Glutathione­iron hybrid nanozyme-based colorimetric sensor for specific and stable detection of thiram pesticide on fruit juices.

Given the potential dangers of thiram to food safety, constructing a facile sensor is significantly critical. Herein, we presented a colorimetric sensor based on glutathione­iron hybrid (GSH-Fe) nanozyme for specific and stable detection of thiram. The GSH-Fe nanozyme exhibits good peroxidase-mimicking activity with comparable Michaelis constant (Km = 0.551 mM) to the natural enzyme. Thiram pesticides can specifically limit the catalytic activity of GSH-Fe nanozyme via surface passivation, causing the change of colorimetric signal. It is worth mentioning that the platform was used to prepare a portable hydrogel kit for rapid qualitative monitoring of thiram. Coupling with an image-processing algorithm, the colorimetric image of the hydrogel reactor is converted into the data information for accurate quantification of thiram with a detection limit of 0.3 µg mL-1. The sensing system has good selectivity and high stability, with recovery rates in fruit juice samples ranging from 92.4% to 106.9%.

The appointment-based model in community pharmacies: Patient demographics and reimbursable clinical services uptake in Ontario.

Background: Community pharmacies typically require patients to request medication refills. The appointment-based model (ABM) is a proactive approach that synchronizes refills and schedules patient-pharmacist appointments. These appointments provide opportunities for medication reviews, medication optimization and health promotion services. The primary aim of this study was to describe the types of patients who received an ABM service in a community pharmacy in Ontario in 2017. The secondary aim was to describe reimbursable clinical service uptake. Methods: In September 2017, the ABM was implemented across 3 Ontario community pharmacies within a Canadian pharmacy banner. Patients who filled at least 1 chronic oral medication and consented to enrolment were eligible. In December 2018, data were extracted from pharmacies using pharmacy management software. Descriptive statistics and frequencies were generated. Results: Analysis of 131 patients (51.1% female; mean ± SD age 70.8 ± 10.5 years) revealed patients were dispensed a mean ± SD of 5.1 ± 2.7 medications, and 73 (55.7%) experienced polypharmacy. Hypertension (87.8%) and dyslipidemia (68.7%) were the most common medical conditions. There were 74 (56.5%) patients who received ≥1 medication review service (MedsCheck). Of 79 unique drug therapy problems (DTPs) identified, the most common categories related to patients needing additional drug therapy and adverse drug reactions. Discussion and conclusion: Patients enrolled in the ABM were generally older adults experiencing polypharmacy. The ABM presented opportunities for DTP identification and delivery of reimbursed services. Findings support continued exploration of the ABM to support integration of clinical services within community practice.

Association of nutritional status and comorbidity with long-term survival among community-dwelling older males.

BACKGROUND: Estimates of survival in the older can be of benefit in various facets, particularly in medical and individual decision-making. We aim to validate the value of a combination of nutrition status evaluation and comorbidity assessment in predicting long-term survival among community-dwelling older. METHODS: The Charlson Comorbidity Index (CCI) was applied for comprehensive evaluation of comorbidities. Participants were classified into CCI score ≤ 2 and ≥ 3 subgroups. Nutritional status was assessed by using Mini Nutritional Assessment-Short Form (MNA-SF) and Geriatric Nutritional Risk Index (GNRI) evaluations. Mortality rates and survival curves over a 5-year period were compared among subgroups classified by CCI and/or MNA-SF/GNRI evaluations. RESULTS: A total of 1033 elderly male participants were enrolled in this study, with an average age of 79.44 ± 8.61 years. 108 deceased participants (10.5%) were identified during a follow-up of 5 years. Cox proportional hazards regression analysis showed that age, CCI, MNA-SF and GNRI were independent predictors of 5-year all-cause death in this cohort. Compared to those with normal nutrition status and CCI ≤ 2, the subgroup at risk of malnutrition and CCI ≥ 3 had a significantly higher 5-year all-cause mortality rate (HR = 4.671; 95% CI:2.613-8.351 for MNA-SF and HR = 7.268; 95% CI:3.401-15.530 for GNRI; P < 0.001 for both). Receiver operating characteristic curve analysis demonstrated that a combination of either MNA-SF or GNRI with CCI had significantly better performance than CCI, MNA-SF or GNRI alone in predicting all-cause death. CONCLUSION: The combination of nutritional assessment (MNA-SF or GNRI) with CCI can significantly improve the predictive accuracy of long-term mortality outcomes among community-dwelling older males.

Narciclasine, a novel topoisomerase I inhibitor, exhibited potent anti-cancer activity against cancer cells.

DNA topoisomerases are essential nuclear enzymes in correcting topological DNA errors and maintaining DNA integrity. Topoisomerase inhibitors are a significant class of cancer chemotherapeutics with a definite curative effect. Natural products are a rich source of lead compounds for drug discovery, including anti-tumor drugs. In this study, we found that narciclasine (NCS), an amaryllidaceae alkaloid, is a novel inhibitor of topoisomerase I (topo I). Our data demonstrated that NCS inhibited topo I activity and reversed its unwinding effect on p-HOT DNA substrate. However, it had no obvious effect on topo II activity. The molecular mechanism of NCS inhibited topo I showed that NCS did not stabilize topo-DNA covalent complexes in cells, indicating that NCS is not a topo I poison. A blind docking result showed that NCS could bind to topo I, suggesting that NCS might be a topo I suppressor. Additionally, NCS exhibited a potent anti-proliferation effect in various cancer cells. NCS arrested the cell cycle at G2/M phase and induced cell apoptosis. Our study reveals the antitumor mechanisms of NCS and provides a good foundation for the development of anti-cancer drugs based on topo I inhibition.

LepR+ niche cell-derived AREG compromises hematopoietic stem cell maintenance under conditions of DNA repair deficiency and aging.

The cross talk between extrinsic niche-derived and intrinsic hematopoietic stem cell (HSC) factors controlling HSC maintenance remains elusive. Here, we demonstrated that amphiregulin (AREG) from bone marrow (BM) leptin receptor (LepR+) niche cells is an important factor that mediates the cross talk between the BM niche and HSCs in stem cell maintenance. Mice deficient of the DNA repair gene Brca2, specifically in LepR+ cells (LepR-Cre;Brca2fl/fl), exhibited increased frequencies of total and myeloid-biased HSCs. Furthermore, HSCs from LepR-Cre;Brca2fl/fl mice showed compromised repopulation, increased expansion of donor-derived, myeloid-biased HSCs, and increased myeloid output. Brca2-deficient BM LepR+ cells exhibited persistent DNA damage-inducible overproduction of AREG. Ex vivo treatment of wild-type HSCs or systemic treatment of C57BL/6 mice with recombinant AREG impaired repopulation, leading to HSC exhaustion. Conversely, inhibition of AREG by an anti-AREG-neutralizing antibody or deletion of the Areg gene in LepR-Cre;Brca2fl/fl mice rescued HSC defects caused by AREG. Mechanistically, AREG activated the phosphoinositide 3-kinases (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, promoted HSC cycling, and compromised HSC quiescence. Finally, we demonstrated that BM LepR+ niche cells from other DNA repair-deficient and aged mice also showed persistent DNA damage-associated overexpression of AREG, which exerts similar negative effects on HSC maintenance. Therefore, we identified an important factor that regulates HSCs function under conditions of DNA repair deficiency and aging.

Discovery of novel thymidylate synthase (TS) inhibitors that influence cancer angiogenesis and metabolic reprogramming in NSCLC cells.

Based on previous work, further search for more effective and less damaging thymidylate synthase (TS) inhibitors was the focus of this study. After further optimization of the structure, in this study, a series of (E)-N-(2-benzyl hydrazine-1-carbonyl) phenyl-2,4-deoxy-1,2,3,4-tetrahydro pyrimidine-5-sulfonamide derivatives were synthesized and reported for the first time. All target compounds were screened by enzyme activity assay and cell viability inhibition assay. On the one hand, the hit compound DG1 could bind directly to TS proteins intracellularly and promote apoptosis in A549 and H1975 cells. Simultaneously, DG1 could inhibit cancer tissue proliferation more effectively than Pemetrexed (PTX) in the A549 xenograft mouse model. On the other hand, the inhibitory effect of DG1 on NSCLC angiogenesis was verified both in vivo and in vitro. In parallel, DG1 was further uncovered to inhibit the expression of CD26, ET-1, FGF-1, and EGF by angiogenic factor antibody microarray. Moreover, RNA-seq and PCR-array assays revealed that DG1 could inhibit NSCLC proliferation by affecting metabolic reprogramming. Collectively, these data demonstrated that DG1as a TS inhibitor could be promising in treating NSCLC angiogenesis, deserving further investigation.

Implementation of the appointment-based model in community pharmacies: An analysis of refills and adherence.

BACKGROUND: Traditionally, much of community pharmacy practice relies on patients to request their own medication refills. These refills are often not aligned, which has been shown to decrease adherence and workflow efficiencies. The appointment-based model (ABM) is designed to proactively synchronize refills and schedule patient-pharmacist appointments. OBJECTIVES: To describe the characteristics of patients enrolled in the ABM; and to compare the number of distinct refill dates, number of refills, and adherence for antihypertensives, oral antihyperglycemics, and statins 6-months and 12-months pre-post ABM implementation. METHODS: In September 2017, the ABM was implemented across independent community pharmacies within a pharmacy banner in Ontario, Canada. In December 2018, a convenience sample of three pharmacies was extracted. Demographic and clinical characteristics were collected on program enrollment (index) date for individual patients and their medication fill histories were used to investigate adherence measures including distinct number of refill dates, number of refills, and proportion of days covered. Descriptive statistics were analyzed using StataCorp. RESULTS: Analysis of 131 patients (48.9% male; mean age 70.8 years ± 10.5 SD) filled on average 5.1 ± 2.7 medications with 73 (55.7%) experiencing polypharmacy. Patients had a significant reduction in mean number of refill dates (6.8 ± 3.8 SD six-months pre-enrollment, 4.9 ± 3.1 SD six-months post-enrollment, p < 0.0001). Adherence to chronic medications remained high (PDC ≥95%). CONCLUSION: The ABM was implemented for a cohort of established users, already highly adherent to their chronic medications. Results demonstrate reduced filling complexity and fewer refill dates while also sustaining the high baseline adherence across all chronic medications studied. Future studies should investigate patient perspectives and potential clinical benefits of the ABM.

Gram-negative bacteria resist antimicrobial agents by a DzrR-mediated envelope stress response.

BACKGROUND: Envelope stress responses (ESRs) are critical for adaptive resistance of Gram-negative bacteria to envelope-targeting antimicrobial agents. However, ESRs are poorly defined in a large number of well-known plant and human pathogens. Dickeya oryzae can withstand a high level of self-produced envelope-targeting antimicrobial agents zeamines through a zeamine-stimulated RND efflux pump DesABC. Here, we unraveled the mechanism of D. oryzae response to zeamines and determined the distribution and function of this novel ESR in a variety of important plant and human pathogens. RESULTS: In this study, we documented that a two-component system regulator DzrR of D. oryzae EC1 mediates ESR in the presence of envelope-targeting antimicrobial agents. DzrR was found modulating bacterial response and resistance to zeamines through inducing the expression of RND efflux pump DesABC, which is likely independent on DzrR phosphorylation. In addition, DzrR could also mediate bacterial responses to structurally divergent envelope-targeting antimicrobial agents, including chlorhexidine and chlorpromazine. Significantly, the DzrR-mediated response was independent on the five canonical ESRs. We further presented evidence that the DzrR-mediated response is conserved in the bacterial species of Dickeya, Ralstonia, and Burkholderia, showing that a distantly located DzrR homolog is the previously undetermined regulator of RND-8 efflux pump for chlorhexidine resistance in B. cenocepacia. CONCLUSIONS: Taken together, the findings from this study depict a new widely distributed Gram-negative ESR mechanism and present a valid target and useful clues to combat antimicrobial resistance.

tRNA modification enzyme MiaB connects environmental cues to activation of Pseudomonas aeruginosa type III secretion system.

Pseudomonas aeruginosa, a major inhabitant of numerous environmental reservoirs, is a momentous opportunistic human pathogen associated with severe infections even death in the patients suffering from immune deficiencies or metabolic diseases. Type III secretion system (T3SS) employed by P. aeruginosa to inject effector proteins into host cells is one of the pivotal virulence factors pertaining to acute infections caused by this pathogen. Previous studies showed that P. aeruginosa T3SS is regulated by various environmental cues such as calcium concentration and the host signal spermidine. However, how T3SS is regulated and expressed particularly under the ever-changing environmental conditions remains largely elusive. In this study, we reported that a tRNA modification enzyme PA3980, designated as MiaB, positively regulated T3SS gene expression in P. aeruginosa and was essential for the induced cytotoxicity of human lung epithelial cells. Further genetic assays revealed that MiaB promoted T3SS gene expression by repressing the LadS-Gac/Rsm signaling pathway and through the T3SS master regulator ExsA. Interestingly, ladS, gacA, rsmY and rsmZ in the LadS-Gac/Rsm signaling pathway seemed potential targets under the independent regulation of MiaB. Moreover, expression of MiaB was found to be induced by the cAMP-dependent global regulator Vfr as well as the spermidine transporter-dependent signaling pathway and thereafter functioned to mediate their regulation on the T3SS gene expression. Together, these results revealed a novel regulatory mechanism for MiaB, with which it integrates different environmental cues to modulate T3SS gene expression in this important bacterial pathogen.

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.

Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.

Gasdermin D-dependent platelet pyroptosis exacerbates NET formation and inflammation in severe sepsis.

Platelets have emerged as key inflammatory cells implicated in the pathology of sepsis, but their contributions to rapid clinical deterioration and dysregulated inflammation have not been defined. Here, we show that the incidence of thrombocytopathy and inflammatory cytokine release was significantly increased in patients with severe sepsis. Platelet proteomic analysis revealed significant upregulation of gasdermin D (GSDMD). Using platelet-specific Gsdmd-deficient mice, we demonstrated a requirement for GSDMD in triggering platelet pyroptosis in cecal ligation and puncture (CLP)-induced sepsis. GSDMD-dependent platelet pyroptosis was induced by high levels of S100A8/A9 targeting toll-like receptor 4 (TLR4). Pyroptotic platelet-derived oxidized mitochondrial DNA (ox-mtDNA) potentially promoted neutrophil extracellular trap (NET) formation, which contributed to platelet pyroptosis by releasing S100A8/A9, forming a positive feedback loop that led to the excessive release of inflammatory cytokines. Both pharmacological inhibition using Paquinimod and genetic ablation of the S100A8/A9-TLR4 signaling axis improved survival in mice with CLP-induced sepsis by suppressing platelet pyroptosis.

A Bacterial Isolate Capable of Quenching Both Diffusible Signal Factor- and N-Acylhomoserine Lactone-Family Quorum Sensing Signals Shows Much Enhanced Biocontrol Potencies.

N-Acylhomoserine lactone (AHL) and diffusible signal factor (DSF) molecules are two families of widely conserved quorum sensing (QS) signals. Quorum quenching (QQ) via enzymatic inactivation of QS signals is a promising strategy of biocontrol. In the search for biocontrol agent quenching both AHL and DSF signals, it has been recently identified that DSF-quenching biocontrol agent Pseudomonas sp. HS-18 contains at least three genes (aigA, aigB, and aigC) encoding AHL-acylases displaying strong AHL-acylase activities on various AHLs. Among them, AigA and AigC presented broad-spectrum enzyme activity against AHLs, while AigB preferred longer AHLs. Interestingly, transcriptional expression of aigC could be significantly induced by AHL signals. Heterologous expression of aigA-C in Burkholderia cenocepacia and Pseudomonas aeruginosa resulted in drastically decreased AHL accumulation, virulence factor production, biofilm formation, motility, and virulence on plants. Significantly, the two types of QQ mechanisms in HS-18 showed a strong and much desired synergistic effect for enhanced biocontrol potency against AHL- and DSF-dependent pathogens.

Hematopoietic stem cell regeneration through paracrine regulation of the Wnt5a/Prox1 signaling axis.

The crosstalk between the BM microenvironment (niche) and hematopoietic stem cells (HSCs) is critical for HSC regeneration. Here, we show that in mice, deletion of the Fanconi anemia (FA) genes Fanca and Fancc dampened HSC regeneration through direct effects on HSCs and indirect effects on BM niche cells. FA HSCs showed persistent upregulation of the Wnt target Prox1 in response to total body irradiation (TBI). Accordingly, lineage-specific deletion of Prox1 improved long-term repopulation of the irradiated FA HSCs. Forced expression of Prox1 in WT HSCs mimicked the defective repopulation phenotype of FA HSCs. WT mice but not FA mice showed significant induction by TBI of BM stromal Wnt5a protein. Mechanistically, FA proteins regulated stromal Wnt5a expression, possibly through modulating the Wnt5a transcription activator Pax2. Wnt5a treatment of irradiated FA mice enhanced HSC regeneration. Conversely, Wnt5a neutralization inhibited HSC regeneration after TBI. Wnt5a secreted by LepR+CXCL12+ BM stromal cells inhibited ß-catenin accumulation, thereby repressing Prox1 transcription in irradiated HSCs. The detrimental effect of deregulated Wnt5a/Prox1 signaling on HSC regeneration was also observed in patients with FA and aged mice. Irradiation induced upregulation of Prox1 in the HSCs of aged mice, and deletion of Prox1 in aged HSCs improved HSC regeneration. Treatment of aged mice with Wnt5a enhanced hematopoietic repopulation. Collectively, these findings identified the paracrine Wnt5a/Prox1 signaling axis as a regulator of HSC regeneration under conditions of injury and aging.

Synthesis and evaluation of novel HER-2 inhibitors to exert anti-breast cancer ability through epithelial-mesenchymal transition (EMT) pathway.

Human epidermal growth factor receptor 2 (HER-2) is an essential member of the receptor tyrosine kinase (RTK) superfamily and has been reported as a critical method for treating HER-2 positive breast cancer. Here, we retained (E)-4-methyl-2-(4-(trifluoromethyl)styryl)oxazole, a fragment of HER-2 inhibitor Mubritinib, and synthesized 32 novel compounds from it. We screened out the most potential compound Q7j with HER-2 positive breast cancer cells through MTT assays, which possessed low toxicity on normal cells (MCF7-10A). Subsequently, wound healing, transwell, western blotting, and immunofluorescence experiments were performed, and it was found that compound Q7j could suppress cell migration by inhibiting the phosphorylation of HER-2 and affecting the expression of EMT-related proteins. Moreover, the SKBR3 orthotopic xenograft model confirmed that compound Q7j was more effective than Mubritinib in inhibiting the proliferation of cancer cells. In general, compound Q7j was a potential HER-2 inhibitor in treating breast cancer, which may be of great significance for developing and improving HER-2 small molecule inhibitors.

Spermidine Is an Intercellular Signal Modulating T3SS Expression in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a vital opportunistic human bacterial pathogen that causes acute and chronic infections. In this study, we set to determine whether the endogenous spermidine biosynthesis plays a role in regulation of type III secretion system (T3SS). The results showed that deletion of speA and speC, which encode putrescine biosynthesis, did not seem to affect cellular spermidine level and the T3SS gene expression. In contrast, mutation of speD and speE encoding spermidine biosynthesis led to significantly decreased spermidine production and expression of T3SS genes. We also showed that endogenous spermidine could auto-induce the transcriptional expression of speE and its full functionality required the transporter SpuDEFGH. Cytotoxicity analysis showed that mutants ΔspeE and ΔspuE were substantially attenuated in virulence compared with their wild-type strain PAO1. Our data imply a possibility that spermidine biosynthesis in P. aeruginosa may not use putrescine as a substrate, and that spermidine signaling pathway may interact with other two T3SS regulatory mechanisms in certain degree, i.e., cAMP-Vfr and GacS/GacA signaling systems. Taken together, these results specify the role of endogenous spermidine in regulation of T3SS in P. aeruginosa and provide useful clues for design and development antimicrobial therapies. IMPORTANCE Type III secretion system (T3SS) is one of the pivotal virulence factors of Pseudomonas aeruginosa responsible for evading phagocytosis, and secreting and translocating effectors into host cells. Previous studies underline the complicated and elaborate regulatory mechanisms of T3SS for the accurate, fast, and malicious pathogenicity of P. aeruginosa. Among these regulatory mechanisms, our previous study indicated that the spermidine from the host was vital to the host-pathogen interaction. However, the role of endogenous spermidine synthesized by P. aeruginosa on the regulation of T3SS expression is largely unknown. Here we reveal the role and regulatory network of endogenous spermidine synthesis in regulation of T3SS and bacterial virulence, showing that the spermidine is an important interspecies signal for modulating the virulence of P. aeruginosa through regulating T3SS expression.

Preventive effect of swim bladder hydrolysates on cyclophosphamide-induced ovarian injury in mice.

AIMS: This study aimed to prepare swim bladder hydrolysate (SBH) with Mn  < 4000 Da, and investigate its effects on cyclophosphamide (CTX)-mediated ovarian injury in mice. METHODS: Hydrolysates were prepared by heating extraction, enzymatic hydrolysis and ultrafiltration. Mn and distribution of SBH were analyzed via gel filtration chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Changes in the mouse oestrus cycle were determined by cytological examination. The number of follicles was examined using histopathology. Enzyme-linked immunosorbent assays (ELISAs) were used to determine the serum sex hormone levels. RESULTS: The Mn of SBH, prepared by heating extraction, enzymatic hydrolysis, ultrafiltration, and from different batches, was below 4000 Da, and the preparation process was stable. Compared with the control group, the low-, middle-, and high-dose SBH treatment groups showed different trends in oestrus duration, serum sex hormone levels, and the number of primordial and secondary follicles. The oestrus cycle duration of the high-dose SBH group was longer than that of the model group. The serum luteinizing hormone, follicle-stimulating hormone, and anti-Müllerian hormone levels in the middle-dose group were the closest to those of control group. The number of primordial and secondary follicles in the medium-dose group was significantly higher than that in the model group and closest to those of control group. CONCLUSION: After heating extraction, trypsin/Flavourzyme hydrolysis and ultrafiltration, a hydrolysate with Mn below 4000 Da could be prepared. We found that a moderate (400 mg/kg) SBH dose resulted in the greatest effect on ovarian injury remission in mice.

The Two-Component System FleS/FleR Represses H1-T6SS via Cyclic di-GMP Signaling in Pseudomonas aeruginosa.

The type VI secretion system (T6SS) is an important translocation apparatus that is widely employed by Gram-negative bacteria to deliver toxic effectors into eukaryotic and prokaryotic target cells, causing host damage and providing competitive advantages in polymicrobial environments. The genome of Pseudomonas aeruginosa harbors three T6SS clusters (H1-T6SS, H2-T6SS, H3-T6SS). Activities of these systems are tightly regulated by a complicated signaling network which remains largely elusive. In this study, we focused on a previously characterized two-component system FleS/FleR, and performed comparative transcriptome analysis between the PAO1 wild-type strain and its isogenic ΔfleR mutant, which revealed the important role of FleS/FleR in regulating multiple physiological pathways including T6SS. Gene expression and bacterial killing assays showed that the expression and activity of H1-T6SS are repressed in the wild-type strain owing to the high intracellular c-di-GMP content. Further explorations demonstrated that c-di-GMP relies on the transcription factor FleQ to repress H1-T6SS and its synthesis is controlled by a global regulator AmrZ which is induced by the active FleS/FleR. Interestingly, repression of H1-T6SS by FleS/FleR in PAO1 is independent of RetS which is known to regulate H1-T6SS by controlling the central post-transcriptional factor RsmA. Together, our results identified a novel regulator of H1-T6SS and provided detailed mechanisms of this signaling pathway in PAO1. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen distributed widely in the environment. The genome of this pathogen contains three T6SS clusters which contribute significantly to its virulence. Understanding the complex regulatory network that controls the activity of T6SS is essential for the development of effective therapeutic treatments for P. aeruginosa infections. In this study, transcriptome analysis led to the identification of a novel regulator FleS/FleR which inversely regulates H1-T6SS and H2-T6SS in P. aeruginosa PAO1. We further revealed a detailed FleS/FleR-mediated regulatory pathway of H1-T6SS in PAO1 which involves two additional transcriptional regulators AmrZ and FleQ and the second messenger c-di-GMP, providing important implications to develop novel anti-infective strategies and antimicrobial drugs.

Discovery of N-(1,3,4-thiadiazol-2-yl)benzamide derivatives containing a 6,7-methoxyquinoline structure as novel EGFR/HER-2 dual-target inhibitors against cancer growth and angiogenesis.

Targeting EGFR and HER-2 is an essential direction for cancer treatment. Here, a series of N-(1,3,4-thiadiazol-2-yl)benzamide derivatives containing a 6,7-methoxyquinoline structure was designed and synthesized to serve as EGFR/HER-2 dual-target inhibitors. The kinase assays verified that target compounds could inhibit the kinase activity of EGFR and HER-2 selectively. The results of CCK-8 and 3D cell viability assays confirmed that target compounds had excellent anti-proliferation ability against breast cancer cells (MCF-7 and SK-BR-3) and lung cancer cells (A549 and H1975), particularly against SK-BR-3 cells, while the inhibitory effect on healthy breast cells (MCF-10A) and lung cells (Beas-2B) was weak. Among them, the hit compound YH-9 binded to EGFR and HER-2 stably in molecular dynamics studies. Further studies found thatYH-9could induce the release of cytochrome c and inhibit proliferation by promoting ROS expression in SK-BR-3 cells. Moreover,YH-9could diminish the secretion of VEGF and bFGF factors in SK-BR-3 cells, then inhibited tube formation and angiogenesis. Notably,YH-9could effectively inhibit breast cancer growth and angiogenesis with little toxicity in the SK-BR-3 cell xenograft model. Taken together,in vitroandin vivoresults revealed that YH-9 had high drug potential as a dual-target inhibitor of EGFR/HER-2 to inhibit breast cancer growth and angiogenesis.

Investigation on Mechanical and Microstructure Properties of Silt Improved by Titanium Gypsum-Based Stabilizer.

Silt in the Yellow River alluvial plain is widely spread, but its uniform particle size and high roundness make it unsuitable as a subgrade filling material, while titanium gypsum (TG) is an industrial solid waste in Shandong Province, not only occupying land resources but also causing water and air pollution. In order to improve the engineering performance of silt, reduce the pollution of solid waste titanium gypsum to the environment and reduce the engineering cost, considering the engineering characteristics of titanium gypsum, it was combined in specific amounts with cement and lime to create a titanium gypsum-based stabilizer (TS) in this study. The effect of curing conditions and TS content on silt improvement was studied through laboratory experiments. The mechanical properties of the stabilized silt were investigated by unconfined compression test (UCT), and the mineral composition and pore structure were analyzed by scanning electron microscopy (SEM) test, X-ray diffraction (XRD) test, and mercury injection pore (MIP) test. The test results show that TS could effectively improve the unconfined compressive strength of silt, and the strength of stabilized silt gradually increases with the curing period and TS content. In terms of the porosity, it decreases with the increase of the curing period and TS content. From the microstructure perspective, this is mainly due to the formation of ettringite and C-S-H during the stabilization of silt by TS.

Quinacrine-CASIN combination overcomes chemoresistance in human acute lymphoid leukemia.

Chemoresistance posts a major hurdle for treatment of acute leukemia. There is increasing evidence that prolonged and intensive chemotherapy often fails to eradicate leukemic stem cells, which are protected by the bone marrow niche and can induce relapse. Thus, new therapeutic approaches to overcome chemoresistance are urgently needed. By conducting an ex vivo small molecule screen, here we have identified Quinacrine (QC) as a sensitizer for Cytarabine (AraC) in treating acute lymphoblastic leukemia (ALL). We show that QC enhances AraC-mediated killing of ALL cells, and subsequently abrogates AraC resistance both in vitro and in an ALL-xenograft model. However, while combo AraC+QC treatment prolongs the survival of primary transplanted recipients, the combination exhibits limited efficacy in secondary transplanted recipients, consistent with the survival of niche-protected leukemia stem cells. Introduction of Cdc42 Activity Specific Inhibitor, CASIN, enhances the eradication of ALL leukemia stem cells by AraC+QC and prolongs the survival of both primary and secondary transplanted recipients without affecting normal long-term human hematopoiesis. Together, our findings identify a small-molecule regimen that sensitizes AraC-mediated leukemia eradication and provide a potential therapeutic approach for better ALL treatment.