Mammalian Commensal Streptococci Utilize a Rare Family of Class VI Lanthipeptide Synthetases to Synthesize Miniature Lanthipeptide-type Ribosomal Peptide Natural Products.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products with remarkable chemical and functional diversities. These peptides are often synthesized as signals or antibiotics and frequently associated with quorum sensing (QS) systems. With the increasing number of available genomes, many hitherto unseen RiPP biosynthetic pathways have been mined, providing new resources for novel bioactive compounds. Herein, we investigated the underexplored biosynthetic potential of Streptococci, prevalent bacteria in mammal-microbiomes that include pathogenic, mutualistic, and commensal members. Using the transcription factor-centric genome mining strategy, we discovered a new family of lanthipeptide biosynthetic loci under the control of potential QS. By in vitro studies, we investigated the reaction of one of these lanthipeptide synthetases and found that it installs only one lanthionine moiety onto its short precursor peptide by connecting a conserved TxxC region. Bioinformatics and in vitro studies revealed that these lanthipeptide synthetases (class VI) are novel lanthipeptide synthetases with a truncated lyase, a kinase, and a truncated cyclase domain. Our data provide important insights into the processing and evolution of lanthipeptide synthetase to tailor smaller substrates. The data are important for obtaining a mechanistic understanding of the post-translational biosynthesis machinery of the growing variety of lanthipeptides.

Development of novel palbociclib-based CDK4/6 inhibitors exploring the back pocket behind the gatekeeper.

CDK4/6 inhibitors plus endocrine therapy is a standard therapy for HR+/HER2- breast cancer. Herein, using structure-based drug design strategy, a novel series of palbociclib derivatives were designed and synthesized as CDK4/6 inhibitors, among which compound 17m exhibited more potent CDK4/6 inhibitory activity and in vitro antiproliferative activity against the phosphorylated Rb-positive cell line MDA-MB-453 than the approved drug palbociclib. Moreover, compound 17m possessed remarkable CDK4/6 selectivity over other CDK family members including CDK1, CDK2, CDK3, CDK5, CDK7 and CDK9. The potent and selective CDK4/6 inhibitory activity endowed compound 17m with robust G1 cell cycle arrest ability in MDA-MB-453 cells. The intracellular inhibition of CDK4/6 by 17m was confirmed by western blot analysis of the levels of phosphorylated Rb in MDA-MB-453 cells. With respect to the metabolic stability, compound 17m possessed longer half-life (t1/2) in mouse liver microsome than palbociclib.

Isolation and genomic analysis of a novel bacteriophage IME278 infecting Enterobacter hormaechei and its biocontrol potential on pork.

Enterobacter hormaechei is an opportunistic pathogen and is found in a large variety of food including animal-derived food. In recent years, bacteria present a severe clinical challenge due to their increasing resistance to antibiotics. Bacteriophages have gained attention as a new antibacterial strategy. In this study, we isolated a novel E. hormaechei bacteriophage IME278 from hospital sewage in Beijing, China. Bacteriophage IME278 had a double-stranded linear DNA genome with 40,164 bp and 51.99% GC content. Whole-genome alignments showed IME278 shared 87% homology with other phages in the National Center for Biotechnology Information (NCBI) database. And phylogenetic analysis demonstrated that IME278 was highly similar to bacteriophages belonging to the genus Kayfunavirus, family Autographiviridae, indicating IME278 can be classified as a new member of the Autographiviridae family. Transmission electron microscopy revealed that IME278 had an icosahedral head 51.72 nm in diameter and a tail 151.28 nm in length. Bacteriophage IME278 was able to survive under high temperature (50 °C-70 °C) and its activity decreased significantly above 70 °C and almost completely inactivated at 80 °C. Bacteriophage IME278 could survive in a wide pH range (4.0-11.0) and it was stable in chloroform (up to 5%). The phage was sensitive to UV irradiation. Bacteriophage IME278 had a latent period of 40 min and reached a plateau stage at 150 min and its cleavage was approximately 8.21 × 108/3.66 × 108 = 2.24. The biocontrol potential of bacteriophage IME278 was evaluated in a model that artificially contaminated pork with E. hormaechei 529 and the result revealed that IME278 could effectively control bacterial contamination on pork. The in-depth analysis of the biological characteristics, whole genome sequencing, and bioinformatics of IME278 has laid the foundation for the biocontrol application and the treatment of bacteria using bacteriophages.

Characterization of the novel temperate Staphylococcus haemolyticus phage IME1365_01.

The presence of a novel functional prophage, IME1365_01, was predicted from bacterial high-throughput sequencing data and then successfully induced from Staphylococcus haemolyticus by mitomycin C treatment. Transmission electron microscopy showed that phage IME1365_01 has an icosahedral head (43 nm in diameter) and a long tail (172 nm long). This phage possesses a double-stranded DNA genome of 44,875 bp with a G+C content of 35.35%. A total of 63 putative open reading frames (ORFs) were identified in its genome. BLASTn analysis revealed that IME1365_01 is similar to Staphylococcus phage vB_SepS_E72, but with a genome homology coverage of only 26%. The phage genome does not have fixed termini. In ORF24 of phage IME1365_01, a conserved Toll-interleukin-1 receptor domain of the TIR_2 superfamily (accession no. c123749) is located at its N-terminus, and this might serve as a component of an anti-bacterial system. In conclusion, we developed a platform to obtain active temperate phage from prediction, identification, and induction from its bacterial host. After mass screening using this platform, numerous temperate phages and their innate anti-bacterial elements can provide extensive opportunities for therapy against bacterial (especially drug-resistant bacterial) infections.

Genetic and Phenotypic Analysis of Phage-Resistant Mutant Fitness Triggered by Phage-Host Interactions.

The emergence of phage-resistant bacterial strains is one of the biggest challenges for phage therapy. However, the emerging phage-resistant bacteria are often accompanied by adaptive trade-offs, which supports a therapeutic strategy called "phage steering". The key to phage steering is to guide the bacterial population toward an evolutionary direction that is favorable for treatment. Thus, it is important to systematically investigate the impacts of phages targeting different bacterial receptors on the fitness of the bacterial population. Herein, we employed 20 different phages to impose strong evolutionary pressure on the host Pseudomonas aeruginosa PAO1 and examined the genetic and phenotypic responses of their phage-resistant mutants. Among these strains with impaired adsorptions, four types of mutations associated with bacterial receptors were identified, namely, lipopolysaccharides (LPSs), type IV pili (T4Ps), outer membrane proteins (OMPs), and exopolysaccharides (EPSs). PAO1, responding to LPS- and EPS-dependent phage infections, mostly showed significant growth impairment and virulence attenuation. Most mutants with T4P-related mutations exhibited a significant decrease in motility and biofilm formation ability, while the mutants with OMP-related mutations required the lowest fitness cost out of the bacterial populations. Apart from fitness costs, PAO1 strains might lose their resistance to antibiotics when counteracting with phages, such as the presence of large-fragment mutants in this study, which may inspire the usage of phage-antibiotic combination strategies. This work provides methods that leverage the merits of phage resistance relative to obtaining therapeutically beneficial outcomes with respect to phage-steering strategies.

Recent developments in maintaining gel properties of surimi products under reduced salt conditions and use of additives.

Salt is a necessary condition to produce a surimi product that is based on the gelation of salt-soluble myofibrillar proteins. Recently, there has been a growing concern among consumers to consume healthy foods due to the threat of several chronic diseases caused by an unhealthy diet. Methods of reducing salt content out of concern for health issues caused by excessive sodium intake may affect the gel properties of surimi, as can many health-oriented food additives. Several studies have investigated different strategies to improve the health characteristics of surimi products without decreasing gel properties. This review reports recent developments in this area and how the gel properties were successfully maintained under reduced-salt conditions and the use of additives. This review of recent studies presents a great deal of progress made in the health benefits of surimi and can be used as a reference for further development in the surimi product processing industry.

Genomic analysis of Acinetobacter phage BUCT629, a newly isolated member of the genus Obolenskvirus.

A new virulent Acinetobacter phage, BUCT629 (GenBank no. MZ712044.1), was isolated from hospital sewage. Next-generation sequencing (NGS) results demonstrated that the double-stranded linear DNA genome of phage BUCT629 is 46,325 bp in length with a G+C content of 38%. The BLASTn analysis showed that the genome sequence shared similarity with Acinetobacter phage vB_AbaM_IME285, with 65% query coverage and 98.23% identity, suggesting that phage BUCT629 is a novel phage. The phage genome contains 89 putative protein-coding genes, and no rRNA or tRNA genes were identified. The results of this study may be helpful for discovering new antibacterial agents and for understanding the evolution and genetic diversity of Acinetobacter phages.

2'-Fucosyllactose Inhibits Coxsackievirus Class A Type 9 Infection by Blocking Virus Attachment and Internalisation.

Coxsackieviruses, a genus of enteroviruses in the small RNA virus family, cause fatal infectious diseases in humans. Thus far, there are no approved drugs to prevent these diseases. Human milk contains various biologically active components against pathogens. Currently, the potential activity of breast milk components against the coxsackievirus remains unclear. In our study, the inhibitory effect of 16 major human milk components was tested on coxsackievirus class A type 9 isolate (CV-A9), BUCT01; 2'-Fucosyllactose (2'-FL) was identified to be effective. Time-of-addition, attachment internalisation assays, and the addition of 2'-FL at different time points were applied to investigate its specific role in the viral life cycle. Molecular docking was used to predict 2'-FL's specific cellular targets. The initial screening revealed a significant inhibitory effect (99.97%) against CV-A9 with 10 mg/mL 2'-FL, with no cytotoxicity observed. Compared with the control group, 2'-FL blocked virus entry (85%) as well as inhibited viral attachment (48.4%) and internalisation (51.3%), minimising its infection in rhabdomyosarcoma (RD) cells. The cell pre-incubation with 2'-FL exhibited significant inhibition (73.2-99.9%). Extended incubation between cells with 2'-FL reduced CV-A9 infection (93.9%), suggesting that 2'-FL predominantly targets cells to block infection. Molecular docking results revealed that 2'-FL interacted with the attachment receptor αvß6 and the internalisation receptor FCGRT and ß2M with an affinity of -2.14, -1.87, and -5.43 kcal/mol, respectively. This study lays the foundation for using 2'-FL as a food additive against CV-A9 infections.

Characterization and Comparative Genomics Analysis of a New Bacteriophage BUCT610 against Klebsiella pneumoniae and Efficacy Assessment in Galleria mellonella Larvae.

The spread of multidrug-resistant Klebsiella pneumoniae (MDR-KP) has become an emerging threat as a result of the overuse of antibiotics. Bacteriophage (phage) therapy is considered to be a promising alternative treatment for MDR-KP infection compared with antibiotic therapy. In this research, a lytic phage BUCT610 was isolated from hospital sewage. The assembled genome of BUCT610 was 46,774 bp in length, with a GC content of 48%. A total of 83 open reading frames (ORFs) and no virulence or antimicrobial resistance genes were annotated in the BUCT610 genome. Comparative genomics and phylogenetic analyses showed that BUCT610 was most closely linked with the Vibrio phage pYD38-A and shared 69% homology. In addition, bacteriophage BUCT610 exhibited excellent thermal stability (4-75 °C) and broad pH tolerance (pH 3-12) in the stability test. In vivo investigation results showed that BUCT610 significantly increased the survival rate of Klebsiella pneumonia-infected Galleria mellonella larvae from 13.33% to 83.33% within 72 h. In conclusion, these findings indicate that phage BUCT610 holds great promise as an alternative agent with excellent stability for the treatment of MDR-KP infection.

Dose-response association of acute-phase quetiapine treatment with risk of new-onset hypothyroidism in schizophrenia patients.

AIMS: To assess association between quetiapine treatment and risk of new-onset hypothyroidism in schizophrenia patients. METHODS: We conducted a retrospective cohort study in a tertiary hospital in China between January 2016 and December 2018. Schizophrenia patients with normal thyroid tests at admission were included. Hypothyroidism, which was defined as thyroid-stimulating hormone >4.20 mU/L and free thyroxine <12.00 pmol/L, or on L-thyroxine prescriptions, was the outcome measure, and quetiapine treatment between admission and subsequent thyroid test was the exposure measure of this study. Adjusted relative risks and 95% confidence intervals were used to assess the independent association of quetiapine treatment with risk of new-onset hypothyroidism. The dose-response association was further analysed by 3 quetiapine doses: low (≤<=0.2 g/d), medium (0.2-0.6 g/d), and high (>0.6 g/d). RESULTS: A total of 2022 eligible patients were included in the final analysis. Sixty patients (15.0%) in the quetiapine group developed hypothyroidism, while 56 patients (3.5%) in the nonquetiapine group developed hypothyroidism. Relative risk (95% confidence interval) of developing hypothyroidism for quetiapine use was 4.01 (2.86-5.64) after adjusting for several potential confounding factors. A strong dose-response association between quetiapine use and risk of developing hypothyroidism was observed: adjusted relative risks (95% confidence intervals) were 1.00 (0.25-2.59), 4.22 (2.80-6.25) and 5.62 (3.66-8.38), respectively, for low-, medium- and high-dose quetiapine, as compared with no quetiapine. CONCLUSION: Acute phase quetiapine treatment for schizophrenia patients was strongly associated with increased risk of developing new-onset hypothyroidism, with a clear dose-response association.

Complete genome sequence of the extreme-pH-resistant Salmonella bacteriophage αα of the family Microviridae.

A novel Salmonella bacteriophage (phage), named αα, was the first reported member of the family Microviridae to exhibit tolerance to both extreme acidic and alkaline conditions (pH 2-12 for 1 h). Phage αα has a circular single-stranded DNA genome of 5,387 nt with a G+C content of 44.66%. A total of 11 putative gene products and no tRNA genes are encoded in the phage αα genome. Whole-genome sequence comparisons revealed that phage αα shares 95% identity with coliphage phiX174 and had a close evolutionary relationship to the phages NC1 and NC7. Phylogenetic analysis of the structural proteins of phage αα and 18 other phiX174-like phages showed that a phylogenetic tree based on protein B sequences had a topology similar to that obtained using whole genome sequences. In addition, variable sites in proteins F and G distributed on the surface of the mature capsid and the conserved protein J were probably involved in maintaining the structural integrity of the phage under extreme pH conditions. Our findings could open up new perspectives for identifying more extreme-pH-resistant phages and their structural proteins and understanding the mechanism of phage adaptation and evolution under extreme environmental stress.

Rapid DNA detection and one-step RNA detection catalyzed by Bst DNA polymerase and narrow-thermal-cycling.

We reported a novel detection method named accelerated strand exchange amplification by employing Bst DNA polymerase and narrow-thermal-cycling for the first time, achieving direct detection of 120 copies of DNA within 15 min and 1.2 × 105 copies of RNA within 20 min and sparking the revolution of the use of routine isothermal polymerases for diverse applications.

Accurate, rapid and low-cost diagnosis of Mycoplasma pneumoniae via fast narrow-thermal-cycling denaturation bubble-mediated strand exchange amplification.

Mycoplasma pneumoniae is a strong infectious pathogen that may cause severe respiratory infections. Since this pathogen may possess a latent period after infection, which sometimes leads to misdiagnosis by traditional diagnosis methods, the establishment of a rapid and sensitive diagnostic method is crucial for transmission prevention and timely treatment. Herein, a novel detection method was established for M. pneumoniae detection. The method, which improves upon a denaturation bubble-mediated strand exchange amplification (SEA) that we developed in 2016, is called accelerated SEA (ASEA). The established ASEA achieved detection of 1% M. pneumoniae genomic DNA in a DNA mixture from multiple pathogens, and the limit of detection (LOD) of ASEA was as low as 1.0 × 10-17 M (approximately 6.0 × 103 copies/mL). Considering that the threshold of an asymptomatic carriage is normally recommended as 1.0 × 104 copies/mL, this method was able to satisfy the requirement for practical diagnosis of M. pneumoniae. Moreover, the detection process was finished within 20.4 min, significantly shorter than real-time PCR and SEA. Furthermore, ASEA exhibited excellent performance in clinical specimen analysis, with sensitivity and specificity of 96.2% and 100%, respectively, compared with the "gold standard" real-time PCR. More importantly, similar to real-time PCR, ASEA requires only one pair of primers and ordinary commercial polymerase, and can be carried out using a conventional fluorescence real-time PCR instrument, which makes this method low-cost and easy to accomplish. Therefore, ASEA has the potential for wide use in the rapid detection of M. pneumoniae or other pathogens in large numbers of specimens. Graphical abstract.

The Vibrio parahaemolyticus-infecting bacteriophage qdvp001: genome sequence and endolysin with a modular structure.

Vibrio parahaemolyticus, a marine pathogen, is a causative agent of gastroenteritis in humans after consumption of contaminated seafood. In recent years, infections with V. parahaemolyticus have become an increasingly frequent factor in microbial food poisoning; therefore, it is urgent to figure out ways to control Vibrio parahaemolyticus. Endolysins, lytic enzymes encoded by bacteriophages, have been regarded as a therapeutic alternative to antibiotics in control of bacterial growth and have been successfully utilized in various areas. Here, we report the full genome sequence of the novel phage qdvp001, which lyses Vibrio parahaemolyticus 17802. The qdvp001 genome consists of a 134,742-bp DNA with a G+C content of 35.35 % and 227 putative open reading frames. Analysis revealed that the qdvp001 open reading frames encoded various putative functional proteins with a putative endolysin gene (ORF 60). No holin genes were identified in qdvp001. ORF 60 was cloned and expressed. The results showed that the purified endolysin Lysqdvp001 had a high hydrolytic activity toward Vibrio parahaemolyticus and a broader spectrum compared to that of the parental bacteriophage qdvp001. Thus, purified endolysin Lysqdvp001 has a potential to be used as an antibacterial agent in the future.

Characterization of the novel T4-like Salmonella enterica bacteriophage STP4-a and its endolysin.

While screening for new antimicrobial agents for multidrug-resistant Salmonella enterica, the novel lytic bacteriophage STP4-a was isolated and characterized. Phage morphology revealed that STP4-a belongs to the family Myoviridae. Bacterial challenge assays showed that different serovars of Salmonella enterica were susceptible to STP4-a infection. The genomic characteristics of STP4-a, containing 159,914 bp of dsDNA with an average GC content of 36.86 %, were determined. Furthermore, the endolysin of STP4-a was expressed and characterized. The novel endolysin, LysSTP4, has hydrolytic activity towards outer-membrane-permeabilized S. enterica and Escherichia coli. These results provide essential information for the development of novel phage-based biocontrol agents against S. enterica.

Cold atmospheric plasma can effectively disinfect SARS-CoV-2 in the wastewater.

COVID-19 is currently pandemic and the detection of SARS-CoV-2 variants in wastewater is causing widespread concern. Herein, cold atmospheric plasma (CAP) is proposed as a novel wastewater disinfection technology that effectively inactivates SARS-CoV-2 transcription- and replication-competent virus-like particles, coronavirus GX_P2V, pseudotyped SARS-CoV-2 variants, and porcine epidemic diarrhoea virus in a large volume of water within 180 s (inhibition rate > 99%). Further, CAP disinfection did not adversely affect the viability of various human cell lines. It is identified that CAP produced peroxynitrite (ONOO-), ozone (O3), superoxide anion radicals (O2 -), and hydrogen peroxide (H2O2) as the major active substances for coronavirus disinfection. Investigation of the mechanism showed that active substances not only reacted with the coronavirus spike protein and affected its infectivity, but also destroyed the nucleocapsid protein and genome, thus affecting virus replication. This method provides an efficient and environmentally friendly strategy for the elimination of SARS-CoV-2 and other coronaviruses from wastewater.

Rapid and highly sensitive colorimetric LAMP assay and integrated device for visual detection of monkeypox virus.

BACKGROUND: The monkeypox virus (MPXV) is a linear double-stranded DNA virus with a large genome that causes tens of thousands of infections and hundreds of deaths in at least 40 countries and regions worldwide. Therefore, timely and accurate diagnostic testing could be an important measure to prevent the ongoing spread of MPXV and widespread epidemics. RESULTS: Here, we designed multiple sets of primers for the target region of MPXV for loop-mediated isothermal amplification (LAMP) detection and identified the optimal primer set. Then, the specificity in fluorescent LAMP detection was verified using the plasmids containing the target gene, pseudovirus and other DNA/RNA viruses. We also evaluated the sensitivity of the colorimetric LAMP detection system using the plasmid and pseudovirus samples, respectively. Besides, we used monkeypox pseudovirus to simulate real samples for detection. Subsequent to the establishment and introduction of a magnetic beads (MBs)-based nucleic acid extraction technique, an integrated device was developed, characterized by rapidity, high sensitivity, and remarkable specificity. This portable system demonstrated a visual detection limit of 137 copies/mL, achieving sample-to-answer detection within 1 h. SIGNIFICANCE: The device has the advantages of integration, simplicity, miniaturization, and visualization, which help promote the realization of accurate, rapid, portable, and low-cost testing. Meanwhile, this platform could facilitate efficient, cost-effective and easy-operable point-of-care testing (POCT) in diverse resource-limited settings in addition to the laboratory.

Long-lasting humoral and cellular memory immunity to vaccinia virus Tiantan provides pre-existing immunity against mpox virus in Chinese population.

Investigating immune memory to vaccinia virus and pre-existing immunity to mpox virus (MPXV) among the population is crucial for the global response to this ongoing mpox epidemic. Blood was sampled from vaccinees inoculated with vaccinia virus Tiantan (VTT) strain born before 1981 and unvaccinated control subjects born since 1982. After at least 40 years of the inoculation, 60% or 5% VTT vaccinees possess neutralizing antibodies (NAbs) to VTT or MPXV, with at least 50% having T cell memory to VTT protein antigens. Notably, 46.7% vaccinees show pre-existing T cell responses to MPXV. Broad pre-existing CD8+ T cell reactivities to MPXV are detected not only against conserved epitopes but also against variant epitopes between VTT and MPXV. Persistent NAbs and T cell memory to VTT among vaccinees, along with pre-existing T cells to MPXV among both vaccinees and the unvaccinated population, indicate a particular immune barrier to mpox.

Complete Genomic Analysis of the Novel Phage BUCT-3589, Infecting Klebsiella pneumoniae.

We report the complete genome sequence of the phage BUCT-3589, infecting multidrug-resistant Klebsiella pneumoniae 3589. It is a new member of the genus Przondovirus in the family Autographiviridae and possesses a double-stranded DNA (dsDNA) genome of 40,757 bp with 53.13% GC content. The genome sequence will support its use as a therapeutic agent.

Characterization, complete genome sequencing, and CRISPR/Cas9 system-based decontamination of a novel Escherichia coli phage TR1 from fermentation substrates.

Phage contamination has become a major concern for industrial bacteria, such as Escherichia coli BL21(DE3), used in fermentation processes. Herein, we report a CRISPR/Cas9 defense system-based strategy to precisely prey and degrade phage DNA to decontaminate target phages. First, we isolated a novel phage from fermentation substrates with BL21(DE3) as the host, named TR1. It showed a typical podovirus morphology with a head diameter of 51.46 ± 2.04 nm and a tail length of 9.31 ± 2.77 nm. The burst size of phage TR1 was 151 PFU/cell, suggesting its strong fecundity in the fermentation system. Additionally, whole-genome sequencing revealed that phage TR1 has a DNA genome of 44,099 bp in length with a 43.8% GC content, encoding a total of 68 open reading frames. Comparative genomics and phylogenetic analysis designated this phage to be a new species of the genus Christensenvirus. To counteract phage TR1, we employed the CRISPR/Cas9 system-based strategy and constructed two phage-resistant E. coli strains, BL21-C and BL21-T, based on conserved genes. Both EOP assays and growth curves indicated strong phage resistance of the recombinant strains, without affecting cell growth. Therefore, this study aimed to provide a resilient strategy to respond to ever-changing phages and ongoing phage-host arm race in industrial fermentation environments by the personalized design of spacers in the recombinant CRISPR/Cas system-containing plasmid. More importantly, our research sparks the use of phage defense mechanism to prevent phage contamination in extensive biotechnological applications.