Extracellular vesicles-encapsulated let-7i shed from bone mesenchymal stem cells suppress lung cancer via KDM3A/DCLK1/FXYD3 axis.

Accumulating evidence has suggested that extracellular vesicles (EVs) play a crucial role in lung cancer treatment. Thus, we aimed to investigate the modulatory role of bone marrow mesenchymal stem cell (BMSC)-EV-derived let-7i and their molecular mechanism in lung cancer progression. Microarray-based analysis was applied to predict lung cancer-related miRNAs and their downstream genes. RT-qPCR and Western blot analyses were conducted to determine Let-7i, lysine demethylase 3A (KDM3A), doublecortin-like kinase 1 (DCLK1) and FXYD domain-containing ion transport regulator 3 (FXYD3) expressions, after which dual-luciferase reporter gene assay and ChIP assay were used to identify the relationship among them. After loss- and gain-of-function assays, the effects of let-7i, KDM3A, DCLK1 and FXYD3 on the biological characteristics of lung cancer cells were assessed. Finally, tumour growth in nude mice was assessed by xenograft tumours in nude mice. Bioinformatics analysis screened out the let-7i and its downstream gene, that is KDM3A. The findings showed the presence of a high expression of KDM3A and DCLK1 and reduced expression of let-7i and FXYD3 in lung cancer. KDM3A elevated DCLK1 by removing the methylation of H3K9me2. Moreover, DCLK1 suppressed the FXYD3 expression. BMSC-EV-derived let-7i resulted in the down-regulation of KDM3A expression and reversed its promoting role in lung cancer development. Consistently, in vivo experiments in nude mice also confirmed that tumour growth was suppressed by the BMSC-EV-derived let-7i. In conclusion, our findings demonstrated that the BMSC-EV-derived let-7i possesses an inhibitory role in lung cancer progression through the KDM3A/DCLK1/FXYD3 axis, suggesting a new molecular target for lung cancer treatment.

Linker Editing of Pneumococcal Lysin ClyJ Conveys Improved Bactericidal Activity.

Streptococcus pneumoniae is a leading human pathogen uniquely characterized by choline moieties on the bacterial surface. Our previous work reported a pneumococcus-specific chimeric lysin, ClyJ, which combines the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) enzymatically active domain (EAD) from the PlyC lysin and the cell wall binding domain (CBD) from the phage SPSL1 lysin, which imparts choline binding specificity. Here, we demonstrate that the lytic activity of ClyJ can be further improved by editing the linker sequence adjoining the EAD and CBD. Keeping the net charge of the linker constant, we constructed three ClyJ variants containing different lengths of linker sequence. Circular dichroism showed that linker editing has only minor effects on the folding of the EAD and CBD. However, thermodynamic examination combined with biochemical analysis demonstrated that one variant, ClyJ-3, with the shortest linker, displayed improved thermal stability and bactericidal activity, as well as reduced cytotoxicity. In a pneumococcal mouse infection model, ClyJ-3 showed significant protective efficacy compared to that of the ClyJ parental lysin or the Cpl-1 lysin, with 100% survival at a single ClyJ-3 intraperitoneal dose of 100 µg/mouse. Moreover, a ClyJ-3 dose of 2 µg/mouse had the same efficacy as a ClyJ dose of 40 µg/mouse, suggesting a 20-fold improvement in vivo Taking these results together, the present study not only describes a promising pneumococcal lysin with improved potency, i.e., ClyJ-3, but also implies for the first time that the linker sequence plays an important role in determining the activity of a chimeric lysin, providing insight for future lysin engineering studies.

2-Methylcitrate cycle: a well-regulated controller of Bacillus sporulation.

Bacillus thuringiensis is the most widely used eco-friendly biopesticide, containing two primary determinants of biocontrol, endospore and insecticidal crystal proteins (ICPs). The 2-methylcitrate cycle is a widespread carbon metabolic pathway playing a crucial role in channelling propionyl-CoA, but with poorly understood metabolic regulatory mechanisms. Here, we dissect the transcriptional regulation of the 2-methylcitrate cycle operon prpCDB and report its unprecedented role in controlling the sporulation process of B. thuringiensis. We found that the transcriptional activity of the prp operon encoding the three critical enzymes PrpC, PrpD, and PrpB in the 2-methylcitrate cycle was negatively regulated by the two global transcription factors CcpA and AbrB, while positively regulated by the LysR family regulator CcpC, which jointly account for the fact that the 2-methylcitrate cycle is specifically and highly active in the stationary phase of growth. We also found that the prpD mutant accumulated 2-methylcitrate, the intermediate metabolite of the 2-methylcitrate cycle, which delayed and inhibited sporulation at the early stage. Thus, our results not only revealed sophisticated transcriptional regulatory mechanisms for the metabolic 2-methylcitrate cycle but also identified 2-methylcitrate as a novel regulator of sporulation in B. thuringiensis.

Construction and characterization of a chimeric lysin ClyV with improved bactericidal activity against Streptococcus agalactiae in vitro and in vivo.

The emergence of antibiotic-resistant beta-hemolytic Streptococcus agalactiae strains poses increasing threat to human beings globally. As an attempt to create a novel lysin with improved activity against S. agalactiae, a chimeric lysin, ClyV, was constructed by fusing the enzymatically active domain (EAD) from PlyGBS lysin (GBS180) and the cell wall binding domain (CBD) from PlyV12 lysin (V12CBD). Plate lysis assay combined with lytic kinetic analysis demonstrated that ClyV has improved activity than its parental enzymatic domain GBS180 against multiple streptococci. Biochemical characterization showed that ClyV is active from pH 7 to 10, with the optimum pH of 9, and is stable under NaCl concentration of < 500 mM. In a S. agalactiae infection model, a single intraperitoneally administration of 0.1 mg/mouse of ClyV protected 100% mice, while it was observed that ~ 29% survive in group that received a single dose of 0.1 mg/mouse of GBS180. Moreover, a high dose of 0.8 mg/mouse ClyV did not show any adverse effects to the health or survival rate of the mice. Considering the robust bactericidal activity and good safety profile of ClyV, it represents a potential candidate for the treatment of S. agalactiae infections.

Effects of Microplate Type and Broth Additives on Microdilution MIC Susceptibility Assays.

The determination of antibiotic potency against bacterial strains by assessment of their minimum inhibitory concentration normally uses a standardized broth microdilution assay procedure developed more than 50 years ago. However, certain antibiotics require modified assay conditions in order to observe optimal activity. For example, daptomycin requires medium supplemented with Ca2+, and the lipoglycopeptides dalbavancin and oritavancin require Tween 80 to be added to the growth medium to prevent the depletion of free drug via adsorption to the plastic microplate. In this report, we examine systematically the effects of several different plate types on microdilution broth MIC values for a set of antibiotics against Gram-positive and Gram-negative bacteria, both in medium alone and in medium supplemented with the commonly used additives Tween 80, lysed horse blood, and 50% human serum. We observed very significant differences in measured MICs (up to 100-fold) for some lipophilic antibiotics, such as the Gram-positive lipoglycopeptide dalbavancin and the Gram-negative lipopeptide polymyxins, and found that nonspecific binding plates can replace the need for surfactant additives. Microtiter plate types and any additives should be specified when reporting broth dilution MIC values, as results can vary dramatically for some classes of antibiotics.

ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain.

Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

Silencing of TRPM8 inhibits aggressive tumor phenotypes and enhances gemcitabine sensitivity in pancreatic cancer.

The transient receptor potential TRPM8 ion channel is required for cellular proliferation in pancreatic epithelia and adenocarcinoma. To elucidate the mechanism that mediates the function of TRPM8, we examined its role in the proliferation and invasion of pancreatic cancer (PC) cells. TRPM8 expression increased in both the PC tissues and cell lines; a high TRPM8 expression was correlated with poorer prognosis in patients with PC. In PC cell lines, PACN-1 and BxPC-3, Ca2+ influxes could be evoked by TRPM8; the sensitivity of PC cells to gemcitabine was increased, while the proliferation and invasion of PC cells were suppressed after RNA interference-mediated silencing of TRPM8. The mechanism of TRPM8 in gemcitabine-based chemotherapy was then investigated. The expression and activity of multidrug resistance-associated proteins, P-gp, MRP-2, LRP, was significantly reduced in response to TRPM8 silence. Moreover, TRPM8 knockdown significantly increased hENT1 protein levels and the ratio of Bax/Bcl-2 while decreased the protein levels of RRM1. Thus, TRPM8 is required for PC cell proliferation and invasion and was closely related to the gemcitabine sensitivity of PC. The modulation of TRPM8 expression may help improve treatment response of PC by combining with traditional chemotherapy.

[Regulation of sporulation by two-component system YvcPQ in Bacillus thuringiensis].

Objective: To study the regulation of sporulation controlled by two-component system (TCS) YvcPQ. Methods: ß-galactosidase experiment was used to verify the regulation of YvcP on kapD expression; bacterial one-hybrid assay, EMSA and RT-qPCR were applied to study the regulation of AbrB on yvcPQ expression; markerless gene deletion coupled with spore count was used to reveal the influence of yvcPQ and kapD expressions on sporulation. Results: transcriptional regulator AbrB up-regulated the expression of yvcPQ; YvcP promoted the expression of kapD to inhibit sporulation. Conclusion: AbrB up-regulated the transcription of yvcPQ operon, then the increased YvcP strengthened the transcriptional acitivation of sporulation inhibitor gene kapD, and subsequently inhibited sporulation.

Identification and functional characteristics of a novel splicing heterozygote variant of COL2A1 associated with Stickler syndrome type I.

Background: Stickler syndrome type I (STL1) is an autosomal dominant disorder characterized by ocular, auditory, orofacial, and skeletal anomalies. The main causes of STL1 are variants in the COL2A1 gene, which encodes a type II collagen precursor protein. The specific focus of this study was on a newborn from China diagnosed with STL1, with the aim of providing novel insights into the effects of a newly identified intronic variant in the COL2A1 gene on pre-mRNA splicing. Methods: Trio whole exome sequencing was used to identify the causative variant in the family. The identified variant was validated using Sanger sequencing. Bioinformatics programs were used to predict the pathogenicity of the candidate variant. Additionally, an in vitro minigene assay was used to investigate the effects of the identified variant on RNA splicing. Results: The proband with STL1 had a novel heterozygous splicing variant in the intron nine acceptor donor site of COL2A1 (c.655-2A>G). This splice junction variant resulted in aberrant COL2A1 mRNA splicing, leading to the skipping of exon 10 and the production of a shorter protein that may lack the last 18 native amino acids. Conclusion: The c.655-2A>G variant in the COL2A1 gene leads to STL1 through abnormal splicing. By expanding the spectrum of variants in the COL2A1 gene, this finding improves the clinical understanding of STL1 and provides guidance for early diagnosis and disease counseling.

Identification of a novel intronic variant in COL4A2 gene associated with fetal severe cerebral encephalomalacia and subdural hemorrhage.

BACKGROUND: Genetic variants in COL4A2 are less common than those of COL4A1 and their fetal clinical phenotype has not been well described to date. We present a fetus from China with an intronic variant in COL4A2 associated with a prenatal diagnosis of severe cerebral encephalomalacia and subdural hemorrhage. METHODS: Whole exome sequencing (WES) was applied to screen potential genetic causes. Bioinformatic analysis was performed to predict the pathogenicity of the variant. In in vitro experiment, the minigene assays were performed to assess the variant's effect. RESULTS: In this proband, we observed ventriculomegaly, subdural hemorrhage, and extensive encephalomalacia that initially suggested cerebral hypoxic-ischemic and/or hemorrhagic lesions. WES identified a de novo heterozygous variant c.549 + 5G > A in COL4A2 gene. This novel variant leads to the skipping of exon 8, which induces the loss of 24 native amino acids, resulting in a shortened COL4A2 protein (p.Pro161_Gly184del). CONCLUSION: Our study demonstrated that c.549 + 5G > A in COL4A2 gene is a disease-causing variant by aberrant splicing. This finding enriches the variant spectrum of COL4A2 gene, which not only improves the understanding of the fetal neurological disorders associated with hypoxic-ischemic and hemorrhagic lesions from a clinical perspective but also provides guidance on genetic diagnosis and counseling.

Metabolomics profiling of maternal and umbilical cord blood in normoglycemia macrosomia.

Background: Macrosomia is a common disorder that occurs during pregnancy. We investigated the comprehensive metabolite profiles of pregnant maternal and fetal sera in normoglycemic macrosomia in a Chinese population. Methods: Forty pregnant women and their fetuses were included in the study (twenty macrosomia patients and twenty normal-weight controls). Maternal and umbilical cord serum metabolites were identified using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Results: In total, 203 metabolites were identified. Lipids and lipid-like molecules were the predominant metabolites. Fifty-three metabolites with significant differences were obtained in the maternal samples. In the macrosomia group, the levels of docosahexaenoic acid, eicosapentaenoic acid, and arachidonic acid were significantly higher than those in the control group. Umbilical cord serum samples were obtained for 24 different metabolites. The maternal-fetal gradient of polyunsaturated fatty acids was decreased in the macrosomia group. Aconitic acid, citric acid, isocitric acid, 2-methylhexanoic acid, and 12-hydroxystearic acid were the common differential metabolites in the maternal and umbilical cord serum samples. Conclusion: There were obvious metabolic abnormalities in the sera of pregnant women and fetuses with macrosomia. Lipids and lipid-like molecules were the predominant differential metabolites but had different classifications in the maternal and umbilical cord serum. These results may provide new insights into the long-term metabolic disorders associated with macrosomia.

LncRNA VPS9D1-AS1 Promotes Malignant Progression of Lung Adenocarcinoma by Targeting miRNA-30a-5p/KIF11 Axis.

Objective: This research probed into the molecular mechanisms of long non-coding RNA (lncRNA) VPS9D1 Antisense RNA 1 (VPS9D1-AS1) in lung adenocarcinoma (LUAD). Methods: lncRNA expression level was evaluated bioinformatically, and its downstream miRNA/mRNA regulatory axis was predicted by bioinformatics methods as well. qRT-PCR was used to measure VPS9D1-AS1, miRNA-30a-5p, and kinesin family member 11 (KIF11) expression. Western blot was performed to measure KIF11 protein expression. Proliferation, migration, and invasion of LUAD cells were all observed by cell biological function experiments. Dual-luciferase assay detected binding between miRNA-30a-5p and VPS9D1-AS1 or KIF11, respectively. RIP experiment detected interaction between VPS9D1-AS1 and miRNA-30a-5p. Results: VPS9D1-AS1 and KIF11 were increased in LUAD, whereas miRNA-30a-5p was decreased. VPS9D1-AS1 promoted the malignant progression of LUAD cells and could sponge miRNA-30a-5p. MiRNA-30a-5p could restore the impact of VPS9D1-AS1 on LUAD cells. KIF11 was a target downstream of miRNA-30a-5p. VPS9D1-AS1 could upregulate KIF11 expression through competitively sponging miRNA-30a-5p, and KIF11 could restore the impact of miRNA-30a-5p on LUAD cells. Conclusion: VPS9D1-AS1 could foster malignant progression of LUAD via regulating miRNA-30a-5p/KIF11 axis, suggesting that VPS9D1-AS1 is key to regulating the malignant progression of LUAD.

Internal cell-penetrating peptide-mediated internalization enables a chimeric lysin to target intracellular pathogens.

Intracellular pathogens pose serious challenges to the public health worldwide. Lysin, peptidoglycan hydrolase from phage, is promising alternative to conventional antibiotics because of its high bactericidal activity and low risk of resistance. However, most proteinaceous lysins cannot penetrate the mammalian cell membrane because of size exclusion. Previously, we reported a broad-spectrum chimeric lysin, ClyR, with a cysteine, histidine-dependent amidohydrolase/peptidase catalytic domain from PlyC lysin and an SH-3b cell-wall binding domain from PlySs2 lysin. Herein, we further report that a novel internal cell-penetrating peptide (CPP) is predicted in the junction region of the two constitutive domains of ClyR, mediated by which ClyR can be internalized by epithelial cells through caveolin-dependent endocytosis to target intracellular pathogens. Residues K153, P154, R169, and R188 of the internal CPP were found to be essential for ClyR-mediated internalization and intracellular killing. RNA-seq analysis further showed that there are minor differences in transcript and metabolic profiles from epithelial cells exposed to 100 µg/ml ClyR for 24 h. Taken together, our findings demonstrate a novel mechanism of internalization by ClyR, providing new insights into the rational designing of the next-generation lysins to target both extracellular and intracellular pathogens.

[Study on the dynamic variation of active components in Geranium carolinianum from different collection time].

OBJECTIVE: To determine the contents of the active components, gallic acid and ellagic acid in Geranium carolinianum from different collection time and to define the best collection time for this herb. METHODS: The contents of gallic acid and ellagic acid in each samples of Geranium carolinianum were determined by HPLC. The HPLC method was performed on a Diamonsil C, 8 column (150 mm x 4.6 mm, 5 microm) with acetonitrile-0.1% H3PO4 as mobile phase. The flow rate was 1.0 mL/min, the detection wavelength was 274 nm and the column temperature was 25 degrees C. RESULTS: The calibration curve of gallic acid and ellagic acid were linear in the range of 0.075-5.00 microg (r = 0.9995) and 0.05-2.00 microg (r = 0.9995), respectively. The average recovery of gallic acid and ellagic acid were 99.88% (RSD = 1.19%) and 99.08% (RSD = 2.81%), respectively. CONCLUSION: The content of gallic acid and ellagic acid in Geranium carolinianum both began to increase in flowering stage and increased to the maximum value in immature-fruit stage.

Clinical Effect of Preservation or Nonpreservation of Left Colic Artery in Total Mesorectal Excision under Laparoscopy: A Meta-analysis.

BACKGROUND AND AIMS: To investigate the clinical effect of preservation or nonpreservation of the left colic artery (LCA) in total mesorectal excision (TME) under laparoscopy. METHODS: The words, like "rectal cancer," "left colonic artery," and "laparoscopy," were used as the retrieval terms, and the keyword retrieval method was adopted. The retrieval period was set as from January 1, 2013, to June 1, 2018. We searched databases including PubMed, Web of Science, and China National Knowledge Infrastructure (CNKI) to collect randomized and controlled trials which compared the effect of preservation or nonpreservation of the LCA in TME under laparoscopy. Two researchers independently carried out literature screening, data extraction, and literature quality evaluation; Review Manager 5.3 was used for the meta-analysis. RESULTS: Seven studies including 1467 cases were identified for the meta-analysis. As showed by the meta-analysis, compared with the LCA nonpreservation group, the LCA preservation group had significantly reduced incidence of anastomotic leakage (OR = 0.44, CI = [0.30, 0.65], P < 0.0001) and postoperative urinary and sexual dysfunction (OR = 0.26, CI = [0.09, 0.78], P = 0.02) and significantly shorter time for intestinal function recovery (WMD = -0.26, CI = [-0.41, -0.11], P = 0.0008). There were no significant differences between the two groups in the duration of surgery, blood loss, number of dissected lymph nodes, or postoperative hospital stay. CONCLUSIONS: From the results, the LCA preservation group seems to achieve comparable success with acceptable safety outcomes. Therefore, this surgical method can be recommended in the clinical practice.

How Does Exercise Improve Implicit Emotion Regulation Ability: Preliminary Evidence of Mind-Body Exercise Intervention Combined With Aerobic Jogging and Mindfulness-Based Yoga.

Purpose: The primary aim of the present study is to examine the effect of 8-week mind-body exercise intervention combining aerobic jogging and mindfulness-based yoga on implicit emotion regulation ability. The secondary aim is to explore the specific potential pathways by which the mind-body exercise intervention fosters implicit emotion regulation. This may help us to understand how the key components of exercise intervention contribute to emotional benefits. Methods: Sixty participants were randomly allocated to one of two parallel groups: (1) the intervention group (n = 29) and (2) the waitlist control group (n = 31). Participants were asked to fill out scales measuring mindfulness and instructed to complete an emotion regulation task to assess implicit emotion regulation ability as well as the PWC 170 Test to evaluate aerobic fitness before and after the intervention. Results: The results of the two-way repeated ANOVA revealed that 8 weeks of intervention improved implicit emotion regulation, mindfulness, and aerobic fitness levels. Path analysis showed that only improved aerobic fitness mediated the intervention effect on implicit emotion regulation ability, controlling for change in negative affect. Notably, the relationship between the effects on implicit emotion regulation ability and aerobic fitness was moderated by improved mindfulness. Conclusion: Eight weeks of mind-body exercise intervention improves implicit emotion regulation ability. The aerobic fitness may be an essential pathway which mediates the efficacy on implicit emotion regulation ability. Furthermore, different components, such as aerobic fitness and mindfulness, may interactively contribute to such emotional benefits.

The role of systemic handling in the pathophysiologic actions of botulinum toxin.

The ability of botulinum toxin to poison cholinergic nerve transmission is a dynamic phenomenon that involves not only the actions of the toxin on the body but also the actions of the body on the toxin. The former has been the subject of intense research, whereas the latter has received almost no attention. Therefore, a series of studies were performed to characterize systemic handling of botulinum toxin. The results indicated that the toxin reaches the general circulation (transcytosis across epithelial cells) without obvious changes in structure or biological activity. The general circulation acts as a holding compartment until there is adequate fractional distribution to neuromuscular junctions to produce blockade of transmission. During its transit through this compartment, the toxin 1) undergoes little biotransformation, 2) does not accumulate significantly in circulating cells, and 3) remains largely in the free state. In naive animals, the t(1/2) for toxin in the general circulation is approximately 10 h, and at any given point in time, there is little uptake in nontarget organs (liver, kidney, heart, and lung). In immunized animals, toxin clearance from the general circulation is rapid, and there is substantial accumulation of antibody-antigen complexes in liver. Thus, enhanced clearance from the circulation is a major mechanism by which active immunization can protect against poisoning.

Multi-Granularity Whole-Brain Segmentation Based Functional Network Analysis Using Resting-State fMRI.

In this work, we systematically analyzed the effects of various nodal definitions, as determined by a multi-granularity whole-brain segmentation scheme, upon the topological architecture of the human brain functional network using the resting-state functional magnetic resonance imaging data of 19 healthy, young subjects. A number of functional networks were created with their nodes defined according to two types of anatomical definitions (Type I and Type II) each of which consists of five granularity levels of whole brain segmentations with each level linked through ontology-based, hierarchical, structural relationships. Topological properties were computed for each network and then compared across levels within the same segmentation type as well as between Type I and Type II. Certain network architecture patterns were observed in our study: (1) As the granularity changes, the absolute values of each node's nodal degree and nodal betweenness change accordingly but the relative values within a single network do not change considerably; (2) The average nodal degree is generally affected by the sparsity level of the network whereas the other topological properties are more specifically affected by the nodal definitions; (3) Within the same ontology relationship type, as the granularity decreases, the network becomes more efficient at information propagation; (4) The small-worldness that we observe is an intrinsic property of the brain's resting-state functional network, independent of the ontology type and the granularity level. Furthermore, we validated the aforementioned conclusions and measured the reproducibility of this multi-granularity network analysis pipeline using another dataset of 49 healthy young subjects that had been scanned twice.

Effects of a Chimeric Lysin against Planktonic and Sessile Enterococcus faecalis Hint at Potential Application in Endodontic Therapy.

Enterococcus faecalis is a commensal opportunistic pathogen found in the intestine, mouth, and vaginal tract of humans. As an invasive pathogen in the oral cavity, E. faecalis is one of the leading causes of periapical endodontic lesions. However, due to the strong biofilm-forming capacity and tolerance of E. faecalis to conventional antibiotics and treatments, limited therapeutic options are available. In the present study, we investigated the activity of ClyR, a chimeric lysin with extended streptococcal lytic spectrum, against planktonic and sessile E. faecalis cells in vitro and in an ex vivo dental model. Our results showed that ClyR has robust and rapid lytic activity against multiple E. faecalis strains, killing >90% planktonic cells within 1 min at a concentration of 50 µg/mL. The biochemical experiments combined with microscopy analysis revealed that ClyR degrades E. faecalis biofilm with high efficacy in a dose-dependent manner, reducing the survival rate to 90% viable bacteria within biofilms at a low dose of 50 µg/mL, which is much better than ampicillin and similar to calcium hydroxide, the extensively used routine intracanal medicament in the treatment of endodontics and dental traumatology. The robust activity of ClyR against both planktonic and sessile E. faecalis suggests the potential of ClyR in treating endodontic infections caused by E. faecalis.

Protein-inspired antibiotics active against vancomycin- and daptomycin-resistant bacteria.

The public health threat posed by a looming 'post-antibiotic' era necessitates new approaches to antibiotic discovery. Drug development has typically avoided exploitation of membrane-binding properties, in contrast to nature's control of biological pathways via modulation of membrane-associated proteins and membrane lipid composition. Here, we describe the rejuvenation of the glycopeptide antibiotic vancomycin via selective targeting of bacterial membranes. Peptide libraries based on positively charged electrostatic effector sequences are ligated to N-terminal lipophilic membrane-insertive elements and then conjugated to vancomycin. These modified lipoglycopeptides, the 'vancapticins', possess enhanced membrane affinity and activity against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive bacteria, and retain activity against glycopeptide-resistant strains. Optimised antibiotics show in vivo efficacy in multiple models of bacterial infection. This membrane-targeting strategy has potential to 'revitalise' antibiotics that have lost effectiveness against recalcitrant bacteria, or enhance the activity of other intravenous-administered drugs that target membrane-associated receptors.