Transoceanic ships as a source of alien dinoflagellate invasions of inland freshwater ecosystems.

Ships' ballast water and sediments have long been linked to the global transport and expansion of invasive species and thus have become a hot research topic and administrative challenge in the past decades. The relevant concerns, however, have been mainly about the ocean-to-ocean invasion and sampling practices have been almost exclusively conducted onboard. We examined and compared the dinoflagellate cysts assemblages in 49 sediment samples collected from ballast tanks of international and domestic routes ships, washing basins associated with a ship-repair yard, Jiangyin Port (PS), and the nearby area of Yangtze River (YR) during 2017-2018. A total of 43 dinoflagellates were fully identified to species level by metabarcoding, single-cyst PCR-based sequencing, cyst germination and phylogenetic analyses, including 12 species never reported from waters of China, 14 HABs-causing, 9 toxic, and 10 not strictly marine species. Our metabarcoding and single-cyst sequencing also detected many OTUs and cysts of dinoflagellates that could not be fully identified, indicating ballast tank sediments being a risky repository of currently unrecognizable invasive species. Particularly important, 10 brackish and fresh water species of dinoflagellate cysts (such as Tyrannodinium edax) were detected from the transoceanic ships, indicating these species may function as alien species potentially invading the inland rivers and adjacent lakes if these ships conduct deballast and other practices in fresh waterbodies. Significantly higher numbers of reads and OTUs of dinoflagellates in the ballast tanks and washing basins than that in PS and YR indicate a risk of releasing cysts by ships and the associated ship-repair yards to the surrounding waters. Phylogenetic analyses revealed high intra-species genetic diversity for multiple cyst species from different ballast tanks. Our work provides novel insights into the risk of bio-invasion to fresh waters conveyed in ship's ballast tank sediments and washing basins of shipyards.

Comparison of Interstitial Lung Disease Between Antineutrophil Cytoplasmic Antibodies Positive and Negative Patients: A Retrospective Cohort Study.

OBJECTIVE: Positive antineutrophil cytoplasmic antibodies (ANCAs) may occur in the setting of interstitial lung disease (ILD), with or without ANCA-associated vasculitis (AAV). We aim to compare the characteristics and clinical course of patients with ILD and positive ANCA (ANCA-ILD) to those with negative ANCA. METHODS: We performed a single-center retrospective cohort study from 2018 to 2021. All patients with ILD and ANCA testing were included. Patient characteristics (symptoms, dyspnea scale, and systemic AAV), test results (pulmonary high-resolution computed tomography and pulmonary function tests), and adverse events were collected from electronic medical records. Descriptive statistics and the Fisher exact test were used to compare the outcomes of patients with ANCA-ILD to those with ILD and negative ANCA. RESULTS: A total of 265 patients with ILD were included. The mean follow-up duration was 69.3 months, 26 patients (9.8%) were ANCA positive, and 69.2% of those with ANCA-ILD had another autoantibody. AAV occurred in 17 patients (65.4%) with ANCA-ILD. In 29.4% of patients, AAV developed following ILD diagnosis. Usual interstitial pneumonia was the most common radiologic pattern in patients with ANCA-ILD. There was no association between ANCA status and the evolution of dyspnea, diffusing capacity of the lungs for carbon monoxide, and lung imaging. Forced vital capacity improved over time in 42% of patients with ANCA-ILD and in 17% of patients with negative ANCA (P = 0.006). Hospitalization occurred in 46.2% of patients with ANCA-ILD and in 21.8% of patients with negative ANCA (P = 0.006). Both groups had similar mortality rates. CONCLUSION: Routine ANCA testing should be considered in patients with ILD. Patients with ANCA-ILD are at risk for AAV. More research is required to better understand and manage patients with ANCA-ILD.

Influencing risk factors of voriconazole-induced liver injury in Uygur pediatric patients undergoing allogeneic hematopoietic stem cell transplantation.

PURPOSE: We aimed to investigated the influencing risk factors of voriconazole-induced liver injury in Uygur pediatric patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). METHODS: This was a prospective cohort design study. High-performance liquid chromatography-mass spectrometry was employed to monitor voriconazole concentration. First-generation sequencing was performed to detect gene polymorphisms. Indicators of liver function were detected at least once before and after voriconazole therapy. RESULTS: Forty-one patients were included in this study, among which, 15 patients (36.6%) had voriconazole-induced liver injury. The proportion of voriconazole trough concentration > 5.5 µg·mL-1 patients within the DILI group (40.0%) was significantly higher compared to the control group (15.4%) (p < 0.05). After administration of voriconazole, the values of ALT (103.3 ± 80.3 U/L) and AST (79.9 ± 60.6 U/L) in the DILI group were higher than that in the control group (24.3 ± 24.8 and 30.4 ± 8.6 U/L) (p < 0.05). There was no significant difference between the two groups in genotype and allele frequencies of CYP2C19*2, CYP2C19*3, CYP2C19*17, and UGT1A4 (rs2011425) (p > 0.05). CONCLUSION: There was a significant correlation between voriconazole-induced liver injury and voriconazole trough concentration in high-risk Uygur pediatric patients with allogeneic HSCT.

Clinical implications of CSF-ctDNA positivity in newly diagnosed diffuse large B cell lymphoma.

The clinical implications of CSF-ctDNA positivity in newly diagnosed diffuse large B cell lymphoma (ND-DLBCL) remains largely unexplored. One hundred ND-DLBCL patients were consecutively enrolled as training cohort and another 26 ND-DLBCL patients were prospectively enrolled in validation cohort. CSF-ctDNA positivity (CSF(+)) was identified in 25 patients (25.0%) in the training cohort and 7 patients (26.9%) in the validation cohort, extremely higher than CNS involvement rate detected by conventional methods. Patients with mutations of CARD11, JAK2, ID3, and PLCG2 were more predominant with CSF(+) while FAT4 mutations were negatively correlated with CSF(+). The downregulation of PI3K-AKT signaling, focal adhesion, actin cytoskeleton, and tight junction pathways were enriched in CSF(+) ND-DLBCL. Furthermore, pretreatment CSF(+) was significantly associated with poor outcomes. Three risk factors, including high CSF protein level, high plasma ctDNA burden, and involvement of high-risk sites were used to predict the risk of CSF(+) in ND-DLBCL. The sensitivity and specificity of pretreatment CSF-ctDNA to predict CNS relapse were 100% and 77.3%. Taken together, we firstly present the prevalence and the genomic and transcriptomic landscape for CSF-ctDNA(+) DLBCL and highlight the importance of CSF-ctDNA as a noninvasive biomarker in detecting and monitoring of CSF infiltration and predicting CNS relapse in DLBCL.

A Case of Bacteremia Caused by Raoultella planticola Direct Identification from Blood Culture by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Background: Raoultella planticola is an uncommon gram-negative organism found in the environment. Patients and Methods: The patient, an 81-year-old female who had undergone total cystectomy and bilateral ureteral stoma surgery, presented to the hospital with a fever. It was determined that Raoultella planticola was responsible for the bacteremia. Results: Rapid identification of bacteria using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in blood culture samples and appropriate antibacterial treatment was begun and the patient was discharged three days later. Conclusions: This case emphasizes the presence of a rare pathogen as the cause of bacteremia and underscores the importance of utilizing rapid methods for bacterial identification to establish an accurate diagnosis.

Multifunctional 2D hemin-bridged MOF for the efficient removal and dual-mode detection of organophosphorus pesticides.

The high-residual and bioaccumulation property of organophosphorus pesticides (OPs) creates enormous risks towards the ecological environment and human health, promoting the research for smart adsorbents and detection methods. Herein, 2D hemin-bridged MOF nanozyme (2D-ZHM) was fabricated and applied to the efficient removal and ultrasensitive dual-mode aptasensing of OPs. On the one hand, the prepared 2D-ZHM contained Zr-OH groups with high affinity for phosphate groups, endowing it with selective recognition and high adsorption capacity for OPs (285.7 mg g-1 for glyphosate). On the other hand, the enhanced peroxidase-mimicking biocatalytic property of 2D-ZHM allowed rapid H2O2-directed transformation of 3,3',5,5'-tetramethylbenzidine to oxidic product, producing detectable colorimetric or photothermal signals. Using aptamers of specific recognition capacity, the rapid quantification of two typical OPs, glyphosate and omethoate, was realized with remarkable sensitivity and selectivity. The limit of detections (LODs) of glyphosate were 0.004 nM and 0.02 nM for colorimetric and photothermal methods, respectively, and the LODs of omethoate were 0.005 nM and 0.04 nM for colorimetric and photothermal methods, respectively. The constructed dual-mode aptasensing platform exhibited outstanding performance for monitoring OPs in water and fruit samples. This work provides a novel pathway to develop MOF-based artificial peroxidase and integrated platform for pollutant removal and multi-mode aptasensing.

Deciphering the influence of salinity stress on the biological aniline degradation system: Pollutants degradation performance and microbial response.

In order to evaluate the impact of salinity gradients on the aniline biodegradation system, six reactors at salinity concentrations (0%-5%) were established. The results presented the salinity except for 5% imposed negligible effects on aniline degradation performance. Nitrification had prominent resistance to salinity (0%-1.5%) while were significantly restrained when salinity increased. The total nitrogen (TN) removal efficiency of Z4 (1.5%) was 20.5% higher than Z1 (0%) during the stable operation phase. Moreover, high throughput sequencing analysis showed that halophilic bacterium, such as Halomonas, Rhodococcus, remained greater survival advantages in high salinity system. The substantial enrichment of Flavobacterium, Dokdonella, Paracoccus observed in Z4 ensured its excellent nitrogen removal performance. The close cooperation among dominant functional bacteria was strengthened when salt content was below 1.5% while exceeding 1.5% led to the collapse of metabolic capacity through integrating the toxicity of aniline and high osmotic pressure.

Diagnostic Role of Metagenomic Next-Generation Sequencing in Tubercular Orthopedic Implant-Associated Infection.

Objective: The diagnosis of tubercular orthopedic implant-associated infection (TB-IAI) is challenging. This study evaluated the value of metagenomic next-generation sequencing (mNGS) for the diagnosis of TB-IAI and developed a standardized diagnostic procedure for TB-IAI. Methods: The records of all patients with TB-IAI diagnosed and treated at our institution between December 2018 and September 2022 were retrospectively reviewed. Patient demographic characteristics, medical history, laboratory test, microbial culture, histopathology, and mNGS results, and time to diagnosis were recorded. The diagnostic efficiency of mNGS for TB-IAI was assessed by comparing the results and diagnostic time with that of other diagnostic modalities. Results: Ten patients were included in the analysis, including eight with prosthetic joint infections and two with fracture-related infections. The mNGS positivity rate was 100% (10/10), which was higher than that of TB-antibody (11%, 1/9), real-time quantitative polymerase chain reaction (22%, 2/9), T-SPOT.TB (25%, 2/8), purified protein derivative (50%, 4/8), microbial culture (50%, 5/10), and histopathology (20%, 2/10). mNGS shortened the time to diagnosis of TB-IAI. A standardized diagnostic procedure for TB-IAI was developed based on the findings. Conclusion: mNGS is useful for the diagnosis of TB-IAI. mNGS is recommended in cases where it is difficult to identify a pathogen using routine diagnostic tests. The standardized diagnostic procedure might improve TB-IAI diagnosis. Importance: TB-IAI is a rare infection, which occurs after orthopedic surgery and hard to diagnose microbiologically. mNGS is a new detection technique not yet discussed in current literature as a means for TB-IAI diagnostics. Here we describe a cohort of patients with TB-IAI diagnosed by mNGS show high efficiency of mNGS for detection of this pathology and present a clinical algorithm supplementing conventional methods for TB-IAI assessment.

NHH promotes Sepsis-associated Encephalopathy with the expression of AQP4 in astrocytes through the gut-brain Axis.

Sepsis-associated encephalopathy (SAE) is a significant cause of mortality in patients with sepsis. Despite extensive research, its exact cause remains unclear. Our previous research indicated a relationship between non-hepatic hyperammonemia (NHH) and SAE. This study aimed to investigate the relationship between NHH and SAE and the potential mechanisms causing cognitive impairment. In the in vivo experimental results, there were no significant abnormalities in the livers of mice with moderate cecal ligation and perforation (CLP); however, ammonia levels were elevated in the hippocampal tissue and serum. The ELISA study suggest that fecal microbiota transplantation in CLP mice can reduce ammonia levels. Reduction in ammonia levels improved cognitive dysfunction and neurological impairment in CLP mice through behavioral, neuroimaging, and molecular biology studies. Further studies have shown that ammonia enters the brain to regulate the expression of aquaporins-4 (AQP4) in astrocytes, which may be the mechanism underlying brain dysfunction in CLP mice. The results of the in vitro experiments showed that ammonia up-regulated AQP4 expression in astrocytes, resulting in astrocyte damage. The results of this study suggest that ammonia up-regulates astrocyte AQP4 expression through the gut-brain axis, which may be a potential mechanism for the occurrence of SAE.

Myricetin Acts as an Inhibitor of Type II NADH Dehydrogenase from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common pathogenic microorganism in humans and animals. Type II NADH oxidoreductase (NDH-2) is the only NADH:quinone oxidoreductase present in this organism and represents a promising target for the development of anti-staphylococcal drugs. Recently, myricetin, a natural flavonoid from vegetables and fruits, was found to be a potential inhibitor of NDH-2 of S. aureus. The objective of this study was to evaluate the inhibitory properties of myricetin against NDH-2 and its impact on the growth and expression of virulence factors in S. aureus. RESULTS: A screening method was established to identify effective inhibitors of NDH-2, based on heterologously expressed S. aureus NDH-2. Myricetin was found to be an effective inhibitor of NDH-2 with a half maximal inhibitory concentration (IC50) of 2 µM. In silico predictions and enzyme inhibition kinetics further characterized myricetin as a competitive inhibitor of NDH-2 with respect to the substrate menadione (MK). The minimum inhibitory concentrations (MICs) of myricetin against S. aureus strains ranged from 64 to 128 µg/mL. Time-kill assays showed that myricetin was a bactericidal agent against S. aureus. In line with being a competitive inhibitor of the NDH-2 substrate MK, the anti-staphylococcal activity of myricetin was antagonized by MK-4. In addition, myricetin was found to inhibit the gene expression of enterotoxin SeA and reduce the hemolytic activity induced by S. aureus culture on rabbit erythrocytes in a dose-dependent manner. CONCLUSIONS: Myricetin was newly discovered to be a competitive inhibitor of S. aureus NDH-2 in relation to the substrate MK. This discovery offers a fresh perspective on the anti-staphylococcal activity of myricetin.

Use of 1.5-Stage Functional Articulating Hip Spacers for Two-Stage Treatment of Hip Infection.

BACKGROUND: A two-stage treatment is commonly used for chronic hip infections. This study compared the clinical efficacy and complications associated with 1.5-stage functional articulating hip spacers (FAHS) and handmade spacers utilized during two-stage treatment. METHODS: This retrospective study included 50 patients who had hip infections, of which 41 were periprosthetic joint infections, 3 were internal fixation infections, and 6 had septic arthritis. They were divided into two groups according to the spacer type: 23 patients treated with handmade spacers comprising 1 to 2 Kirschner wires as an endoskeleton (group A) and 27 patients treated with 1.5-stage FAHS comprising a cemented femoral stem, metal femoral head, and polyethylene acetabular liner or cemented acetabular cup (group B). Clinical characteristics, surgical data, infection control rate, spacer complications, modified Harris hip, visual analog scale, and 36-item short-form physical functioning scale scores were compared between the groups. All patients were followed up for at least 24 months after the last surgical procedure. RESULTS: No significant differences were noted in the infection eradication rate between the two groups (100 versus 96.30%, P = 1.0). The incidence of mechanical complications, especially spacer fracture, was significantly lower in group B than in group A (P = .044). Hip function and quality of life were significantly better in group B during the interim period. Group B patients had a longer interval time (median 7.40 versus 4.30 months, P = .004) and a lower reimplantation rate than group A patients (42.31 versus 82.61%, P = .004). CONCLUSIONS: The 1.5-stage FAHS surgical technique is feasible for the treatment of hip infection, with a lower mechanical complication rate, better hip function, and better quality of life during the interim period compared to that of handmade spacers. The 1.5-stage FAHS with maintained function could delay or negate the need for second-stage revision.

Prognostic analysis of related factors of adverse reactions to immunotherapy in advanced gastric cancer and establishment of a nomogram model.

BACKGROUND: Immunotherapy for advanced gastric cancer has attracted widespread attention in recent years. However, the adverse reactions of immunotherapy and its relationship with patient prognosis still need further study. In order to determine the association between adverse reaction factors and prognosis, the aim of this study was to conduct a systematic prognostic analysis. By comprehensively evaluating the clinical data of patients with advanced gastric cancer treated by immunotherapy, a nomogram model will be established to predict the survival status of patients more accurately. AIM: To explore the characteristics and predictors of immune-related adverse reactions (irAEs) in advanced gastric cancer patients receiving immunotherapy with programmed death protein-1 (PD-1) inhibitors and to analyze the correlation between irAEs and patient prognosis. METHODS: A total of 140 patients with advanced gastric cancer who were treated with PD-1 inhibitors in our hospital from June 2021 to October 2023 were selected. Patients were divided into the irAEs group and the non-irAEs group according to whether or not irAEs occurred. Clinical features, manifestations, and prognosis of irAEs in the two groups were collected and analyzed. A multivariate logistic regression model was used to analyze the related factors affecting the occurrence of irAEs, and the prediction model of irAEs was established. The receiver operating characteristic (ROC) curve was used to evaluate the ability of different indicators to predict irAEs. A Kaplan-Meier survival curve was used to analyze the correlation between irAEs and prognosis. The Cox proportional risk model was used to analyze the related factors affecting the prognosis of patients. RESULTS: A total of 132 patients were followed up, of whom 63 (47.7%) developed irAEs. We looked at the two groups' clinical features and found that the two groups were statistically different in age ≥ 65 years, Ki-67 index, white blood cell count, neutrophil count, and regulatory T cell (Treg) count (all P < 0.05). Multivariate logistic regression analysis showed that Treg count was a protective factor affecting irAEs occurrence (P = 0.030). The ROC curve indicated that Treg + Ki-67 + age (≥ 65 years) combined could predict irAEs well (area under the curve = 0.753, 95% confidence interval: 0.623-0.848, P = 0.001). Results of the Kaplan-Meier survival curve showed that progression-free survival (PFS) was longer in the irAEs group than in the non-irAEs group (P = 0.001). Cox proportional hazard regression analysis suggested that the occurrence of irAEs was an independent factor for PFS (P = 0.006). CONCLUSION: The number of Treg cells is a separate factor that affects irAEs in advanced gastric cancer patients receiving PD-1 inhibitor immunotherapy. irAEs can affect the patients' PFS and result in longer PFS. Treg + Ki-67 + age (≥ 65 years old) combined can better predict the occurrence of adverse reactions.

Utilizing collimated aperture with proton pencil beam scanning (PBS) for stereotactic radiotherapy.

PURPOSE: Proton stereotactic radiosurgery (PSRS) has emerged as an innovative proton therapy modality aimed at achieving precise dose delivery with minimal impact on healthy tissues. This study explores the dosimetric outcomes of PSRS in comparison to traditional intensity-modulated proton therapy (IMPT) by focusing on cases with small target volumes. A custom-made aperture system designed for proton therapy, specifically tailored to small target volumes, was developed and implemented for this investigation. METHODS: A prerequisite mechanical validation through an isocentricity test precedes dosimetric assessments, ensuring the seamless integration of mechanical and dosimetry analyses. Five patients were enrolled in the study, including two with choroid melanoma and three with arteriovenous malformations (AVM). Two treatment plans were meticulously executed for each patient, one utilizing a collimated aperture and the other without. Both plans were subjected to robust optimization, maintaining identical beam arrangements and consistent optimization parameters to account for setup errors of 2 mm and range uncertainties of 3.5%. Plan evaluation metrics encompassing the Heterogeneity Index (HI), Paddick Conformity Index (CIPaddick), Gradient Index (GI), and the R50% index to evaluate alterations in low-dose volume distribution. RESULTS: The comparative analysis between PSRS and traditional PBS treatment revealed no significant differences in plan outcomes, with both modalities demonstrating comparable target coverage. However, collimated apertures resulted in discernible improvements in dose conformity, dose fall-off, and reduced low-dose volume. CONCLUSIONS: This study underscores the advantageous impact of the aperture system on proton therapy, particularly in cases involving small target volumes.

Global quantification of emerging and legacy per- and polyfluoroalkyl substances in indoor dust: Levels, profiles and human exposure.

This study investigated the occurrence and distribution of per- and polyfluoroalkyl substances (PFASs) in house dust samples from six regions across four continents. PFASs were detected in all indoor dust samples, with total median concentrations ranging from 17.3 to 197 ng/g. Among the thirty-one PFAS analytes, eight compounds, including emerging PFASs, exhibited high detection frequencies in house dust from all six locations. The levels of PFASs varied by region, with higher concentrations found in Adelaide (Australia), Tianjin (China), and Carbondale (United States, U.S.). Moreover, PFAS composition profiles also differed among regions. Dust from Australia and the U.S. contained high levels of 6:2 fluorotelomer phosphate ester (6:2 diPAP), while perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) were predominant in other regions. Furthermore, our results indicate that socioeconomic factors impact PFAS levels. The assessment of human exposure through dust ingestion and dermal contact indicates that toddlers may experience higher exposure levels than adults. However, the hazard quotients of PFASs for both toddlers and adults were below one, indicating significant health risks are unlikely. Our study highlights the widespread occurrence of PFASs in global indoor dust and the need for continued monitoring and regulation of these chemicals.

PRMT5 activates lipid metabolic reprogramming via MYC contributing to the growth and survival of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable and aggressive subtype of non-Hodgkin B-cell lymphoma. Increased lipid uptake, storage, and lipogenesis occur in a variety of cancers and contribute to rapid tumor growth. However, no data has been explored for the roles of lipid metabolism reprogramming in MCL. Here, we identified aberrant lipid metabolism reprogramming and PRMT5 as a key regulator of cholesterol and fatty acid metabolism reprogramming in MCL patients. High PRMT5 expression predicts adverse outcome prognosis in 105 patients with MCL and GEO database (GSE93291). PRMT5 deficiency resulted in proliferation defects and cell death by CRISPR/Cas9 editing. Moreover, PRMT5 inhibitors including SH3765 and EPZ015666 worked through blocking SREBP1/2 and FASN expression in MCL. Furthermore, PRMT5 was significantly associated with MYC expression in 105 MCL samples and the GEO database (GSE93291). CRISPR MYC knockout indicated PRMT5 can promote MCL outgrowth by inducing SREBP1/2 and FASN expression through the MYC pathway.

Enzyme-integrated metal-organic framework platform for cascade detection of α-amylase.

As one of the most important industrial enzymes, α-amylase is widely used in food processing, such as starch sugar and fermentation, bringing high added value to industry of more than a trillion dollars. We developed a multi-enzyme system (Glu&Gox@Cu-MOF-74) prepared by embedding α-glucosidase (Glu) and glucose oxidase (Gox) into the biomimetic metal-organic framework Cu-MOF-74 using in situ encapsulation within 15 min at room temperature for efficient and sensitive detection of α-amylase activity. Benefitting from the remarkable peroxidase-mimicking property and rigid skeleton of Cu-MOF-74, the biocatalytic platform exhibited excellent cascade activity and tolerance in various extremely harsh environments compared to natural enzymes. On this basis, a cascade biocatalytic platform was constructed for the detection of α-amylase activity with wide linear range (5-100 U/L) and low limit of detection (1.45 U/L). The colorimetric cascade scheme is important for the sensitive and selective determination of α-amylase in complex fermentation samples, and the detection time is short (∼0.5 h). This work provides new ideas for the detection of α-amylase based on the cascade amplification method.

De novo design of pH-responsive self-assembling helical protein filaments.

Biological evolution has led to precise and dynamic nanostructures that reconfigure in response to pH and other environmental conditions. However, designing micrometre-scale protein nanostructures that are environmentally responsive remains a challenge. Here we describe the de novo design of pH-responsive protein filaments built from subunits containing six or nine buried histidine residues that assemble into micrometre-scale, well-ordered fibres at neutral pH. The cryogenic electron microscopy structure of an optimized design is nearly identical to the computational design model for both the subunit internal geometry and the subunit packing into the fibre. Electron, fluorescent and atomic force microscopy characterization reveal a sharp and reversible transition from assembled to disassembled fibres over 0.3 pH units, and rapid fibre disassembly in less than 1 s following a drop in pH. The midpoint of the transition can be tuned by modulating buried histidine-containing hydrogen bond networks. Computational protein design thus provides a route to creating unbound nanomaterials that rapidly respond to small pH changes.

Directed Evolution of Acoustic Reporter Genes Using High-Throughput Acoustic Screening.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 µm. 1 Within the past decade, reporter genes for US have been introduced 2,3 and engineered 4,5 to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). 6 Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semi-automated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologs of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

Design, Synthesis, and Biological Evaluation of Novel EGFR PROTACs Targeting C797S Mutation.

The epidermal growth factor receptor (EGFR) tertiary C797S mutation is an important cause of resistance to Osimertinib, which seriously hinders the clinical application of Osimertinib. Developing proteolysis-targeting chimeras (PROTACs) targeting EGFR mutants can offer a promising strategy to overcome drug resistance. In this study, some novel PROTACs targeting C797S mutation were designed and synthesized based on a new EGFR inhibitor and displayed a potent degradation effect in H1975-TM cells harboring EGFRL858R/T790M/C797S. The representative compound C6 exhibited a DC50 of 10.2 nM against EGFRL858R/T790M/C797S and an IC50 of 10.3 nM against H1975-TM. Furthermore, C6 also showed potent degradation activity against various main EGFR mutants, including EGFRDel19/T790M/C797S. Mechanistic studies revealed that the protein degradation was achieved through the ubiquitin-proteasome system. Finally, C6 inhibited tumor growth in the H1975-TM xenograft tumor model effectively and safely. This study identifies a novel and potent EGFR PROTAC to overcome Osimertinib resistance mediated by C797S mutation.

Phosphorus deficiency-induced cell wall pectin demethylesterification enhances cadmium accumulation in roots of Salix caprea.

Regions affected by heavy metal contamination frequently encounter phosphorus (P) deficiency. Numerous studies highlight crucial role of P in facilitating cadmium (Cd) accumulation in woody plants. However, the regulatory mechanism by which P affects Cd accumulation in roots remains ambiguous. This study aims to investigate the effects of phosphorus (P) deficiency on Cd accumulation, Cd subcellular distribution, and cell wall components in the roots of Salix caprea under Cd stress. The results revealed that under P deficiency conditions, there was a 35.4% elevation in Cd content in roots, coupled with a 60.1% reduction in Cd content in shoots, compared to the P sufficiency conditions. Under deficient P conditions, the predominant response of roots to Cd exposure was the increased sequestration of Cd in root cell walls. The sequestration of Cd in root cell walls increased from 37.1% under sufficient P conditions to 66.7% under P deficiency, with pectin identified as the primary Cd binding site under both P conditions. Among cell wall components, P deficiency led to a significant 31.7% increase in Cd content within pectin compared to P sufficiency conditions, but did not change the pectin content. Notably, P deficiency significantly increased pectin methylesterase (PME) activity by regulating the expression of PME and PMEI genes, leading to a 10.4% reduction in the degree of pectin methylesterification. This may elucidate the absence of significant changes in pectin content under P deficiency conditions and the concurrent increase in Cd accumulation in pectin. Fourier transform infrared spectroscopy (FTIR) results indicated an increase in carboxyl groups in the root cell walls under P deficiency compared to sufficient P treatment. The results provide deep insights into the mechanisms of higher Cd accumulation in root mediated by P deficiency.