Screening of a potential probiotic Lactiplantibacillus plantarum NUC08 and its synergistic effects with yogurt starter.

This study aims to identify lactic acid bacteria (LAB) isolates possessing physiological characteristics suitable for use as probiotics in yogurt fermentation. Following acid and bile salt tolerance tests, Lactiplantibacillus plantarum (NUC08 and NUC101), Lacticaseibacillus rhamnosus (NUC55 and NUC201), and Lacticaseibacillus paracasei (NUC159, NUC216, and NUC351) were shortlisted based on intraspecies distribution for further evaluation. Their physiological probiotic properties, including transit tolerance, adhesion, autoaggregation, surface hydrophobicity, biofilm formation, and antibacterial activity, were assessed. Principal component analysis indicated that Lactiplantibacillus plantarum NUC08 was the preferred choice among the evaluated strains. Subsequent investigations revealed that co-culturing Lactiplantibacillus plantarum NUC08 with 2 yogurt starter strains resulted in a cooperative and synergistic effect, enhancing the growth of mixed strains and increasing their tolerance to simulated gastric and intestinal conditions. Additionally, when Vibrio harveyi bioluminescent reporter strain was used, the 3 cocultured strains cooperated to induce the activity of a quorum sensing (QS) molecule autoinducer-2 (AI-2), hinting a potential connection between phenotypic traits and QS in the cocultured strains. Importantly, LAB viable counts were significantly higher in yogurt co-fermented with Lactiplantibacillus plantarum NUC08, consistently throughout the storage period. In conclusion, the study demonstrates that the probiotic strain Lactiplantibacillus plantarum NUC08 can be employed in synergy with yogurt starter strains, affirming its potential for use in the development of functional fermented dairy products.

Ferrocene-Based Polymeric Nanoparticles Carrying Doxorubicin for Oncotherapeutic Combination of Chemotherapy and Ferroptosis.

Mono-chemotherapy has significant side effects and unsatisfactory efficacy, limiting its clinical application. Therefore, a combination of multiple treatments is becoming more common in oncotherapy. Chemotherapy combined with the induction of ferroptosis is a potential new oncotherapy. Furthermore, polymeric nanoparticles (NPs) can improve the antitumor efficacy and decrease the toxicity of drugs. Herein, a polymeric NP, mPEG-b-PPLGFc@Dox, is synthesized to decrease the toxicity of doxorubicin (Dox) and enhance the efficacy of chemotherapy by combining it with the induction of ferroptosis. First, mPEG-b-PPLGFc@Dox is oxidized by endogenous H2 O2 and releases Dox, which leads to an increase of H2 O2 by breaking the redox balance. The Fe(II) group of ferrocene converts H2 O2 into ·OH, inducing subsequent ferroptosis. Furthermore, glutathione peroxidase 4, a biomarker of ferroptosis, is suppressed and the lipid peroxidation level is elevated in cells incubated with mPEG-b-PPLGFc@Dox compared to those treated with Dox alone, indicating ferroptosis induction by mPEG-b-PPLGFc@Dox. In vivo, the antitumor efficacy of mPEG-b-PPLGFc@Dox is higher than that of free Dox. Moreover, the loss of body weight in mice treated mPEG-b-PPLGFc@Dox is lower than in those treated with free Dox, indicating that mPEG-b-PPLGFc@Dox is less toxic than free Dox. In conclusion, mPEG-b-PPLGFc@Dox not only has higher antitumor efficacy but it reduces the damage to normal tissue.

Deletion of cox7c Results in Pan-Azole Resistance in Aspergillus fumigatus.

In Aspergillus fumigatus, the most prevalent resistance to azoles results from mutational modifications of the azole target protein Cyp51A, but there are non-cyp51A mutants resistant to azoles, and the mechanisms underlying the resistance of these strains remain to be explored. Here, we identified a novel cytochrome c oxidase, cox7c (W56*), nonsense mutation in the laboratory and found that it caused reduced colony growth and resistance to multiantifungal agents. Meanwhile, we revealed that cold storage is responsible for increased tolerance of conidia to itraconazole (ITC) stress, which further advances azole-resistant mutations (cryopreservation→ITC tolerance→azole resistance). The deletion or mutation of cox7c results explicitly in resistance to antifungal-targeting enzymes, including triazoles, polyenes, and allylamines, required for ergosterol synthesis, or resistance to fungal ergosterol. A high-performance liquid chromatography (HPLC) assay showed that the cox7c knockout strain decreased intracellular itraconazole concentration. In addition, the lack of Cox7c resulted in the accumulation of intracellular heme B. We validated that an endogenous increase in, or the exogenous addition of, heme B was capable of eliciting azole resistance, which was in good accordance with the phenotypic resistance analysis of cox7c mutants. Furthermore, RNA sequencing verified the elevated transcriptional expression levels of multidrug transport genes. Additionally, lower itraconazole-induced reactive oxygen species generation in mycelia of a cox7c-deletion strain suggested that this reduction may, in part, contribute to drug resistance. These findings increase our understanding of how A. fumigatus's direct responses to azoles promote fungal survival in the environment and address genetic mutations that arise from patients or environments.

Ofloxacin-loaded HMPB NPs for Klebsiella pneumoniae eradication in the surgical wound with the combination of PTT.

Klebsiella pneumoniae (K. pneumoniae) is a common bacterium whose drug-resistant can cause surgical failures and incurable infections in hospital patients. Thus, how to reverse or delay the resistance induction has become a great challenge for development antiresistant drug. Recently, the combination of nanomaterial-loaded antibiotics with photothermal therapy showed the efficient antibacteria ability under a low dosage of antibiotics. In this study, a nanocomposite of HMPB NPs with inherent photothermal therapy capability was used to eradicate K. pneumoniae after loading with Ofloxacin, an antibiotic against K. pneumoniae in vitro and in vivo. The nanocomplexes named as Ofloxacin@HMPB@HA NPs showed a higher effect against K. pneumoniae by destroying cell integrity and inducing ATP leakage with the assistance of laser irradiation, compared with sole Ofloxacin@HMPB@HA NPs or laser irradiation. Surgical wound infection assay further demonstrated the efficient killing K. pneumoniae and promoting the formation of new tissues, as well, which was reflected by the rapid healing of surgical wound. In summary, these results indicate the great potential of this combinational tactic based on Ofloxacin@HMPB@HA NPs for preventing the failure caused by K. pneumoniae infection.

Long-chain unsaturated fatty acids are involved in the viability and itraconazole susceptibility of Aspergillus fumigatus.

The prevalence of invasive aspergillosis with azole resistance is increasing, but the mechanisms underlying the development of resistance and treatment strategies are still limited. The present work is focused on finding a relationship between long-chain unsaturated fatty acids (LCUFAs), Aspergillus fumigatus development, and antifungal resistance. The effects of LCUFAs on antifungal agents in vitro were determined, and the stearic acid desaturase gene (sdeA) of A. fumigatus was characterized. In in vitro antifungal tests, LCUFAs antagonized the antifungal activity of itraconazole by extracting it from media, thereby preventing it from entering cells. The OA auxotrophic phenotype caused by an sdeA deletion confirmed that SdeA was required for OA biosynthesis in A. fumigatus. Furthermore, several low-level sdeA-overexpressing mutants with impaired vegetative growth phenotypes were successfully constructed. Additionally, an sdeA-overexpressing mutant, OEsdeA-5, showed lowered sensitivity levels to itraconazole. Moreover, RNA sequencing of OEsdeA-5 revealed that the altered gene-expression pattern. Through targeted metabolomics, decreased palmitic acid and stearic acid contents, accompanied by higher palmitoleic acid, margaroleic acid, and OA production levels, were found in OEsdeA-5. This study provides a novel insight of understanding of azole resistance and a potential target for drug development.

The OxrA Protein of Aspergillus fumigatus Is Required for the Oxidative Stress Response and Fungal Pathogenesis.

An efficient reactive oxygen species (ROS) detoxification system is vital for the survival of the pathogenic fungus Aspergillus fumigatus within the host high-ROS environment of the host. Therefore, identifying and targeting factors essential for oxidative stress response is one approach to developing novel treatments for fungal infections. The oxidation resistance 1 (Oxr1) protein is essential for protection against oxidative stress in mammals, but its functions in pathogenic fungi remain unknown. The present study aimed to characterize the role of an Oxr1 homolog in A. fumigatus. The results indicated that the OxrA protein plays an important role in oxidative stress resistance by regulating the catalase function in A. fumigatus, and overexpression of catalase can rescue the phenotype associated with OxrA deficiency. Importantly, the deficiency of oxrA decreased the virulence of A. fumigatus and altered the host immune response. Using the Aspergillus-induced lung infection model, we demonstrated that the ΔoxrA mutant strain induced less tissue damage along with decreased levels of lactate dehydrogenase (LDH) and albumin release. Additionally, the ΔoxrA mutant caused inflammation at a lower degree, along with a markedly reduced influx of neutrophils to the lungs and a decreased secretion of cytokine usually associated with recruitment of neutrophils in mice. These results characterize the role of OxrA in A. fumigatus as a core regulator of oxidative stress resistance and fungal pathogenesis. IMPORTANCE Knowledge of ROS detoxification in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of oxidative stress resistance mechanisms. In this study, we demonstrate that OxrA protein localizes to the mitochondria and functions to protect against oxidative damage. We demonstrate that OxrA contributes to oxidative stress resistance by regulating catalase function, and overexpression of catalase (CatA or CatB) can rescue the phenotype that is associated with OxrA deficiency. Remarkably, a loss of OxrA attenuated the fungal virulence in a mouse model of invasive pulmonary aspergillosis and altered the host immune response. Therefore, our finding indicates that inhibition of OxrA might be an effective approach for alleviating A. fumigatus infection. The present study is, to the best of our knowledge, a pioneer in reporting the vital role of Oxr1 protein in pathogenic fungi.

Aspergillus fumigatus Afssn3-Afssn8 Pair Reverse Regulates Azole Resistance by Conferring Extracellular Polysaccharide, Sphingolipid Pathway Intermediates, and Efflux Pumps to Biofilm.

Antifungal treatment is often ineffectual, partly because of biofilm formation. In this study, by using a combined forward and reverse genetic strategy, we identified that nucleus-localized AfSsn3 and its partner AfSsn8, which constitute a Cdk8-cyclin pair, are required for azole resistance in Aspergillus fumigatus Deletion of Afssn3 led to increased absorption and utilization of glucose and amino acids. Interestingly, absorption and utilization of glucose accelerated the extracellular polysaccharide formation, while utilization of the amino acids serine, threonine, and glycine increased sphingolipid pathway intermediate accumulation. In addition, the absence of Afssn3 induced the activity of the efflux pump proteins. These factors indicate the mature biofilm is responsible for the major mechanisms of A. fumigatus resistance to azoles in the ΔAfssn3 mutant. Collectively, the loss of Afssn3 led to two "barrier" layers between the intracellular and extracellular spaces, which consequently decreased drug penetration into the cell.

The function of the three phosphoribosyl pyrophosphate synthetase (Prs) genes in hyphal growth and conidiation in Aspergillus nidulans.

Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyses the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three PRPP synthase-homologous genes (AnprsA, AnprsB and AnprsC), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, AnprsB and AnprsC are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.

Protein phosphatase 2A (PP2A) regulatory subunits ParA and PabA orchestrate septation and conidiation and are essential for PP2A activity in Aspergillus nidulans.

Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. Different activities of PP2A and subcellular localization are determined by its regulatory subunits. Here we identified and characterized the functions of two protein phosphatase regulatory subunit homologs, ParA and PabA, in Aspergillus nidulans. Our results demonstrate that ParA localizes to the septum site and that deletion of parA causes hyperseptation, while overexpression of parA abolishes septum formation; this suggests that ParA may function as a negative regulator of septation. In comparison, PabA displays a clear colocalization pattern with 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei, and deletion of pabA induces a remarkable delayed-septation phenotype. Both parA and pabA are required for hyphal growth, conidiation, and self-fertilization, likely to maintain normal levels of PP2A activity. Most interestingly, parA deletion is capable of suppressing septation defects in pabA mutants, suggesting that ParA counteracts PabA during the septation process. In contrast, double mutants of parA and pabA led to synthetic defects in colony growth, indicating that ParA functions synthetically with PabA during hyphal growth. Moreover, unlike the case for PP2A-Par1 and PP2A-Pab1 in yeast (which are negative regulators that inactivate the septation initiation network [SIN]), loss of ParA or PabA fails to suppress defects of temperature-sensitive mutants of the SEPH kinase of the SIN. Thus, our findings support the previously unrealized evidence that the B-family subunits of PP2A have comprehensive functions as partners of heterotrimeric enzyme complexes of PP2A, both spatially and temporally, in A. nidulans.

Dermal extracellular matrix gelatin delivering Prussian blue nanoparticles to relieve skin flap ischemia.

The survival rate of flap is a crucial factor for determining the success of tissue repair and reconstruction. Flap transplantation surgery often leads to ischemic and reperfusion injury, causing apoptosis and tissue necrosis, which significantly reduces the survival rate of flap. To address this issue, we developed a porcine skin decellularized matrix gel nanocomplex loaded with alprostadil (Alp) in Prussian blue nanoparticles (PB NPs) called Alp@PB-Gel. This gel not only maintained the cell affinity of the extracellular scaffold but also exhibited a high degree of plasticity. In vitro assays demonstrated that Alp@PB-Gel possessed antioxidant activity, scavenging ROS ability, and effectively promoted the angiogenesis and migration of human vascular endothelial cells (HUVECs) by stimulating the proliferation of vascular epithelial cells and fibroblasts. In vivo assays further confirmed that Alp@PB-Gel could effectively alleviate necrosis in the early and late stages after surgery, downregulate the levels of NLRP3 and CD68 to inhibit apoptosis and attenuate inflammation, while upregulate the levels of VEGF and CD31 to promote vascular tissue regeneration. Moreover, Alp@PB-Gel exhibited excellent cell affinity and biocompatibility, highlighting its potential for clinical application.

U2-Net and ResNet50-Based Automatic Pipeline for Bacterial Colony Counting.

In this paper, an automatic colony counting system based on an improved image preprocessing algorithm and convolutional neural network (CNN)-assisted automatic counting method was developed. Firstly, we assembled an LED backlighting illumination platform as an image capturing system to obtain photographs of laboratory cultures. Consequently, a dataset was introduced consisting of 390 photos of agar plate cultures, which included 8 microorganisms. Secondly, we implemented a new algorithm for image preprocessing based on light intensity correction, which facilitated clearer differentiation between colony and media areas. Thirdly, a U2-Net was used to predict the probability distribution of the edge of the Petri dish in images to locate region of interest (ROI), and then threshold segmentation was applied to separate it. This U2-Net achieved an F1 score of 99.5% and a mean absolute error (MAE) of 0.0033 on the validation set. Then, another U2-Net was used to separate the colony region within the ROI. This U2-Net achieved an F1 score of 96.5% and an MAE of 0.005 on the validation set. After that, the colony area was segmented into multiple components containing single or adhesive colonies. Finally, the colony components (CC) were innovatively rotated and the image crops were resized as the input (with 14,921 image crops in the training set and 4281 image crops in the validation set) for the ResNet50 network to automatically count the number of colonies. Our method achieved an overall recovery of 97.82% for colony counting and exhibited excellent performance in adhesion classification. To the best of our knowledge, the proposed "light intensity correction-based image preprocessing→U2-Net segmentation for Petri dish edge→U2-Net segmentation for colony region→ResNet50-based counting" scheme represents a new attempt and demonstrates a high degree of automation and accuracy in recognizing and counting single-colony and multi-colony targets.

A Unique Approach: Biomimetic Graphdiyne-Based Nanoplatform to Treat Prostate Cancer by Combining Cuproptosis and Enhanced Chemodynamic Therapy.

Purpose: Current treatment approaches for Prostate cancer (PCa) often come with debilitating side effects and limited therapeutic outcomes. There is urgent need for an alternative effective and safe treatment for PCa. Methods: We developed a nanoplatform to target prostate cancer cells based on graphdiyne (GDY) and a copper-based metal-organic framework (GDY-CuMOF), that carries the chemotherapy drug doxorubicin (DOX) for cancer treatment. Moreover, to provide GDY-CuMOF@DOX with homotypic targeting capability, we coated the PCa cell membrane (DU145 cell membrane, DCM) onto the surface of GDY-CuMOF@DOX, thus obtaining a biomimetic nanoplatform (DCM@GDY-CuMOF@DOX). The nanoplatform was characterized by using transmission electron microscope, atomic force microscope, X-ray diffraction, etc. Drug release behavior, antitumor effects in vivo and in vitro, and biosafety of the nanoplatform were evaluated. Results: We found that GDY-CuMOF exhibited a remarkable capability to load DOX mainly through π-conjugation and pore adsorption, and it responsively released DOX and generated Cu+ in the presence of glutathione (GSH). In vivo experiments demonstrated that this nanoplatform exhibits remarkable cell-killing efficiency by generating lethal reactive oxygen species (ROS) and mediating cuproptosis. In addition, DCM@GDY-CuMOF@DOX effectively suppresses tumor growth in vivo without causing any apparent side effects. Conclusion: The constructed DCM@GDY-CuMOF@DOX nanoplatform integrates tumor targeting, drug-responsive release and combination with cuproptosis and chemodynamic therapy, offering insights for further biomedical research on efficient PCa treatment.

Phosphoribosyl pyrophosphate synthetase, as a suppressor of the sepH mutation in Aspergillus nidulans, is required for the proper timing of septation.

Timely cytokinesis/septation is essential for hyphal growth and conidiation in Aspergillus nidulans. Genetic analyses have identified that A. nidulans has components of the septum initiation network (SIN) pathway; one of these, SEPH, is a key player for early events during cytokinesis. However, little is known about how the SEPH kinase cascade is regulated by other components. Here, we demonstrate that the phosphoribosyl pyrophosphate synthetase family acts antagonistically against the SIN so that the downregulation of AnPRS family can bypass the requirements of the SIN for septum formation and conidiation. The transcription defect of the Anprs gene family accompanied with the reduction of AnPRS activity causes the formation of hyper-septation as well as the restoration of septation and conidiation in the absence of SEPH. Clearly, the timing and positioning of septation is related to AnPRS activity. Moreover, with the extensive yeast two-hybrid analysis and rescue combination experiments, it demonstrated that AnPRS members are able to form the heterodimers for functional interacting entities but they appear to contribute so unequally that Anprs1 mutant display relatively normal septation, but Anprs2 deletion is lethal. Thus, compared to in yeast, the AnPRS family may have a unique regulation mechanism during septation in filamentous fungi.

Use of external control arms in immune-mediated inflammatory diseases: a systematic review.

OBJECTIVES: External control arms (ECAs) provide useful comparisons in clinical trials when randomised control arms are limited or not feasible. We conducted a systematic review to summarise applications of ECAs in trials of immune-mediated inflammatory diseases (IMIDs). DESIGN: Systematic review with an appraisal of ECA source quality rated across five domains (data collection, study populations, outcome definitions, reliability and comprehensiveness of the dataset, and other potential limitations) as high, low or unclear quality. DATA SOURCES: Embase, Medline and Cochrane Central Register of Controlled Trial were searched through to 12 September 2023. ELIGIBILITY CRITERIA: Eligible studies were single-arm or randomised controlled trials (RCTs) of inflammatory bowel disease, pouchitis, rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis, psoriasis and atopic dermatitis in which an ECA was used as the comparator. DATA EXTRACTION AND SYNTHESIS: Two authors independently screened the search results in duplicate. The characteristics of included studies, external data source(s), outcomes and statistical methods were recorded, and the quality of the ECA data source was assessed by two independent authors. RESULTS: Forty-three studies met the inclusion criteria (inflammatory bowel disease: 16, pouchitis: 1, rheumatoid arthritis: 12, juvenile idiopathic arthritis: 1, ankylosing spondylitis: 5, psoriasis: 3, multiple indications: 4). The majority of these trials were single-arm (33/43) and enrolled adult patients (34/43). All included studies used a historical control rather than a contemporaneous ECA. In RCTs, ECAs were most often derived from the placebo arm of another RCT (6/10). In single-arm trials, historical case series were the most common ECA source (19/33). Most studies (31/43) did not employ a statistical approach to generate the ECA from historical data. CONCLUSIONS: Standardised ECA methodology and reporting conventions are lacking for IMIDs trials. The establishment of ECA reporting guidelines may enhance the rigour and transparency of future research.

The C-22 sterol desaturase Erg5 is responsible for ergosterol biosynthesis and conidiation in Aspergillus fumigatus.

Aspergillus fumigatus is the most prevalent saprophytic fungi and can cause severe invasive aspergillosis in immunocompromised individuals. For infection of A. fumigatus, the small hydrophobic conidia have been shown to play a dominant role. In this study, we found that deletion of erg5, a C-22 sterol desaturase gene which function in the last two steps of ergosterol biosynthesis, was sufficient to block ergosterol biosynthesis and conidiation. The deletion phenotype was further verified by a conditional expression strain of erg5 using the inducible tet-on system. Strikingly, erg5 mutant displays increased susceptibility to antifungal azoles itraconazole. RNA sequencing analysis showed that erg5 deficiency resulted in changes in transcription mainly related to lipid, carbohydrate, and amino acid metabolism. Genes encoding ergosterol biosynthesis-related enzymes were found to be up-regulated in erg5 null mutants. However, genes involved in asexual development, including upstream regulators, melanin biosynthesis enzymes, heterotrimeric G proteins, and MAPK signaling, were down-regulated to various degrees. Furthermore, metabolomic study revealed that erg5 deficiency also resulted in altered lipid and amino acid metabolism, which was consistent with our transcriptomics analysis. Collectively, our study established a link between ergosterol biosynthesis and asexual development at the transcriptomics and metabolomics level in A. fumigatus.

A phosphopantetheinyl transferase gene restricted to Porphyromonas.

The phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of carrier proteins (CPs) from fatty acid synthases (FASs) in primary metabolism and from polyketide synthases (PKSs) and non-ribosomal polypeptide synthases (NRPSs) in secondary metabolism. Based on the conserved sequence motifs and substrate specificities, two types (AcpS-type and Sfp-type) of PPTases have been identified in prokaryotes. We present here that Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, harbors merely one PPTase, namely PptP. Complementation and gene deletion experiments clearly show that PptP can replace the function of Escherichia coli AcpS and is essential for the growth of P. gingivalis. Purified PptP transfers the 4-phosphopantetheine moiety of CoA to inactive apo-acyl carrier protein (ACP) to form holo-ACP, which functions as an active carrier of the acyl intermediates of fatty acid synthesis. Moreover, PptP exhibits broad substrate specificity, modifying all ACP substrates tested and catalyzing the transfer of coenzyme A (CoA) derivatives. The lack of sequence alignment with known PPTases together with phylogenetic analyses revealed PptP as a new class of PPTases. Identification of the new PPTase gene pptP exclusive in Porphyromonas species reveals a potential target for treating P. gingivalis infections.

mRNA vaccine-a desirable therapeutic strategy for surmounting COVID-19 pandemic.

As an acute respiratory infectious disease, COVID-19 threatens the safety of global public health. Given the current lack of specific treatment against this disease, research and development of vaccines have become sharp weapons for overcoming the pandemic. mRNA vaccines have become the lead in COVID-19 vaccination strategies due to their advantages, such as rapid industrial production and efficacy. A total of 137 COVID-19 vaccines have entered the clinical trial stage, among which 23 are mRNA vaccines, accounting for 17% of the total vaccines. Herein, we summarize the research and developmental processes of mRNA vaccines as well as the approach for protecting the human body against infection. Focusing on the latest clinical trial data of two COVID-19 mRNA vaccines from Pfizer and Modena, we discuss their effectiveness and safety. Finally, we analyze the challenges and problems that mRNA vaccines face in controlling the COVID-19 pandemic.

Heat-Treated Meat Origin Tracing and Authenticity through a Practical Multiplex Polymerase Chain Reaction Approach.

Meat adulteration have become a global issue, which has increasingly raised concerns due to not only economic losses and religious issues, but also public safety and its negative effects on human health. Using optimal primers for seven target species, a multiplex PCR method was developed for the molecular authentication of camel, cattle, dog, pig, chicken, sheep and duck in one tube reaction. Species-specific amplification from the premixed total DNA of seven species was corroborated by DNA sequencing. The limit of detection (LOD) is as low as 0.025 ng DNA for the simultaneous identification of seven species in both raw and heat-processed meat or target meat: as little as 0.1% (w/w) of the total meat weight. This method is strongly reproducible even while exposed to intensively heat-processed meat and meat mixtures, which renders it able to trace meat origins in real-world foodstuffs based on the authenticity assessment of commercial meat samples. Therefore, this method is a powerful tool for the inspection of meat adulterants and has broad application prospects.

Detection and characterization of meat adulteration in various types of meat products by using a high-efficiency multiplex polymerase chain reaction technique.

Identification of meat authenticity is a matter of increasing concerns due to religious, economical, legal, and public health reasons. However, little is known about the inspection of eight meat species in one tube reaction due to technological challenge of multiplex polymerase chain reaction (PCR) techniques. Here, a developed multiplex PCR method can simultaneously authenticate eight meat species including ostrich (753 bp), cat (564 bp), goose (391 bp), duck (347 bp), chicken (268 bp), horse (227 bp), dog (190 bp), and sheep (131 bp). The detectable deoxyribonucleic acid (DNA) contents for each target species was as low as 0.01 ng in both raw and heat-treated meat or target meat down to 0.1% (w/w) of total meat weight reflecting high stability of the assay in heat processing condition, indicating that this method is adequate for tracing meat origin in real-world meat products, which has been further validated by authenticity assays of commercial meat products. Overall, this method is a powerful tool for accurate evaluation of meat origin with a good application foreground.

A Heptaplex PCR Assay for Molecular Traceability of Species Origin With High Efficiency and Practicality in Both Raw and Heat Processing Meat Materials.

Frequent meat frauds have become a global issue because adulteration risks the food safety, breaches market rules, and even threatens public health. Multiplex PCR is considered to be a simple, fast, and inexpensive technique that can be applied for the identification of meat products in food industries. However, relatively less is known about a multiplex PCR method authenticating seven animal species simultaneously in one reaction due to technological challenge. Through screening new species-specific primers and optimizing PCR system, a heptaplex PCR method was established, which could simultaneously detect seven meat ingredients of camel (128 bp), pigeon (157 bp), chicken (220 bp), duck (272 bp), horse (314 bp), beef (434 bp), and pork (502 bp) in a single-tube reaction. DNA sequencing solidly validated that each set of primers specifically amplified target species from total DNA mixtures of seven meat species. The developed multiplex assay was stable and sensitive enough to detect 0.01-0.025 ng DNA from various meat treatments including raw, boiled, and autoclaved meat samples or target meat content of 0.1% total meat weight, suggesting the suitability of the heptaplex PCR technique for tracing target meats with high accuracy and precision. Most importantly, a market survey validated the availability of this multiplex PCR technique in real-world meat products with a good application foreground.