify entry inhibitors and neutralizing antibodies. Methods: To generate SARS-CoV-2 pseudovirus, a lentiviral packaging plasmid p8.91, a luciferase expression plasmid pCSFLW, and SARS-CoV-2 Spike expression plasmids (Wild-type (D614G) or Delta strain) were co-transfected into HEK293 cells to produce a luciferase-expressing non-replicating pseudovirus which expresses SARS-CoV-2 spike protein on the surface. For relative quantitation, HEK293 cells expressing ACE2 (ACE2-HEK293) were infected with the pseudovirus, after which luciferase activity in the cells was measured as a relative luminescence unit. The ACE2-HEK293/Pseudovirus infection system was used to assess the antiviral effects of some compounds and plasma from COVID-19 patients to demonstrate the utility of this assay for drug discovery and neutralizing antibody screening. Results: We successfully produced lentiviral-based SARS-CoV2 pseudoviruses and ACE2-expressing HEK293 cells. The system was used to screen compounds for SARS-CoV-2 entry inhibitors and identified two compounds with potent activity against SARS-CoV-2 pseudovirus entry into cells. The assay was also employed to screen patient plasma for neutralizing antibodies against SARS-CoV-2, as a precursor to live virus screening, using successful hits. Significance: This assay is scalable and can perform medium-to high-throughput screening of antiviral compounds with neither severe biohazard risks nor the need for higher-level containment facilities. Now fully deployed in our resource-limited laboratory, this system can be applied to other highly infectious viruses by swapping out the envelope proteins in the plasmids used in pseudovirus production.

Considerations on the scoring of telomere aberrations in vertebrate cells detected by telomere or telomere plus centromere PNA-FISH.

Given that telomeres play a fundamental role in maintaining genomic stability, the study of the chromosomal aberrations involving telomeric sequences is a topic of considerable research interest. In recent years, the scoring of these types of aberrations has been used in vertebrate cells, particularly human cells, to evaluate the effects of genotoxic agents on telomeres and the involvement of telomeric sequences on chromosomal aberrations. Currently, chromosomal aberrations involving telomeric sequences are evaluated in peripheral blood lymphocytes or immortalized cell lines, using telomere or telomere plus centromere fluorescence in situ hybridization (FISH) with Peptide Nucleic Acid (PNA) probes (PNA-FISH). The telomere PNA probe is more efficient in the detection of telomeric sequences than conventional FISH with a telomere DNA probe. In addition, the intensity of the telomeric PNA-FISH probe signal is directly correlated with the number of telomeric repeats. Therefore, use of this type of probe can identify chromosomal aberrations involving telomeres as well as determine the telomere length of the sample. There are several mistakes and inconsistencies in the literature regarding the identification of telomere aberrations, which prevent accurate scoring and data comparison between different publications concerning these types of aberrations. The aim of this review is to clarify these issues, and provide proper terminology and criteria for the identification, scoring, and analysis of telomere aberrations.

K-ras mutation detected by peptide nucleic acid-clamping polymerase chain reaction, Ki-67, S100P, and SMAD4 expression can improve the diagnostic accuracy of inconclusive pancreatic EUS-FNB specimens.

OBJECTIVES: We aimed to assess the diagnostic utility of an immunohistochemical panel including calcium-binding protein P, p53, Ki-67, and SMAD family member 4 and K-ras mutation for diagnosing pancreatic solid lesion specimens obtained by endoscopic ultrasound-guided fine-needle biopsy and to confirm their usefulness in histologically inconclusive cases. METHODS: Immunohistochemistry and peptide nucleic acid-clamping polymerase chain reaction for K-ras mutation were performed on 96 endoscopic ultrasound-guided fine-needle biopsy specimens. The diagnostic efficacy of each marker and the combination of markers was calculated. The diagnostic performances of these markers were evaluated in 27 endoscopic ultrasound-guided fine-needle biopsy specimens with histologically inconclusive diagnoses. A classification tree was constructed. RESULTS: K-ras mutation showed the highest accuracy and consistency. Positivity in more than two or three of the five markers showed high diagnostic accuracy (94.6 % and 93.6 %, respectively), and positivity for more than three markers showed the highest accuracy for inconclusive cases (92.0 %). A classification tree using K-ras mutation, Ki-67, S100P, and SMAD4 showed high diagnostic performance, with only two misclassifications in inconclusive cases. CONCLUSIONS: K-ras mutation detection via peptide nucleic acid-clamping polymerase chain reaction is a stable and accurate method for distinguishing between pancreatic ductal adenocarcinoma and non-pancreatic ductal adenocarcinoma lesions. A classification tree using K-ras mutation, Ki-67, S100P, and SMAD4 helps increase the diagnostic accuracy of cases that are histologically difficult to diagnose.

Achieving high nitrogen and antibiotics removal efficiency by nZVI-C in partial nitritation/anammox system with a single-stage membrane-aerated biofilm reactor.

This study innovated constructed an activated carbon-loaded nano-zero-valent iron (nZVI-C) enhanced membrane aerated biofilm reactor (MABR) coupled partial nitritation/anammox (PN/A) system for optimizing nitrogen and antibiotics removal. Results showed that nitrogen and antibiotic removal efficiencies of 88.45 ± 0.14% and 89.90 ± 3.07% were obtained by nZVI-C, respectively. nZVI-C hastened Nitrosomonas enrichment (relative abundance raised from 2.85% to 12.28%) by increasing tryptophan content in EPS. Furthermore, nZVI-C proliferated amo gene by 3.92 times and directly generated electrons, stimulating Ammonia monooxygenase (AMO) co-metabolism activity. Concurrently, via antibiotic resistance genes (ARGs) horizontal transfer, Nitrosomonas synergized with Arenimonas and Comamonadaceae for efficient antibiotic removal. Moreover, nZVI-C mitigated antibiotics inhibition of electron transfer by proliferating genes for PN and anammox electron production (hao, hdh) and utilization (amo, hzs, nir). That facilitated electron transfer and synergistic substrate conversion between ammonia oxidizing bacteria (AOB) and anaerobic ammonia oxidizing bacteria (AnAOB). Finally, the high nitrogen removal efficiency of the MABR-PN/A system was achieved.

Optimizing hydraulic retention time of high-rate activated sludge designed for potential integration with partial nitritation/anammox in municipal wastewater treatment.

The integration of high-rate activated sludge (HRAS), an effective carbon redirection technology, with partial nitritation/anammox (PN/A) is a novel AB treatment process for municipal wastewater. In this study, an airlift HRAS reactor was operated in the continuous inflow mode for 200 d at a wastewater treatment plant. The balance between potential PN/A system stability and peak HRAS performance under decreasing hydraulic retention time (HRT) was optimized. Energy consumption and recovery and CO2 emissions were calculated. The results showed that the optimal HRT suitable with the PN/A process was 3 h, achieving 2-3 g/L mixed liquor volatile suspended solid, 67.8 % chemical oxygen demand (COD) recovery, 81 % total COD removal efficiency, 2.27 ± 1.03 g COD/L/d organic loading rate, 62 % aeration reduction, and 0.24 kWh/m3 power recovery potential. Such findings hold practical value and contribute to the development of the optimal AB process capable of achieving energy autonomy and carbon neutrality.

Impact of Iron Supplementation on Hospital Length of Stay for Pneumonia or Skin and Skin Structure Infections: A Retrospective Cohort Study.

Objectives: Pathogenic organisms utilize iron to survive and replicate and have evolved many processes to extract iron from human hosts. The goal of this study was to elucidate the impact of iron supplementation given in the setting of acute infection. Methods: This was a retrospective cohort study of Veterans Affairs patients who received intravenous antibiotics for pneumonia or skin and skin structure infections. Five-thousand subjects were included in each of the 2 cohorts: iron-receiving and non-iron-receiving. Data was analyzed using Fischer's Exact test if categorical and independent t-tests if continuous. Primary and secondary objectives analyzed with Cox proportional hazard regression and outcome rates estimated utilizing Kaplan-Meier method. Results: Five-thousand patients were included in each cohort. The iron cohort was significantly older (Mean-years: Iron = 71.6, No-iron = 68.9; mean-difference = 2.7, P < .0001) with reduced renal function (Mean-eGFR[mL/min/1.73 m²]: Iron = 67.2, No-iron = 77.4; mean-difference = 10.2, P < .0001). For the primary outcome, the iron cohort had a significantly longer mean length of hospital stay (10.4 days) compared to the no-iron cohort (8.7 days) (mean difference 1.7 days, P < .0001). Secondary outcome analysis showed the iron cohort received intravenous antibiotics for longer (Iron = 8.2 days, No-iron = 7.1 days; mean-difference = 1.1 days, P < .0001) with a higher proportion of 30-day readmissions (Iron = 15.6%, No-iron = 12.8%; proportion difference = 2.8%, P < .0001). No significant difference was found between cohort proportions for 30-day mortality (Iron = 12.7%, No-iron = 11.3%, proportion difference = 1.4%, P = .052). Conclusions: Baseline characteristic differences between cohorts is representative of patients who would be expected to require iron replacement therapy. Given the magnitude of primary and secondary-outcomes, further studies controlling for these factors would be warranted.

Temperature-resilient superior performances by coupling partial nitritation/anammox and iron-based denitrification with granular formation.

Partial nitritation-anammox (PN/A), an energy-neutral process, is widely employed in the treatment of nitrogen-rich wastewater. However, the intrinsic nitrate accumulation limits the total nitrogen (TN) removal, and the practical application of PN/A continues to face a significant challenge at low temperatures (<15 °C). Here, an integrated partial nitritation-anammox and iron-based denitrification (PNAID) system was developed to address the concern. Two up-flow bioreactors were set up and operated for 400 days, with one as the control group and the other as the experiment group with the addition of Fe0. In comparison to the control group, the experiment group with the Fe0 supplement showed better nitrogen removal during the entire course of the experiment at different temperature levels. Specifically, the TN removal efficiency of the control group decreased from 82.9 % to 53.9 % when the temperature decreased from 30 to 12 °C, while in stark contrast, the experiment group consistently achieved 80 % of TN removal in the same condition. Apart from the enhanced nitrogen removal, the experiment group also exhibited better phosphorus removal (10.6 % versus 74.1 %) and organics removal (49.5 % versus 65.1 %). The enhanced and resilient nutrient removal performance of the proposed integrated process under low temperatures appeared to be attributed to the compact structure of granules and the increased microbial metabolism with Fe0 supplement, elucidated by a comprehensive analysis including microbial-specific activity, apparent activation energy, characteristics of granular sludge, and metagenomic sequencing. These results clearly confirmed that Fe0 supplement not only improved nitrogen removal of PN/A process, but also conferred a certain degree of robustness to the system in the face of temperature fluctuations.

Clinical utility of the Oncomine Dx Target Test multi-CDx system and the possibility of utilizing those original sequence data.

BACKGROUND: Companion diagnostic tests play a crucial role in guiding treatment decisions for patients with non-small cell lung cancer (NSCLC). The Oncomine Dx Target Test (ODxTT) Multi-CDx System has emerged as a prominent companion diagnostic method. However, its efficacy in detecting driver gene mutations, particularly rare mutations, warrants investigation. AIMS: This study aimed to assess the performance of the ODxTT in detecting driver gene mutations in NSCLC patients. Specifically, we aimed to evaluate its sensitivity in detecting epidermal growth factor receptor (EGFR) mutations, a key determinant of treatment selection in NSCLC. MATERIALS AND METHODS: We conducted a retrospective analysis of NSCLC patients who underwent testing with the ODxTT at Keio University Hospital between May 2020 and March 2022. Patient samples were subjected to both DNA and RNA tests. Driver gene mutation status was assessed, and instances of missed mutations were meticulously examined. RESULTS: Of the 90 patients, five had nucleic acid quality problems, while 85 underwent both DNA and RNA tests. Driver gene mutations were detected in 56/90 (62.2%) patients. Of the 34 patient specimens, driver mutations were not detected using the ODxTT; however, epidermal growth factor receptor (EGFR) mutations were detected using polymerase chain reaction-based testing in two patients, and a KRAS mutation was detected by careful examination of the sequence data obtained using the ODxTT in one patient. For the above three cases, carefully examining the gene sequence information obtained using the ODxTT could identify driver mutations that were not mentioned in the returned test results. Additionally, we confirmed comparable instances of overlook results for EGFR mutations in the dataset from South Korea, implying that this type of oversight could occur in other countries using the ODxTT. EGFR mutation was missed in ODxTT in Japan (6.3%, 2/32), South Korea (2.0%, 1/49), and overall (3.7%, 3/81). CONCLUSION: Even if sufficient tumor samples are obtained, rare EGFR mutations (which are excluded from the ODxTT's genetic mutation list) might not be detected using the current ODxTT system due to the program used for sequence analysis. However, such rare EGFR mutations can still be accurately detected on ODxTT's sequence data using next-generation sequencing.

A Biotinylated cpFIT-PNA Platform for the Facile Detection of Drug Resistance to Artemisinin in Plasmodium falciparum.

The evolution of drug resistance to many antimalarial drugs in the lethal strain of malaria (Plasmodium falciparum) has been a great concern over the past 50 years. Among these drugs, artemisinin has become less effective for treating malaria. Indeed, several P. falciparum variants have become resistant to this drug, as elucidated by specific mutations in the pfK13 gene. This study presents the development of a diagnostic kit for the detection of a common point mutation in the pfK13 gene of P. falciparum, namely, the C580Y point mutation. FIT-PNAs (forced-intercalation peptide nucleic acid) are DNA mimics that serve as RNA sensors that fluoresce upon hybridization to their complementary RNA. Herein, FIT-PNAs were designed to sense the C580Y single nucleotide polymorphism (SNP) and were conjugated to biotin in order to bind these molecules to streptavidin-coated plates. Initial studies with synthetic RNA were conducted to optimize the sensing system. In addition, cyclopentane-modified PNA monomers (cpPNAs) were introduced to improve FIT-PNA sensing. Lastly, total RNA was isolated from red blood cells infected with P. falciparum (WT strain - NF54-WT or mutant strain - NF54-C580Y). Streptavidin plates loaded with either FIT-PNA or cpFIT-PNA were incubated with the total RNA. A significant difference in fluorescence for mutant vs WT total RNA was found only for the cpFIT-PNA probe. In summary, this study paves the way for a simple diagnostic kit for monitoring artemisinin drug resistance that may be easily adapted to malaria endemic regions.

Response mechanism of a highly efficient partial nitritation-anammox (PN/A) process under antibiotic stress: Extracellular polymers, microbial community, and functional genes.

The Partial nitritation-Anammox (PN/A) process can be restricted when treating high ammonia nitrogen wastewater containing antibiotics. This study aims to explore the response mechanism of the PN/A process under antibiotic stress. Results showed the PN/A process achieved a nitrogen removal rate higher than 1.01 ± 0.03 kg N/m3/d under long-term sulfamethazine stress. The increase of extracellular polymers from 22.52 to 43.96 mg/g VSS was conducive to resisting antibiotic inhibitory. The increase of Denitratisoma and SM1A02 abundance as well as functional genes nirS and nirK indicated denitrifiers should play an important role in the stability of the PN/A system under sulfamethazine stress. In addition, antibiotic-resistant genes (ARGs) sul1 and intI1 significantly increased by 8.78 and 5.12 times of the initial values to maintain the resistance of PN/A process to sulfamethazine stress. This study uncovers the response mechanism of the PN/A process under antibiotic stress, offering a scientific basis and guidance for further application in the future.

Effect of sludge redistribution strategy on stability of partial nitrification-anammox process: Further exploration of the potential value of sludge.

The stability of the two-stage partial nitrification-anammox (PN/A) system was compromised by the inappropriate conversion of insoluble organic matter. In response, a sludge redistribution strategy was implemented. Through the redistribution of PN sludge and anammox sludge in the two-stage PN/A system, a transition was made to the Anammox-single stage PN/A (A-PN/A) system. This specific functional reorganization, facilitated by the rapid reorganization of microbial communities, has the potential to significantly decrease the current risk of suppression. The results of the study showed that implementing the sludge redistribution strategy led to a substantial enhancement in the total nitrogen removal rate (TNRR) by 87.51%, accompanied by a significant improvement of 34.78% in the chemical oxygen demand removal rate (CRR). Additionally, this approach resulted in a remarkable two-thirds reduction in the aeration requirements. High-throughput sequencing revealed that the strategy enriched anammox and ammonia-oxidizing bacteria while limiting denitrifying bacteria, as confirmed by quantitative polymerase chain reaction analysis. Furthermore, the principal component analysis revealed that the location and duration of aeration had direct and indirect effects on functional gene expression and the evolution of microbial communities. This study emphasizes the potential benefits of restructuring microbial communities through a sludge redistribution strategy, especially in integrated systems that encounter challenges with suppression.

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.