Experimental and molecular dynamics simulation studies on the physical properties of three HBc-VLP derivatives as nanoparticle protein vaccine candidates.

Self-assembling virus-like particles (VLPs) are promising platforms for vaccine development. However, the unpredictability of the physical properties, such as self-assembly capability, hydrophobicity, and overall stability in engineered protein particles fused with antigens, presents substantial challenges in their downstream processing. We envision that these challenges can be addressed by combining more precise computer-aided molecular dynamics (MD) simulations with experimental studies on the modified products, with more to-date forcefield descriptions and larger models closely resembling real assemblies, realized by rapid advancement in computing technology. In this study, three chimeric designs based on the hepatitis B core (HBc) protein as model vaccine candidates were constructed to study and compare the influence of inserted epitopes as well as insertion strategy on HBc modifications. Large partial VLP models containing 17 chains for the HBc chimeric model vaccines were constructed based on the wild-type (wt) HBc assembly template. The findings from our simulation analysis have demonstrated good consistency with experimental results, pertaining to the surface hydrophobicity and overall stability of the chimeric vaccine candidates. Furthermore, the different impact of foreign antigen insertions on the HBc scaffold was investigated through simulations. It was found that separately inserting two epitopes into the HBc platform at the N-terminal and the major immunogenic regions (MIR) yields better results compared to a serial insertion at MIR in terms of protein structural stability. This study substantiates that an MD-guided design approach can facilitate vaccine development and improve its manufacturing efficiency by predicting products with extreme surface hydrophobicity or structural instability.

Pore-blocking steric mass-action model for adsorption of bioparticles.

The steric mass-action (SMA) model has been widely reported to describe the adsorption of proteins in different types of chromatographic adsorbents. Here in the present work, a pore-blocking steric mass-action model (PB-SMA) was developed for the adsorption of large-size bioparticles, which usually exhibit the unique pore-blocking characteristic on the adsorbent and thus lead to a fraction of ligands in the deep channels physically inaccessible to bioparticles adsorption, instead of being shielded due to steric hindrance by adsorbed bioparticles. This unique phenomenon was taken into account by introducing an additional parameter, Lin, which is defined as the inaccessible ligand densities in the physically blocked pore area, into the PB-SMA model. This fraction of ligand densities (Lin) will be deducted from the total ligand (Lt) for model development, thus the steric factor (σ) in the proposed PB-SMA will reflect the steric shielding effect on binding sites by adsorbed bioparticles more accurately than the conventional SMA model, which assumes that all ligands on the adsorbent have the same accessibility to the bioparticles. Based on a series of model assumptions, a PB-SMA model was firstly developed for inactivated foot-and-mouth disease virus (iFMDV) adsorption on immobilized metal affinity chromatography (IMAC) adsorbents. Model parameters for static adsorption including equilibrium constant (K), characteristic number of binding sites (n), and steric factor (σ) were determined. Compared with those derived from the conventional SMA model, the σ values derived from the PB-SMA model were dozens of times smaller and much closer to the theoretical maximum number of ligands shielded by a single adsorbed iFMDV, indicating the modified model was more accurate for bioparticles adsorption. The applicability of the PB-SMA model was further validated by the adsorption of hepatitis B surface antigen virus-like particles (HBsAg VLPs) on an ion exchange adsorbent with reasonably improved accuracy. Thus, it is considered that the PB-SMA model would be more accurate in describing the adsorption of bioparticles on different types of chromatographic adsorbents.

Integration of Zn2+, ATP, and bFGF to Nanodressing with Core-Shell Structure Fabricated by Emulsion Electrospinning for Wound Healing.

Basic fibroblast growth factor (bFGF) plays an important role in active wound repair. However, the existing dosage forms in clinical applications are mainly sprays and freeze-dried powders, which are prone to inactivation and cannot achieve a controlled release. In this study, a bioactive wound dressing named bFGF-ATP-Zn/polycaprolactone (PCL) nanodressing with a "core-shell" structure was fabricated by emulsion electrospinning, enabling the sustained release of bFGF. Based on the coordination and electrostatic interactions among bFGF, ATP, and Zn2+, as well as their synergistic effect on promoting wound healing, a bFGF-ATP-Zn ternary combination system was prepared with higher cell proliferation activity and used as the water phase for emulsion electrospinning. The bFGF-ATP-Zn/PCL nanodressing demonstrated improved mechanical properties, sustained release of bFGF, cytocompatibility, and hemocompatibility. It increased the proliferation activity of human dermal fibroblasts (HDFs) and enhanced collagen secretion by 1.39 and 3.45 times, respectively, while reducing the hemolysis rate to 3.13%. The application of the bFGF-ATP-Zn/PCL nanodressing in mouse full-thickness skin defect repair showed its ability to accelerate wound healing and reduce wound scarring within 14 days. These results provide a research basis for the development and application of this bioactive wound dressing product.

In situ bio-mineralized Mn nanoadjuvant enhances anti-influenza immunity of recombinant virus-like particle vaccines.

Virus like particles (VLPs) have been well recognized as one of the most important vaccine platforms due to their structural similarity to natural viruses to induce effective humoral and cellular immune responses. Nevertheless, lack of viral nucleic acids in VLPs usually leads the vaccine candidates less efficient in provoking innate immune against viral infection. Here, we constructed a biomimetic dual antigen hybrid influenza nanovaccines THM-HA@Mn with robust immunogenicity via in situ synthesizing a stimulator of interferon genes (STING) agonist Mn3O4 inside the cavity of a recombinant Hepatitis B core antigen VLP (HBc VLP) having fused SpyTag and influenza M2e antigen peptides (Tag-HBc-M2e, THM for short), followed by conjugating a recombinant hemagglutinin (rHA) antigen on the surface of the nanoparticles through SpyTag/SpyCatcher ligating. Such inside Mn3O4 immunostimulator-outside rHA antigen design, together with the chimeric M2e antigen on the HBc skeleton, enabled the synthesized hybrid nanovaccines THM-HA@Mn to well imitate the spatial distribution of M2e/HA antigens and immunostimulant in natural influenza virus. In vitro cellular experiments indicated that compared with the THM-HA antigen without Mn3O4 and a mixture vaccine consisting of THM-HA + MnOx, the THM-HA@Mn hybrid nanovaccines showed the highest efficacies in dendritic cells uptake and in promoting BMDC maturation, as well as inducing expression of TNF-α and type I interferon IFN-ß. The THM-HA@Mn also displayed the most sustained antigen release at the injection site, the highest efficacies in promoting the DC maturation in lymph nodes and germinal center B cells activation in the spleen of the immunized mice. The co-delivery of immunostimulant and antigens enabled the THM-HA@Mn nanovaccines to induce the highest systemic antigen-specific antibody responses and cellular immunogenicity in mice. Together with the excellent colloid dispersion stability, low cytotoxicity, as well as good biosafety, the synthetic hybrid nanovaccines presented in this study offers a promising strategy to design VLP-based vaccine with robust natural and adaptive immunogenicity against emerging viral pathogens.

Global WWTP Microbiome-based Integrative Information Platform: From experience to intelligence.

Domestic and industrial wastewater treatment plants (WWTPs) are facing formidable challenges in effectively eliminating emerging pollutants and conventional nutrients. In microbiome engineering, two approaches have been developed: a top-down method focusing on domesticating seed microbiomes into engineered ones, and a bottom-up strategy that synthesizes engineered microbiomes from microbial isolates. However, these approaches face substantial hurdles that limit their real-world applicability in wastewater treatment engineering. Addressing this gap, we propose the creation of a Global WWTP Microbiome-based Integrative Information Platform, inspired by the untapped microbiome and engineering data from WWTPs and advancements in artificial intelligence (AI). This open platform integrates microbiome and engineering information globally and utilizes AI-driven tools for identifying seed microbiomes for new plants, providing technical upgrades for existing facilities, and deploying microbiomes for accidental pollution remediation. Beyond its practical applications, this platform has significant scientific and social value, supporting multidisciplinary research, documenting microbial evolution, advancing Wastewater-Based Epidemiology, and enhancing global resource sharing. Overall, the platform is expected to enhance WWTPs' performance in pollution control, safeguarding a harmonious and healthy future for human society and the natural environment.

Metagenomics reveals the influence of small microplastics on microbial communities in coastal sediments.

The ecological impact of microplastics (MPs) in coastal environments has been widely studied. However, the influence of small microplastics in the actual environment is often overlooked due to measurement challenges. In this study, Hangzhou Bay (HZB), China, was selected as our study area. High-throughput metagenomic sequencing and micro-Raman spectrometry were employed to analyze the microbial communities and microplastics of coastal sediment samples, respectively. We aimed to explore the ecological impact of MPs with small sizes (≤ 100 µm) in real coastal sediment environments. Our results revealed that as microplastic size decreased, the environmental behavior of MPs underwent alterations. In the coastal sediments, no significant correlations were observed between the detected MPs and the whole microbial communities, but small MPs posed potential hazards to eukaryotic communities. Moreover, these small MPs were more prone to microbial degradation and significantly affected carbon metabolism in the habitat. This study is the first to reveal the comprehensive impact of small MPs on microbial communities in a real coastal sediment environment.

On-column capping of poly dT media-tethered mRNA accomplishes high capping efficiency, enhanced mRNA recovery, and improved stability against RNase.

The messenger RNA (mRNA) 5'-cap structure is indispensable for mRNA translation initiation and stability. Despite its importance, large-scale production of capped mRNA through in vitro transcription (IVT) synthesis using vaccinia capping enzyme (VCE) is challenging, due to the requirement of tedious and multiple pre-and-post separation steps causing mRNA loss and degradation. Here in the present study, we found that the VCE together with 2'-O-methyltransferase can efficiently catalyze the capping of poly dT media-tethered mRNA to produce mRNA with cap-1 structure under an optimized condition. We have therefore designed an integrated purification and solid-based capping protocol, which involved capturing the mRNA from the IVT system by using poly dT media through its affinity binding for 3'-end poly-A in mRNA, in situ capping of mRNA 5'-end by supplying the enzymes, and subsequent eluting of the capped mRNA from the poly dT media. Using mRNA encoding the enhanced green fluorescent protein as a model system, we have demonstrated that the new strategy greatly simplified the mRNA manufacturing process and improved its overall recovery without sacrificing the capping efficiency, as compared with the conventional process, which involved at least mRNA preseparation from IVT, solution-based capping, and post-separation and recovering steps. Specifically, the new process accomplished a 1.76-fold (84.21% over 47.79%) increase in mRNA overall recovery, a twofold decrease in operation time (70 vs. 140 min), and similar high capping efficiency (both close to 100%). Furthermore, the solid-based capping process greatly improved mRNA stability, such that the integrity of the mRNA could be well kept during the capping process even in the presence of exogenously added RNase; in contrast, mRNA in the solution-based capping process degraded almost completely. Meanwhile, we showed that such a strategy can be operated both in a batch mode and in an on-column continuous mode. The results presented in this work demonstrated that the new on-column capping process developed here can accomplish high capping efficiency, enhanced mRNA recovery, and improved stability against RNase; therefore, can act as a simple, efficient, and cost-effective platform technology suitable for large-scale production of capped mRNA.

Study of self-assembling properties of HBc-VLP derivatives aided by molecular dynamic simulations from a thermodynamic perspective.

Self-assembling protein nanoparticles showed promise for vaccine design due to efficient antigen presentations and safety. However, the unpredictable formations of epitopes-fused protein assemblies remain challenging in the upstream design. This study suggests employing molecular dynamic (MD) simulations to investigate the assembly properties of Hepatitis B core protein (HBc) from thermodynamic perspectives. Eight HBc derivatives were expressed in E. coli, with their self-assembly properties characterised by high-performance liquid chromatography and transmission electron microscopy. MD simulations on the dimers, based on AlphaFold-predicted 3D structures, analysed the derivative at the atomic level. Results revealed that HBc derivatives can form dissociative polymers or large multi-subunit structures due to assembly failures. The instability of the dimer in aqueous solvents or inappropriate intradimer distances could cause major assembly failures. Polar solvation energies played a vital role too in forming assemble-incompetent dimers. Importantly, our study demonstrated that MD simulations on dimers can provide preliminary predictions on the assembly properties of HBc derivatives, thus aiding vaccine design by lowering the risk of self-assembling failures in engineered proteins.Communicated by Ramaswamy H. Sarma.

[Quantification of complete viral particles in inactivated avian influenza virus antigen by high performance size exclusion chromatography coupled with multi-angle laser light scattering].

We developed a method for accurate quantification of the intact virus particles in inactivated avian influenza virus feedstocks. To address the problem of impurities interference in the detection of inactivated avian influenza virus feedstocks by direct high performance size exclusion chromatography (HPSEC), we firstly investigated polyethylene glycol (PEG) precipitation and ion exchange chromatography (IEC) for H5N8 antigen purification. Under the optimized conditions, the removal rate of impurity was 86.87% in IEC using DEAE FF, and the viral hemagglutination recovery was 100%. HPSEC was used to analyze the pretreated samples. The peak of 8.5-10.0 min, which was the characteristic adsorption of intact virus, was analyzed by SDS-PAGE and dynamic light scattering. It was almost free of impurities and the particle size was uniform with an average particle size of 127.7 nm. After adding antibody to the IEC pretreated samples for HPSEC detection, the characteristic peak disappeared, indicating that IEC pretreatment effectively removed the impurities. By coupling HPSEC with multi-angle laser scattering technique (MALLS), the amount of intact virus particles in the sample could be accurately quantified with a good linear relationship between the number of virus particles and the chromatographic peak area (R2=0.997). The established IEC pretreatment-HPSEC-MALLS assay was applied to accurate detection of the number of intact virus particles in viral feedstocks of different subtypes (H7N9), different batches and different concentrations, all with good applicability and reproducibility, Relative standard deviation < 5%, n=3.

Moderate anthropogenic disturbance stimulates versatile microbial taxa contributing to denitrification and aromatic compound degradation.

Wastewater treatment plants (WWTPs) effluent often contains a significant amount of residual organic pollutants and nutrients, causing disturbance to the coastal effluent receiving areas (ERA). Microbial communities in coastal ERA sediments may benefit from the coexistence of organic pollutants and nutrients, promoting the emergence of versatile taxa that are capable of eliminating these substances simultaneously. However, the identification and exploration of versatile taxa in natural environments under anthropogenic disturbances remain largely uncharted territory. In this study, we specifically focused on the versatile taxa coupled by the degradation of aromatic compounds (ACs) and denitrification, using Hangzhou Bay in China as our study area. We explored how WWTPs effluent disturbance would affect the versatile taxa, and particularly examined the role of disturbance intensity in shaping their composition. Intriguingly, we found that versatile taxa were mainly derived from denitrifiers like Pseudomonas, suggesting the fulfilled potential of denitrifiers regarding ACs degradation. We also discovered that moderate disturbance stimulated the diversity of versatile taxa, resulting in strengthened functional redundancy. Through correlation network analysis, we further demonstrated that moderate disturbance enhanced the community-level cooperation. Thus, moderate disturbance serves as a catalyst for versatile taxa to maintain community function, making them more resilient to effluent disturbances. Additionally, we identified COD and NO3--N concentrations as significant environmental factors influencing the versatile taxa. Overall, our findings reveal the role of effluent disturbances in the promotion of versatile taxa, and highlight moderate disturbance can foster more robust versatile taxa that are better equipped to handle effluent disturbances.

Messenger RNA chromatographic purification: advances and challenges.

Messenger RNA (mRNA) technologies have shown great potential in prophylactic vaccines and therapeutic medicines due to their adaptability, rapidity, efficacy, and safety. The purity of mRNA determines the efficacy and safety of mRNA drugs. Though chromatographic technologies are currently employed in mRNA purification, they are facing challenges, mainly arising from the large size, relatively simple chemical composition, instability, and high resemblance of by-products to the target mRNA. In this review, we will first make a comprehensive analysis of physiochemical properties differences between mRNA and proteins, then the major challenges facing in mRNA purification and general considerations are highlighted. A detailed summary of the state-of-arts in mRNA chromatographic purification will be provided, which are mainly classified into physicochemical property-based (size, charge, and hydrophobicity) and chemical structure-based (phosphate backbone, bases, cap structure, and poly A tail) technologies. Efforts in eliminating dsRNA byproducts via post in vitro transcript (IVT) purification and by manipulating the IVT process to reduce the generation of dsRNA are highlighted. Finally, a brief summary of the current status of chromatographic purification of the emerging circular mRNA (circRNA) is provided. We hope this review will provide some useful guidance for the Quality by Design (QbD) of mRNA downstream process development.

Screening of priority antibiotics in Chinese seawater based on the persistence, bioaccumulation, toxicity and resistance.

Antibiotics are emerging pollutants that have detrimental effects on both target and non-target organisms in the environment. However, current methods for environmental risk assessment primarily focus on the risk to non-target organisms in ecosystems, overlooking a crucial risk of antibiotics - the induction of resistance in targeted bacteria. To address this oversight, we have incorporated resistance (R) risk with persistence, bioaccumulation and toxicity (PBT) to establish a more comprehensive PBTR (persistence, bioaccumulation, toxicity, and resistance) framework for antibiotic-specific risk assessment. Using the PBTR framework, we evaluated 74 antibiotics detected in Chinese seawater from 2000 to 2021, and identified priority antibiotics. Our analysis revealed that the priority antibiotics with R risk accounted for the largest proportion (50% to 70%), followed by P risk (40% to 58%), T risk (16% to 35%) and B risk (0 to 13%). To further categorize these priority antibiotics, we assigned them a risk level according to their fulfillment of criteria related to P, B, T, and R. Antibiotics meeting all four indicators were classified as Grade I, representing the highest risk level. Grade II and Grade III were assigned to antibiotics meeting three or two indicators, respectively. Antibiotics meeting only one indicator were classified as Grade IV, representing the lowest risk level. The majority of priority antibiotics fell into Grade IV, indicating low risk (55% to 79%), followed by Grade III (16% to 45%). The highest risk antibiotic identified in this study was clindamycin (CLIN), categorized as Grade II, in the East China Sea. Our findings aligned with previous studies for 25 antibiotics, affirming the validity of the PBTR framework. Moreover, we identified 13 new priority antibiotics, highlighting the advancement of this approach. This study provides a feasible screening strategy and monitoring recommendations for priority antibiotics in Chinese seawater.

The position of Spy Tag/Catcher system in hepatitis B core protein particles affects the immunogenicity and stability of the synthetic vaccine.

Presenting exogenous antigens on virus-like particles (VLPs) through "plug-and-display" decoration strategies based on SpyTag/SpyCatcher isopeptide bonding have emerged as attractive technology for vaccine synthesis. However, whether the position of ligation site in VLPs will impose effects on immunogenicity and physiochemical properties of the synthetic vaccine remains rarely investigated. Here in the present work, the well-established hepatitis B core (HBc) protein was used as chassis to construct dual-antigen influenza nanovaccines, with the conserved epitope peptides derived from extracellular domain of matrix protein M2 (M2e) and hemagglutinin (HA) as target antigens. The M2e antigen was genetically fused to the HBc in the MIR region, together with the SpyTag peptide, which was fused either in the MIR region or at the N-terminal of the protein, so that a recombinant HA antigen (rHA) linked to SpyCatcher can be displayed on it, at two different localizations. Both synthetic nanovaccines showed ability in inducing strong M2e and rHA-specific antibodies and cellular immunogenicity; nevertheless, the one in which rHA was conjugated by N-terminal Tag ligation, was superior to another one synthesized by linking the rHA to MIR region SpyTagged-HBc in all aspects, including higher antigen-specific immunogenicity responses, lower anti-HBc carrier antibody, as well as better dispersion stability. Surface charge and hydrophobicity properties of the two synthetic nanovaccines were analyzed, results revealed that linking the rHA to MIR region SpyTagged-HBc lead to more significant and disadvantageous alteration in physiochemical properties of the HBc chassis. This study will expand our knowledge on "plug-and-display" decoration strategies and provide helpful guidance for the rational design of HBc-VLPs based modular vaccines by using SpyTag/Catcher synthesis.

Thermal-triggered loading and GSH-responsive releasing property of HBc particles for drug delivery.

Hepatitis B core protein virus-like particles (HBc VLPs) have attracted wide attentions using as drug delivery vehicles, due to its excellent stability and easy in large scale production. Here in the present work, we report unique thermal-triggered loading and glutathione-responsive releasing property of the HBc particles for anticancer drug delivery. Through reversible temperature-dependent hole gating of the HBc particle capsid, about 4248 doxorubicin (DOX) were successfully encapsulated inside nanocage of a single nanoparticle at high HBc recovery of 83.2%, by simply incubating the DOX with HBc at 70 °C for 90 min. The new strategy was significantly superior to the disassembly-reassembly methods, which can only yield 3556 DOX loading at 52.3% HBc recovery. The thermal-sensitive drug entry channel in HBc was analyzed by molecular dynamic simulations, and the G113, G117 and R127 were identified as the key amino acid residues that are not conducive to the entrance of DOX but sensitive to temperature. Especially, the ΔGbind of R127 become even higher at high temperature, mutation of the R127 would be the first choice to make the drug entry thermodynamically easier. Due to plenty of disulfide bonds linking the HBc subunits, the HBc particles loaded with DOX exhibited intrinsic glutathione (GSH) responsivity for efficient controlled release in tumor sites. To further increase the tumor-targeting effect of the drug, Cyclo(Arg-Gly-Asp-d-Tyr-Lys) peptide was conjugated to the surface of HBc through a PEG linker. The prepared HBc-based anticancer drug showed significantly improved stability, tumor specificity, and in vivo anticancer activity on MCF7-bearing Balb/c-nu mice. Overall, our work demonstrated that the HBc VLPs can be an ideal drug carrier to fulfill requirement of the intelligent loading and "on demand" release of the therapeutic agents for efficient cancer therapy with minimal adverse effects.

Synthesis of a crystalline zeolitic imidazole framework-8 nano-coating on single environment-sensitive viral particles for enhanced immune responses.

Encapsulating antigens with zeolitic imidazole framework-8 (ZIF-8) exhibits many advantages in vaccine development. However, most viral antigens with complex particulate structures are sensitive to pH or ionic strength, which cannot tolerate harsh synthesis conditions of ZIF-8. Balancing the viral integrity and the growth of ZIF-8 crystals is crucial for the successful encapsulation of these environment-sensitive antigens in ZIF-8. Here, we explored the synthesis of ZIF-8 on inactivated foot and mouth disease virus (known as 146S), which is easily disassociated into no immunogenic subunits under the existing ZIF-8 synthesis conditions. Our results showed that intact 146S could be encapsulated into ZIF-8 with high embedding efficiency by lowering the pH of the 2-MIM solution to 9.0. The size and morphology of 146S@ZIF-8 could be further optimized by increasing the amount of Zn2+ or adding cetyltrimethylammonium bromide (CTAB). 146S@ZIF-8 with a uniform diameter of about 49 nm could be synthesized by adding 0.01% CTAB, which was speculated to be composed of single 146S armored with nanometer-scale ZIF-8 crystal networks. Plenty of histidine on the 146S surface forms a unique His-Zn-MIM coordination in the near vicinity of 146S particles, which greatly increases the thermostability of 146S by about 5 °C, and the nano-scale ZIF-8 crystal coating exhibited extraordinary stability to resist EDTE-treatment. More importantly, the well-controlled size and morphology enabled 146S@ZIF-8(0.01% CTAB) to facilitate antigen uptake. The immunization of 146S@ZIF-8(4×Zn2+) or 146S@ZIF-8(0.01% CTAB) significantly enhanced the specific antibody titers and promoted the differentiation of memory T cells without adding another immunopotentiator. This study reported for the first time the strategy of the synthesis of crystalline ZIF-8 on an environment-sensitive antigen and demonstrated that the nano-size and appropriate morphology of ZIF-8 are crucial to exert adjuvant effects, thus expanding the application of MOFs in vaccine delivery.

PTB Regulates the Metabolic Pathways and Cell Function of Keloid Fibroblasts through Alternative Splicing of PKM.

Keloids, benign fibroproliferative cutaneous lesions, are characterized by abnormal growth and reprogramming of the metabolism of keloid fibroblasts (KFb). However, the underlying mechanisms of this kind of metabolic abnormality have not been identified. Our study aimed to investigate the molecules involved in aerobic glycolysis and its exact regulatory mechanisms in KFb. We discovered that polypyrimidine tract binding (PTB) was significantly upregulated in keloid tissues. siRNA silencing of PTB decreased the mRNA levels and protein expression levels of key glycolytic enzymes and corrected the dysregulation of glucose uptake and lactate production. In addition, mechanistic studies demonstrated that PTB promoted a change from pyruvate kinase muscle 1 (PKM1) to PKM2, and silencing PKM2 substantially reduced the PTB-induced increase in the flow of glycolysis. Moreover, PTB and PKM2 could also regulate the key enzymes in the tricarboxylic acid (TCA) cycle. Assays of cell function demonstrated that PTB promoted the proliferation and migration of KFb in vitro, and this phenomenon could be interrupted by PKM2 silencing. In conclusion, our findings indicate that PTB regulates aerobic glycolysis and the cell functions of KFb via alternative splicing of PKM.

Wastewater treatment plant effluent discharge decreases bacterial community diversity and network complexity in urbanized coastal sediment.

The wastewater treatment plant (WWTP) effluent discharge affects the microorganisms in the receiving water bodies. Despite the ecological significance of microbial communities in pollutant degradation and element cycling, how the community diversity is affected by effluent remains obscure. Here, we compared the sediment bacterial communities exposed to different intensities of WWTP effluent discharge in Hangzhou Bay, China: i) a severely polluted area that receives effluent from an industrial WWTP, ii) a moderately polluted area that receives effluent from a municipal WWTP, and iii) less affected area that inner the bay. We found that the sediment bacterial diversity decreased dramatically with pollution levels of inorganic nutrients, heavy metals, and organic halogens. Microbial community assembly model analysis revealed increased environmental selection and decreased species migration rate in the severely polluted area, resulting in high phylogenetic clustering of the bacterial communities. The ecological networks were less complex in the two WWTP effluent receiving areas than in the inner bay area, as suggested by the smaller network size and lower modularity. Fewer negative network associations were detected in the severely (6.7%) and moderately (8.3%) polluted areas than in the less affected area (16.7%), indicating more collaborative inter-species behaviors are required under stressful environmental conditions. Overall, our results reveal the fundamental impacts of WWTP effluents on the ecological processes shaping coastal microbial communities and point to the potential adverse effects of diversity loss on ecosystem functions.

A New Technique for Chondrolaryngoplasty in Patients Assigned Female at Birth.

BACKGROUND: Chondrolaryngoplasty is a classical facial feminization surgery for transgender women. In recent years, however, an increasing number of patients assigned female at birth are seeking chondrolaryngoplasty for esthetic purposes. Traditional chondrolaryngoplasty can no longer cope with problems of the growing group whose leading cause of laryngeal prominence differs from the transgender population. METHODS: A modified technique is designed as a supplement to the classical procedure. After the cartilage reduction process, paired platysma flaps are raised and advanced successively, resulting in an overlapped area over the thyroid notch, to further camouflage the thyroid prominence. To evaluate the efficiency of the new technique, a retrospective survey of 34 patients (5 men and 29 women) who underwent the surgery from 2016 to 2021 was performed, via a 5-point Likert scale including 7 questions. Physician assessment was also accomplished to provide an extra estimation. Complications were followed up and analyzed to evaluate the safety of modified surgery as well. RESULTS: Although only half of the patients graded prominence changes more than "moderately changed," as many as 75.0% of them still expressed "completely satisfied" or "satisfied very much" with the outcome. Similarly, physician assessment indicated a satisfactory result in appearance improvement. No severe and irreversible complications occurred after surgery, but lasting scar-related issues were reported by 4 patients and should be paid more attention to. CONCLUSIONS: Generally speaking, the new technique is both safe, efficient, and satisfying for most patients, especially ones assigned females at birth with esthetic demand.

Industrial effluents boosted antibiotic resistome risk in coastal environments.

Wastewater treatment plants (WWTPs) have been regarded as an important source of antibiotic resistance genes (ARGs) in environment, but out of municipal domestic WWTPs, few evidences show how environment is affected by industrial WWTPs. Here we chose Hangzhou Bay (HZB), China as our study area, where land-based municipal and industrial WWTPs discharged their effluent into the bay for decades. We adopted high-throughput metagenomic sequencing to examine the antibiotic resistome of the WWTP effluent and coastal sediment samples. And we proposed a conceptual framework for the assessment of antibiotic resistome risk, and a new bioinformatic pipeline for the evaluation of the potential horizontal gene transfer (HGT) frequency. Our results revealed that the diversity and abundance of ARGs in the WWTP's effluent were significantly higher than those in the sediment. Furthermore, the antibiotic resistome in the effluent-receiving area (ERA) showed significant difference from that in HZB. For the first time, we identified that industrial WWTP effluent boosted antibiotic resistome risk in coastal sediment. The crucial evidences included: 1) the proportion of ARGs derived from WWTP activated sludge (WA) was higher (14.3 %) and two high-risky polymyxin resistance genes (mcr-4 and mcr-5) were enriched in the industrial effluent receiving area; 2) the HGT potential was higher between resistant microbiome of the industrial effluent and its ERA sediment; and 3) the highest resistome risk was determined in the industrial effluent, and some biocide resistance genes located on high-risky contigs were related to long-term stress of industrial chemicals. These findings highlight the important effects of industrial activities on the development of environmental antimicrobial resistance.

Mechanism and inhibition of abnormal chromatographic behavior of serotype type a inactivated foot and mouth disease virus in high-performance size-exclusion chromatography.

High-performance size-exclusion chromatography (HPSEC) has been developed for the rapid and quantitative analysis of inactivated foot and mouth disease virus (FMDV) and adopted by regulatory agencies and vaccine manufacturers. However, strong non-specific adsorption of type A/AKT III FMDV was found on some batches of TSK G4000 SWXL column, which significantly affected the analysis accuracy. The adsorption mechanism was studied by investigating the charge and hydrophobicity of A/AKT III FMDV and another serotype O/Mya 98, as well as several model proteins, by zeta potential and hydrophobic interaction chromatography analysis. Adsorption was related to both the FMDV strain and column lots. Some specific amino acids residues on the A/AKT III FMDV surface may strongly interact with the column if the silica-based stationary phase was not completely diol-modified. Several amino acids and chaotropic salts were screened as additives in the mobile phase to suppress the non-specific adsorption of AKT III FMDV in HPSEC analysis. Results showed that adding 0.4 M of arginine (Arg), lysine (Lys), NaClO4, or NaSCN achieved 100% FMDV recovery and normal retention time. Suppression of interaction between FMDV and the backbone of the silica matrix through competitive binding with residues of FMDV or the matrix is considered as the main mechanism by which these four additives act as suppressors. The addition of Arg, NaClO4, or NaSCN led to an apparent decrease in the thermal dissociation temperature Tm of FMDV, whereas Lys slightly increased viral stability. Finally, the mobile phase comprising 0.4 M Lys was screened as optimum that allowed accurate quantification of both two serotypes of FMDV according to method validation; particularly, a relative standard deviation (RSD) < 5% was achieved for AKT III FMDV using three different lots of columns.