Isaridin E Protects against Sepsis by Inhibiting Von Willebrand Factor-Induced Endothelial Hyperpermeability and Platelet-Endothelium Interaction.

Endothelial hyperpermeability is pivotal in sepsis-associated multi-organ dysfunction. Increased von Willebrand factor (vWF) plasma levels, stemming from activated platelets and endothelium injury during sepsis, can bind to integrin αvß3, exacerbating endothelial permeability. Hence, targeting this pathway presents a potential therapeutic avenue for sepsis. Recently, we identified isaridin E (ISE), a marine-derived fungal cyclohexadepsipeptide, as a promising antiplatelet and antithrombotic agent with a low bleeding risk. ISE's influence on septic mortality and sepsis-induced lung injury in a mouse model of sepsis, induced by caecal ligation and puncture, is investigated in this study. ISE dose-dependently improved survival rates, mitigating lung injury, thrombocytopenia, pulmonary endothelial permeability, and vascular inflammation in the mouse model. ISE markedly curtailed vWF release from activated platelets in septic mice by suppressing vesicle-associated membrane protein 8 and soluble N-ethylmaleide-sensitive factor attachment protein 23 overexpression. Moreover, ISE inhibited healthy human platelet adhesion to cultured lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs), thereby significantly decreasing vWF secretion and endothelial hyperpermeability. Using cilengitide, a selective integrin αvß3 inhibitor, it was found that ISE can improve endothelial hyperpermeability by inhibiting vWF binding to αvß3. Activation of the integrin αvß3-FAK/Src pathway likely underlies vWF-induced endothelial dysfunction in sepsis. In conclusion, ISE protects against sepsis by inhibiting endothelial hyperpermeability and platelet-endothelium interactions.

Melatonin attenuates cellular senescence and apoptosis in diabetic nephropathy by regulating STAT3 phosphorylation.

AIMS: Melatonin is an endogenous hormone related to the regulation of biorhythm. Previous researchers have found that melatonin can ameliorate diabetic nephropathy (DN), but the mechanism remains to be elucidated. To discover the possible mechanism by which melatonin prevents DN, we investigated the potential effects of melatonin on signal transducer and activator of transcription 3 (STAT3) on the progression of cellular senescence and apoptosis. MAIN METHODS: Cellular senescence, apoptosis and the underlying mechanism of melatonin were investigated both in vivo and in vitro. C57BL/6 mice were intraperitoneally injected with streptozotocin (STZ) to establish DN. For an in vitro model of DN, human renal cortex proximal epithelial tubule (HK-2) cells were exposed to high glucose conditions. KEY FINDINGS: Melatonin inhibited the phosphorylation of STAT3, decreased the expression of senescence proteins p53, p21 and p16INK4A. Melatonin also downregulated the expression of apoptotic proteins, including cleaved PARP1, cleaved caspase-9 and -3. Melatonin treatment decreased the positive area of senescence-associated galactosidase (SA-ß-gal) staining and the number of TUNEL-positive cells in kidneys of DN mice. In vitro, melatonin inhibited STAT3 phosphorylation and lowered cellular senescence and apoptosis markers, in a manner similar to the STAT3 inhibitor S3I-201. In addition, the inhibition effect of melatonin on cellular senescence and apoptosis in HK-2 cells was reversed by the usage of recombinant IL-6 (rIL-6), which can induce STAT3 phosphorylation. SIGNIFICANCE: We, for the first time, demonstrate that melatonin inhibits STAT3 phosphorylation, which is involved in alleviating the cellular senescence and apoptosis in DN.

Resveratrol alleviates high glucose-induced oxidative stress and apoptosis in rat cardiac microvascular endothelial cell through AMPK/Sirt1 activation.

Diabetic cardiomyopathy (DCM) is a common complication of diabetes. DCM causes extensive lesions on cardiac microvasculature that is predominantly cardiac microvascular endothelial cells (CMECs). Reducing high glucose (HG)-induced damage such as oxidative damage and apoptosis could alleviate the development of DCM. The natural polyphenol resveratrol (RSV) is widely suggested as a cardioprotective agent that protect against DCM. However, limited evidence supports the protection of RSV against oxidative damage and apoptosis and study on the direct effects of RSV in CMEC is missing. Therefore, the current paper aimed to illustrate if RSV could attenuate oxidative stress and apoptosis in CMEC and to investigate the underlying mechanisms. Our data showed that HG elevated reactive oxygen species, malondialdehyde, decreased superoxide dismutase activity, increased apoptotic cell percentage in CMEC, which were reversed by RSV administration. In addition, RSV demonstrated antioxidative and anti-apoptotic effects in CMEC through AMPK/Sirt1 activation, further confirmed by AMPK inhibition or Sirt1 silencing. This study provides new evidence to support RSV as a potential cardioprotective alternative in treating DCM.

Verapamil Alleviates Myocardial Ischemia/Reperfusion Injury by Attenuating Oxidative Stress via Activation of SIRT1.

Myocardial ischemia/reperfusion (I/R) injury is a potential complication of ischemic heart disease after recanalization. One of the primary reasons for I/R injury is the excessive accumulation of reactive oxygen species (ROS) in cardiomyocytes. Verapamil, a classic calcium channel blocker, has the potential to mitigate I/R-evoked oxidative stress. However, the underlying mechanisms have not been fully elucidated. SIRT1 is an essential regulator of I/R and offers resistance to oxidative stress arising from I/R. It is still inconclusive if verapamil can reduce myocardial I/R-triggered oxidative damage through modulating SIRT1 antioxidant signaling. To verify our hypothesis, the H9c2 cardiomyocytes and the mice were treated with verapamil and then exposed to hypoxia/reoxygenation (H/R) or I/R in the presence or absence of the SIRT1 inhibitor EX527. As expected, verapamil stimulated SIRT1 antioxidant signaling evidenced by upregulation of SIRT1, FoxO1, SOD2 expressions and downregulation of Ac-FoxO1 expression in vitro and in vivo. In addition, verapamil remarkably suppressed H/R and I/R-induced oxidative stress proven by declined ROS level and MDA content. The cardioprotective actions of verapamil via SIRT1 were further confirmed in the experiments with the presence of the specific SIRT1 inhibitor EX527. We demonstrated that verapamil alleviated myocardial I/R-evoked oxidative stress partially via activation of SIRT1 antioxidant signaling. Subsequently, verapamil protected against cardiac dysfunction and myocardial infarction accompanied by oxidative stress.

Environmental Bacteriophages of the Emerging Enterobacterial Phytopathogen, Dickeya solani, Show Genomic Conservation and Capacity for Horizontal Gene Transfer between Their Bacterial Hosts.

Dickeya solani is an economically important phytopathogen widespread in mainland Europe that can reduce potato crop yields by 25%. There are no effective environmentally-acceptable chemical systems available for diseases caused by Dickeya. Bacteriophages have been suggested for use in biocontrol of this pathogen in the field, and limited field trials have been conducted. To date only a small number of bacteriophages capable of infecting D. solani have been isolated and characterized, and so there is a need to expand the repertoire of phages that may have potential utility in phage therapy strategies. Here we describe 67 bacteriophages from environmental sources, the majority of which are members of the viral family Myoviridae. Full genomic sequencing of two isolates revealed a high degree of DNA identity with D. solani bacteriophages isolated in Europe in the past 5 years, suggesting a wide ecological distribution of this phage family. Transduction experiments showed that the majority of the new environmental bacteriophages are capable of facilitating efficient horizontal gene transfer. The possible risk of unintentional transfer of virulence or antibiotic resistance genes between hosts susceptible to transducing phages cautions against their environmental use for biocontrol, until specific phages are fully tested for transduction capabilities.

Environmental T4-Family Bacteriophages Evolve to Escape Abortive Infection via Multiple Routes in a Bacterial Host Employing "Altruistic Suicide" through Type III Toxin-Antitoxin Systems.

Abortive infection is an anti-phage mechanism employed by a bacterium to initiate its own death upon phage infection. This reduces, or eliminates, production of viral progeny and protects clonal siblings in the bacterial population by an act akin to an "altruistic suicide." Abortive infection can be mediated by a Type III toxin-antitoxin system called ToxINPa consisting of an endoribonuclease toxin and RNA antitoxin. ToxINPa is a heterohexameric quaternary complex in which pseudoknotted RNA inhibits the toxicity of the toxin until infection by certain phages causes destabilization of ToxINPa, leading to bacteriostasis and, eventually, lethality. However, it is still unknown why only certain phages are able to activate ToxINPa. To try to address this issue we first introduced ToxINPa into the Gram-negative enterobacterium, Serratia sp. ATCC 39006 (S 39006) and then isolated new environmental S 39006 phages that were scored for activation of ToxINPa and abortive infection capacity. We isolated three T4-like phages from a sewage treatment outflow point into the River Cam, each phage being isolated at least a year apart. These phages were susceptible to ToxINPa-mediated abortive infection but produced spontaneous "escape" mutants that were insensitive to ToxINPa. Analysis of these resistant mutants revealed three different routes of escaping ToxINPa, namely by mutating asiA (the product of which is a phage transcriptional co-activator); by mutating a conserved, yet functionally unknown, orf84; or by deleting a 6.5-10 kb region of the phage genome. Analysis of these evolved escape mutants may help uncover the nature of the corresponding phage product(s) involved in activation of ToxINPa.

Evolution of Pectobacterium Bacteriophage ΦM1 To Escape Two Bifunctional Type III Toxin-Antitoxin and Abortive Infection Systems through Mutations in a Single Viral Gene.

Some bacteria, when infected by their viral parasites (bacteriophages), undergo a suicidal response that also terminates productive viral replication (abortive infection [Abi]). This response can be viewed as an altruistic act protecting the uninfected bacterial clonal population. Abortive infection can occur through the action of type III protein-RNA toxin-antitoxin (TA) systems, such as ToxINPa from the phytopathogen Pectobacterium atrosepticum Rare spontaneous mutants evolved in the generalized transducing phage ΦM1, which escaped ToxINPa-mediated abortive infection in P. atrosepticum ΦM1 is a member of the Podoviridae and a member of the "KMV-like" viruses, a subset of the T7 supergroup. Genomic sequencing of ΦM1 escape mutants revealed single-base changes which clustered in a single open reading frame. The "escape" gene product, M1-23, was highly toxic to the host bacterium when overexpressed, but mutations in M1-23 that enabled an escape phenotype caused M1-23 to be less toxic. M1-23 is encoded within the DNA metabolism modular section of the phage genome, and when it was overexpressed, it copurified with the host nucleotide excision repair protein UvrA. While the M1-23 protein interacted with UvrA in coimmunoprecipitation assays, a UvrA mutant strain still aborted ΦM1, suggesting that the interaction is not critical for the type III TA Abi activity. Additionally, ΦM1 escaped a heterologous type III TA system (TenpINPl) from Photorhabdus luminescens (reconstituted in P. atrosepticum) through mutations in the same protein, M1-23. The mechanistic action of M1-23 is currently unknown, but further analysis of this protein may provide insights into the mode of activation of both systems.IMPORTANCE Bacteriophages, the viral predators of bacteria, are the most abundant biological entities and are important factors in driving bacterial evolution. In order to survive infection by these viruses, bacteria have evolved numerous antiphage mechanisms. Many of the studies involved in understanding these interactions have led to the discovery of biotechnological and gene-editing tools, most notably restriction enzymes and more recently the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems. Abortive infection is another such antiphage mechanism that warrants further investigation. It is unique in that activation of the system leads to the premature death of the infected cells. As bacteria infected with the virus are destined to die, undergoing precocious suicide prevents the release of progeny phage and protects the rest of the bacterial population. This altruistic suicide can be caused by type III toxin-antitoxin systems, and understanding the activation mechanisms involved will provide deeper insight into the abortive infection process.

Viunalikeviruses are environmentally common agents of horizontal gene transfer in pathogens and biocontrol bacteria.

Bacteriophages have been used as natural biocontrol and therapeutic agents, but also as biotechnological tools for bacterial engineering. We showed recently that the transducing bacteriophage ϕMAM1 is a ViI-like phage and a member of the new genus, 'Viunalikevirus'. Here, we show that four additional ViI-like phages and three new environmentally isolated viunalikeviruses, all infecting plant and human pathogens, are very efficient generalised transducers capable of transducing chromosomal markers at frequencies of up to 10(-4) transductants per plaque-forming unit. We also demonstrate the interstrain transduction of plasmids and chromosomal markers, including genes involved in anabolism, genes for virulence and genes encoding secondary metabolites involved in biocontrol. We propose that all viunalikeviruses are likely to perform efficient horizontal gene transfer. Viunalikeviruses therefore represent useful agents for functional genomics and bacterial engineering, and for chemical and synthetic biology studies, but could be viewed as inappropriate choices for phage therapy.