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Phage-encoded endolysins are emerging antibacterial agents based on their ability to efficiently degrade peptidoglycan on Gram-positive bacteria, but the envelope characteristics of Gram-negative bacteria limit their application. Engineering modifications of endolysins can improve the optimization of their penetrative and antibacterial properties. This study constructed a screening platform to screen for engineered Artificial-Bp7e (Art-Bp7e) endolysins with extracellular antibacterial activity against Escherichia coli. An oligonucleotide of 20 repeated NNK codons was inserted upstream of the endolysin gene Bp7e to construct a chimeric endolysin library in the pColdTF vector. The chimeric Art- Bp7e proteins were expressed by transforming the plasmid library into E. coli BL21 and released by chloroform fumigation, and the protein activities were evaluated by the spotting method and the colony-counting method to screen for promising proteins. Sequence analysis showed that all screened proteins with extracellular activities had a chimeric peptide with a positive charge and an a-helical structure. Also, a representative protein, Art-Bp7e6, was further characterized. It exhibited broad antibacterial activity against E. coli (7/21), Salmonella enterica serovar Enteritidis (4/10), Pseudomonas aeruginosa (3/10), and even Staphylococcus aureus (1/10). In the transmembrane process, the chimeric peptide of Art-Bp7e6 depolarized the host cell envelope, increased the permeability of the cell, and facilitated the movement of Art-Bp7e6 across the envelope to hydrolyze the peptidoglycan. In conclusion, the screening platform successfully screened for chimeric endolysins with extracellular antibacterial activities against Gram-negative bacteria, which provides methodological support for the further screening of engineered endolysins with high extracellular activities against Gram-negative bacteria. Also, the established platform showed broad application prospects and can be used to screen various proteins. [ABSTRACT FROM AUTHOR]

Chicken colibacillosis is caused by some pathogenic Escherichia coli strains. Thirty-five pathogenic antibiotic-resistant E. coli strains were used in the host range detection of bacteriophage Bp7. The phage showed a wide range of E. coli hosts (46%). The complete genome of bacteriophage Bp7 was sequenced, assembled, and analyzed. The results revealed a linear double-stranded DNA sequence of 168,066 bp harboring 791 open reading frames. The major findings from its annotation are described. [ABSTRACT FROM AUTHOR]

Metastatic diseases of the spine are becoming increasingly common with an aging population and improvements in systemic cancer therapies. Microwave and vertebroplasty are the mainstay modalities for treating painful spine metastases. Most early spinal metastases predominantly attack the adnexa, but there are few reports on its treatment. This report presents a case of a 56-year-old female who had experienced severe thoracic back pain for several days and was diagnosed with a metastatic tumor of the right transverse process of T7. Percutaneous microwave ablation in combination with bone cement injection was used to treat the metastatic tumor under CT guidance. The postoperative pain on the Visual Analogue Scale was 1/10, without nerve or vessel damage and bone cement leakage during the operation. [ABSTRACT FROM AUTHOR]

Viral myocarditis (VMC) is a type of cardiovascular disease caused by viral infection of myocardial cells characterized by myocardial interstitital inflammatory cell infiltration, myocardial fiber necrosis or figrinolysis. Ivabradine (IVA) is a commonly known drug used to control heart rate, resist inflammation and oxidative stress, particularly in VMC. Here, we have tried to evaluate the protective effects of IVA on mice with VMC and understand the possible mechanism behind this process. Eighty male mice aged 6 weeks old were randomly divided into normal control, VMC model, low-dose IVA (L-IVA) and high-dose IVA (H-IVA) groups. Half an hour after modeling, IVA aqueous solution was administered intragastrically into L-IVA and H-IVA groups at 5 mg·kg-1·d-1 and 20 mg·kg-1·d-1, respectively for 14 consecutive days. Another 120 mice of the same batch were grouped and treated as described above. At 7 and 14 d, 6 mice in each group were sacrificed to obtain blood and heart samples. Body wt./heart wt. (BW/HW) was calculated, hematoxylin-eosin staining was performed to observe the pathological changes of myocardium, and the level of cardiac troponin I (cTnI) was measured by ELISA. Related kits were employed to measure superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and catalase (CAT) activities in myocardial homogenate, and the levels of interleukin-6 (IL-6), IL-18, IL-1ß and tumor necrosis factor-a (TNF-a) were determined by double-antibody sandwich ELISA. TUNEL assay was performed to detect the apoptosis of myocardial cells. The protein expressions of Bcl-2, Bax and Caspase-3 in myocardial cells were measured by Western blotting. Compared to the VMC model group, the heart/body weight ratio, myocardial pathological score, cTnI level, MDA activity, and levels of IL-6, IL-18, IL-1ß and TNF-a were found decreased, while survival rate and activity of SOD, GSH-Px and CAT increased in L-IVA and H-IVA groups (P <0.05). The apoptosis rate of myocardial cells declined in L-IVA and HIVA groups (P <0.05), especially in H-IVA group. IVA downregulated the protein expressions of Bax and Caspase-3 and upregulated that of Bcl-2 (P <0.05). Thus, it has been found that IVA elevates the survival rate of VMC mice and relieves myocardial damage possibly by enhancing antioxidant capacity and modulating apoptosis-related proteins to suppress apoptosis. [ABSTRACT FROM AUTHOR]

Cellular responses elicited by cell surface receptors differ according to stimulus strength. We investigated how the high-affinity receptor for immunoglobulin E (IgE) modulates the response of mast cells to a high- or low-affinity stimulus. Both high- and low-affinity stimuli elicited similar receptor phosphorylation; however, differences were observed in receptor cluster size, mobility, distribution, and the cells' effector responses. Low-affinity stimulation increased receptor association with the Src family kinase Fgr and shifted signals from the adapter LAT1 to the related adapter LAT2. LAT1-dependent calcium signals required for mast cell degranulation were dampened, but the role of LAT2 in chemokine production was enhanced, altering immune cell recruitment at the site of inflammation. These findings uncover how receptor discrimination of stimulus strength can be interpreted as distinct in vivo outcomes. [ABSTRACT FROM AUTHOR]

Chicken-pathogenic Escherichia coli is severely endangering the poultry industry in China and worldwide, and antibiotic therapy is facing an increasing problem of antibiotic resistance. Bacteriophages can kill bacteria with no known activity in human or animal cells, making them an attractive alternative to antibiotics. In this study, we present the characteristics of a novel virulent bacteriophage, Bp7, specifically infecting pathogenic multidrug-resistant E. coli. Phage Bp7 was isolated from chicken feces. Bp7 belongs to the family Myoviridae, possessing an elongated icosahedral head and contractile sheathed tail. It has a 168-kb double-stranded DNA genome. For larger yields, its optimal multiplicity of infection (MOI) to infect E. coli was about 0.001. The latent period was 10 to 15 min, and the burst size was 90 PFU/infected cell. It was stable both at pH 5.0 to 10.0 and at 40°C or 50°C for at least 1 h. Bp7 could infect 46% of pathogenic clinical E. coli strains. Bp7 harbored 791 open reading frames (ORFs) and 263 possible genes. Among the 263 genes, 199 possessed amino acid sequence identities with ORFs of phage T4,62 had identities with other T4-like phages, and only one lacked any database match. The genome of Bp7 manifested obvious division and rearrangement compared to phages T4, JS98, and IME08. Bp7 is a new member of the "T4-like" genus, family Myoviridae. Its wide host range, strong cell-killing activity, and high stability to pH make it an alternative to antimicrobials for controlling drug-resistant E. coli in chickens. [ABSTRACT FROM AUTHOR]

Mutations of parkin are linked to early onset Parkinson disease. Here we show that stable transfection of parkin in the human dopaminergic neuroblastoma cell line SH-SY5Y markedly reduced the activities of both monoamine oxidase (MAO) A and B. The amount of 3,4-dihydroxyphenylacetic acid, which is produced during dopamine oxidation by MAO, was greatly reduced by parkin overexpression. Radioligand binding assays showed that MAO binding sites were decreased accordingly. Consistent with these, MAO-B protein level was much lower, whereas the amount of MAO-A protein was not determined due to the lack of a suitable antibody. Co-transfection of either MAO with parkin in HEK293 cells did not significantly alter ubiquitination and degradation of each MAO. When we measured MAO expression by real-time quantitative reverse transcription-PCR, marked reductions were seen in SH-SY5Y cells stably expressing parkin compared with the parental cells or a control line stably transfected with luciferase. In addition, parkin mutants defective in E3 ligase activity exhibited different effects on MAO expression. We found that parkin also significantly decreased mRNA levels of both MAOs in the mouse fibroblast cell line NIH3T3. Furthermore, MAO expression was significantly increased in human B lymphocyte cell lines derived from Parkinson disease patients with homozygous but not heterozygous deletion of exon 4 of parkin. Together these results suggest that parkin suppresses MAO expression. This function may limit the production of reactive oxygen species generated by MAO in dopamine oxidation and would, thus, be beneficial to the survival of dopaminergic neurons. [ABSTRACT FROM AUTHOR]

Importance: For patients with large vessel occlusion strokes, it is unknown whether endovascular treatment alone compared with intravenous thrombolysis plus endovascular treatment (standard treatment) can achieve similar functional outcomes.Objective: To investigate whether endovascular thrombectomy alone is noninferior to intravenous alteplase followed by endovascular thrombectomy for achieving functional independence at 90 days among patients with large vessel occlusion stroke.Design, Setting, and Participants: Multicenter, randomized, noninferiority trial conducted at 33 stroke centers in China. Patients (n = 234) were 18 years or older with proximal anterior circulation intracranial occlusion strokes within 4.5 hours from symptoms onset and eligible for intravenous thrombolysis. Enrollment took place from May 20, 2018, to May 2, 2020. Patients were enrolled and followed up for 90 days (final follow-up was July 22, 2020).Interventions: A total of 116 patients were randomized to the endovascular thrombectomy alone group and 118 patients to combined intravenous thrombolysis and endovascular thrombectomy group.Main Outcomes and Measures: The primary end point was the proportion of patients achieving functional independence at 90 days (defined as score 0-2 on the modified Rankin Scale; range, 0 [no symptoms] to 6 [death]). The noninferiority margin was -10%. Safety outcomes included the incidence of symptomatic intracerebral hemorrhage within 48 hours and 90-day mortality.Results: The trial was stopped early because of efficacy when 234 of a planned 970 patients had undergone randomization. All 234 patients who were randomized (mean age, 68 years; 102 women [43.6%]) completed the trial. At the 90-day follow-up, 63 patients (54.3%) in the endovascular thrombectomy alone group vs 55 (46.6%) in the combined treatment group achieved functional independence at the 90-day follow-up (difference, 7.7%, 1-sided 97.5% CI, -5.1% to ∞)P for noninferiority = .003). No significant between-group differences were detected in symptomatic intracerebral hemorrhage (6.1% vs 6.8%; difference, -0.8%; 95% CI, -7.1% to 5.6%) and 90-day mortality (17.2% vs 17.8%; difference, -0.5%; 95% CI, -10.3% to 9.2%).Conclusions and Relevance: Among patients with ischemic stroke due to proximal anterior circulation occlusion within 4.5 hours from onset, endovascular treatment alone, compared with intravenous alteplase plus endovascular treatment, met the prespecified statistical threshold for noninferiority for the outcome of 90-day functional independence. These findings should be interpreted in the context of the clinical acceptability of the selected noninferiority threshold.Trial Registration: Chinese Clinical Trial Registry: ChiCTR-IOR-17013568. [ABSTRACT FROM AUTHOR]

Bp7 is a T-even phage with a broad host range specific to Escherichia coli, including E. coli K-12. The receptor binding protein (RBP) of bacteriophages plays an important role in the phage adsorption process and determines phage host range, but the molecular mechanism involved in host recognition of phage Bp7 remains unknown. In this study, the interaction between phage Bp7 and E. coli K-12 was investigated. Based on homology alignment, amino acid sequence analysis, and a competitive assay, gp38, located at the tip of the long tail fiber, was identified as the RBP of phage Bp7. Using a combination of in vivo and in vitro approaches, including affinity chromatography, gene knockout mutagenesis, a phage plaque assay, and phage adsorption kinetics analysis, we identified the LamB and OmpC proteins on the surface of E. coli K-12 as specific receptors involved in the first step of reversible phage adsorption. Genomic analysis of the phage-resistant mutant strain E. coli K-12-R and complementation tests indicated that HepI of the inner core of polysaccharide acts as the second receptor recognized by phage Bp7 and is essential for successful phage infection. This observation provides an explanation of the broad host range of phage Bp7 and provides insight into phage-host interactions. IMPORTANCE The RBPs of T4-like phages are gp37 and gp38. The interaction between phage T4 RBP gp37 and its receptors has been clarified by many reports. However, the interaction between gp38 and its receptors during phage adsorption is still not completely understood. Here, we identified phage Bp7, which uses gp38 as an RBP, and provided a good model to study the phage-host interaction mechanisms in an enterobacteriophage. Our study revealed that gp38 of phage Bp7 recognizes the outer membrane proteins (OMPs) LamB and OmpC of E. coli K-12 as specific receptors and binds with them reversibly. HepI of the inner-core oligosaccharide is the second receptor and binds with phage Bp7 irreversibly to begin the infection process. Determining the interaction between the phage and its receptors will help elucidate the mechanisms of phage with a broad host range and help increase understanding of the phage infection mechanism based on gp38. [ABSTRACT FROM AUTHOR]

The monoamine system in prefrontal cortex (PFC) has been implicated in various mental disorders and has been the major target of anxiolytics and antidepressants. Clinical studies show that serotonin and norepinephrine reuptake inhibitors (SNRIs) produce better therapeutic effects than single selective reuptake inhibitors, while the underlying mechanisms are largely unknown. Here, we found that low-dose SNRIs, by acting on 5-HT1A and α2-adrenergic receptors (α2-AR) receptors, synergistically reduced AMPAR-mediated excitatory postsynaptic currents (EPSC) and AMPAR surface expression in PFC pyramidal neurons, via a mechanism involving Rab5/dynamin-mediated endocytosis of AMPARs. The synergistic effect of SNRIs on AMPARs was blocked by inhibition of Activator of G protein Signaling 3 (AGS3), a G-protein modulator that prevents re-association of Gi protein α subunit and prolongs βγ-mediated signaling pathway. Moreover, the depression of AMPAR-EPSC by SNRIs required p38 kinase activity, which was increased by 5-HT1A and α2-AR co-activation in an AGS3-dependent manner. These results have revealed a potential mechanism for the synergy between serotonin and norepinephrine systems on the regulation of glutamatergic transmission in cortical neurons. [ABSTRACT FROM AUTHOR]

Skeletal muscle microtubules (MTs) form a nonclassic grid-like network, which has so far been documented in static images only. We have now observed and analyzed dynamics of GFP constructs of MT and Golgi markers in single live fibers and in the whole mouse muscle in vivo. Using confocal, intravital, and superresolution microscopy, we find that muscle MTs are dynamic, growing at the typical speed of ~9 µm/min, and forming small bundles that build a durable network. We also show that static Golgi elements, associated with the MT-organizing center proteins γ-tubulin and pericentrin, are major sites of muscle MT nucleation, in addition to the previously identified sites (i.e., nuclear membranes). These data give us a framework for understanding how muscle MTs organize and how they contribute to the pathology of muscle diseases such as Duchenne muscular dystrophy. [ABSTRACT FROM AUTHOR]

T-cell responses may be shaped by sterile "danger signals" that are constituted by damage-associated molecular patterns (DAMP). However, whether and what type of adaptive immune responses are triggered in vivo by DAMPs induced by tumor progression are not well characterized. In this study, we report that the production of HMGB1, an established DAMP released by dying cells, was critical for tumor progression in an established mouse model of prostate cancer. HMGB1 was required for the activation and intratumoral accumulation of T cells that expressed cytokine lymphotoxinα1β2 (LT) on their surface. Intriguingly, these tumor-activated T cells recruited macro- phages to the lesion and were essential to promote the preneoplasia to invasive carcinoma in an LTβ receptor (LTβR)-dependent manner. Taken together, our findings suggest that the release of HMGB1 as an endogenous danger signal is important for priming an adaptive immune response that promotes malignant progression, with implications for cancer prevention and therapy. [ABSTRACT FROM AUTHOR]

Objectives. To compare the epidemic prevention ability of COVID-19 of each province in China and to evaluate the existing prevention and control capacity of each province. Methods. We established a quasi-Poisson linear mixed-effects model using the case data in cities outside Wuhan in Hubei Province, China. We adapted this model to estimate the number of potential cases in Wuhan and obtained epidemiological parameters. We estimated the initial number of cases in each province by using passenger flowrate data and constructed the extended susceptible-exposed-infectious-recovered model to predict the future disease transmission trends. Results. The estimated potential cases in Wuhan were about 3 times the reported cases. The basic reproductive number was 3.30 during the initial outbreak. Provinces with more estimated imported cases than reported cases were those in the surrounding provinces of Hubei, including Henan and Shaanxi. The regions where the number of reported cases was closer to the predicted value were most the developed areas, including Beijing and Shanghai. Conclusions. The number of confirmed cases in Wuhan was underestimated in the initial period of the outbreak. Provincial surveillance and emergency response capabilities vary across the country.