Ten percent of adult, outpatient visits result in an antibiotic prescription (Rx). At the start of our intervention, our VA healthcare system consisted of 13 community-based outpatient clinics (CBOCs), 9 of which did not have an onsite pharmacy but utilized automated dispensing cabinets (ADCs) for prepackaged outpatient Rxs. ADC antibiotic orders are generated from electronic medical record (EMR) order sets. The stewardship team shortened the durations of 5 antibiotics in the ADC order sets to make them consistent with current literature and guidelines. We assessed the impact of these changes on antibiotic prescribing habits. We compared outpatient antibiotic Rx data between 10/1/2018-9/30/2019 (pre-intervention) and 10/1/19-9/30/20 (post-intervention) from 8 CBOCs with ADCs (1 closed during the pandemic). Amoxicillin-clavulanate 875/125mg (AMC), cephalexin 500mg (CPH), levofloxacin 500mg and 750mg (LEV 500 and LEV 750), and sulfamethoxazole-trimethoprim 800/160mg (SXT) prescription durations were all reduced by 3 days. Process metrics included days supplied/1000 prescriptions (DS/1000 Rx), median DS, and ADC utilization rates. We used Mann-Whitney U and correlation statistical analyses to assess differences and associations.The DS/1000 Rx of antibiotics with a default duration change decreased in the post-intervention phase for CBOCs with ADCs . The median DS for these antibiotics all reduced by 3 days in concordance with new ADC prescriptions defaults . Due to COVID-19, 7/8 ADC CBOCs closed for in-person visits from 3/20/20-5/4/20. ADC utilization was inversely proportional to DS/1000 Rx for most antibiotics (R: -0.51 to -0.77) except SXT.EMR-driven reductions in ADC default Rx durations led to a corresponding decrease in overall outpatient antibiotic prescribing. Higher DS/1000 Rx were often associated with lower ADC utilization. Informatics-driven antibiotic interventions may be potential outpatient stewardship tools to increase guideline-concordant prescribing across multisite healthcare systems.Sharanie Sims, PharmD, AbbVie (formerly Allergan) (Speaker’s Bureau)


A β-catenin-driven switch in TCF/LEF transcription factor binding to DNA target sites promotes commitment of mammalian nephron progenitor cells. Published 15, February 2021We overlooked an important published study by Ramalingam et al (2018) which draws the conclusion that different levels of ß-catenin, resulting from different levels of Wnt9b ligand, determine nephron progenitor cell (NPC) outcomes. Though our study focuses on defined NPC cultures and genomic interactions amongst ß-catenin and Tcf/Lef transcriptional mediators that were not explored in Ramalingham et al., our approaches agree on the importance of ß-catenin levels in modifying the state of NPCs. Accordingly, we have corrected our manuscript below to acknowledge Ramalingam et al (2018) were appropriate and we apologize to the authors for the oversight.#1Original text:Genomic analysis of β-catenin engagement at TCF/LEF recognition motifs within enhancers linked to genes driving NPC differentiation , and subsequent transgenic studies demonstrating TCF/LEF-dependent activity of cis regulatory elements, provides strong evidence for a canonical Wnt/β-catenin/Tcf regulatory axis .Corrected text:Genetic and chemical modification of Wnt pathway components suggest different levels of β-catenin distinguish maintenance and commitment of NPCs . Genomic analysis of β-catenin engagement at TCF/LEF recognition motifs within enhancers linked to genes driving NPC differentiation , and subsequent transgenic studies demonstrating TCF/LEF-dependent activity of cis regulatory elements, provides strong evidence for a canonical Wnt/β-catenin/Tcf regulatory axis .#2Original text:Within 3 days of elevating CHIR levels (3 µM), aggregate NPC cultures show a robust signature of nephron differentiation Corrected text:Within 3 days of elevating CHIR levels (3 µM), aggregate NPC cultures show a robust signature of nephron differentiation #3Original text:Wnt4 Genetic analysis of mouse models points to a requirement for β-catenin in both the maintenance  and differentiation  of NPCs. Previous studies directly examining β-catenin association in differentiating NPCs showed direct engagement of β-catenin at enhancers regulating expression of differentiation promoting genes such as Corrected text:Wnt4 .Genetic analysis of mouse models points to a requirement for β-catenin in both the maintenance  and differentiation  of NPCs. Genetically modulating Wnt inputs and chemically modifying Wnt-pathway activity suggest Wnt-modulation of β-catenin levels is critical to these alternative outcomes . Previous studies directly examining β-catenin association in differentiating NPCs showed direct engagement of β-catenin at enhancers regulating expression of differentiation promoting genes such as #4Insert reference below into reference list:Ramalingam H, Fessler AR, Das A, Valerius MT, Basta J, Robbins L, Brown AC, Oxburgh L, McMahon AP, Rauchman M and Carroll TJ. (2018). Disparate levels of beta-catenin activity determine nephron progenitor cell fate. Developmental Biology440(1):13-21.#5 (an independent typographic error)Original text:Sub section title - Elevating β-catenin leads to activation of Tcftt/Lef-bound enhancersCorrected text:Sub-section title - Elevating β-catenin leads to activation of Tcf/Lef-bound enhancers


Next-generation sequencing has deepened our understanding of meningiomas, particularly those that behave aggressively. Classifications using either DNA methylation profiling or RNA-sequencing predict tumor behavior more reliably than WHO grade, and segregate common meningioma features similarly, implying possible overlap between classifications. In this study, we performed DNA methylation profiling, RNA-sequencing, and whole-exome sequencing on 110 primary meningiomas . Unsupervised non-negative matrix factorization demonstrated three epigenetic types which were highly concordant with our published transcriptional types (87.3% concordance). Two additional classifications  were also highly concordant and an overall meningioma group (MenG) classification was assigned integrating all four together. MenG A and B rarely recur, while MenG C behave aggressively  of 3.1 years), even after gross total resection (median RFS 4.2 years). MenG A tumors retain Merlin expression (no chromosome loss or NF2 mutation) and harbor mutations in TRAF7, AKT1, or KLF4. Both MenG B and C are merlin-deficient, but MenG B demonstrate low rates of CNV and MenG C high rates of CNV, particularly loss of chromosome 1p. Using partial least squares regression (PLS), we explored how gene expression correlated with promoter methylation and CNV, thereby classifying genes which correlated closely as ‘methylation-driven’ or ‘CNV-driven’. Overall, there were more methylation-driven (5.7%) than CNV-driven (2.9%) genes. Differentially expressed genes (DEGs) were enriched for both methylation- and CNV-driven genes at similar proportions (10.6% and 5.8%), but DEGs unique to MenG C were significantly enriched for CNV-driven (23.7%), but not methylation-driven (4.7%) genes, primarily due many MenG C DEGs on chromosome 1p. Overall, this work suggests three underlying meningioma groups which are identifiable through methylation, transcriptional, or genetic/cytogenetic profiling and warrants exploration of the role of chromosome 1p in group that behaves aggressively.


PCK1 mRNA and protein levels are significantly elevated in human pancreatic cancer tissues and cells. In established and primary pancreatic cancer cells, PCK1 silencing (by shRNA) or CRISPR/Cas9-induced PCK1 knockout potently inhibited cell growth, proliferation, migration and invasion, and induced robust apoptosis activation. Conversely, ectopic overexpression of PCK1 in pancreatic cancer cells accelerated cell proliferation and migration. RNA-seq analyzing of differentially expressed genes (DEGs) in PCK1-silenced pancreatic cancer cells implied that DEGs were enriched in the PI3K-Akt-mTOR cascade. In pancreatic cancer cells, Akt-mTOR activation was largely inhibited by PCK1 shRNA, but was augmented after ectopic PCK1 overexpression. In vivo, the growth of PCK1 shRNA-bearing PANC-1 xenografts was largely inhibited in nude mice. Akt-mTOR activation was suppressed in PCK1 shRNA-expressing PANC-1 xenograft tissues. Collectively, PCK1 is a potential therapeutic target for pancreatic cancer.Pancreatic cancer is the third leading cause of cancer-related mortalities and is characterized by rapid disease progression. Identification of novel therapeutic targets for this devastating disease is important. Phosphoenolpyruvate carboxykinase 1 (PCK1) is the rate-limiting enzyme of gluconeogenesis. The current study tested the expression and potential functions of PCK1 in pancreatic cancer. We show that  Abnormal activation of multiple signaling cascades are closely associated with the initiation and progression of pancreatic cancer . Dysregu2 [PCK1 gene locates at chromosome 20q13.31 and PCK1 protein mainly accumulates in the cytoplasm under unstimulated condition. Activated PCK1 can translocate into the endoplasmic reticulum [Phosphoenolpyruvate carboxykinase (PCK) is the first rate-limiting enzyme of gluconeogenesis that converts oxaloacetate and GTP into phosphoenolpyruvate (PEP) and CO2 . It play2 . There a2 . PCK1 geeticulum .Recent studies have reported upregulation and/or increased phosphorylation of PCK1 or PCK2 in different human malignancies, including colon cancer, non-small cell lung cancer (NSCLC), melanoma, and lymphoma, as well as metastatic breast cancer and hepatocellular carcinoma (HCC) –15. ThesAll sequences and viral constructs were provided by Shanghai Genechem Co. .Fresh pancreatic cancer tissues and adjacent normal pancreatic tissues from five primary pancreatic cancer patients  were obtained. None of these patients received chemotherapy or radiotherapy before surgery. Written informed consent was obtained from each patient. The protocols were approved by the Ethics Board of Jiangsu University BR2015021), according to the Declaration of Helsinki. The characteristic of the patients are summarized in Table 5021, acc6 cells per mouse, in 0.2 mL 10% FBS DMEM/Matrigel solution) with indicated genetic modifications were subcutaneously injected into the flanks of the nude mice. The mice body weights and tumor volumes were measured every 5 days with digital calipers [Animal protocols have been approved by IACUC and the Ethics Review Board of Jiangsu University. Five-to six-week-old female BALB/c nude mice (18–19 g), purchased from the Animal Center of Soochow University, were raised indoors at standard conditions. PANC-1 cells . Statistical analysis was performed using SPSS 23.0 . Unpaired student’s PCK1 expression in pancreatic ductal adenocarcinoma (PDAC). As shown PCK1 mRNA levels in pancreatic cancer tissues  were significantly higher than those in normal pancreatic tissues   were significantly higher than those in normal pancreatic tissues (“N”)  cohort was consulted to examine  4) Fig. . In addiN”) Fig. .Fig. 1PCn = 5) primary pancreatic cancer patients (Table PCK1 mRNA levels in pancreatic cancer tissues (“T”) were again significantly higher than those in adjacent normal tissues (“N”)  Fig. . WesternN”) Fig. . IncreasPCK1 mRNA expression levels were elevated in the pancreatic cancer cells when compared to low expression in primary pancreatic epithelial cells (“pEpi”)  as well as the primary human pancreatic cancer cells derived from three patients (namely “pPC1/pPC2/pPC3”). The qRT-PCR assay results, Fig. i”) Fig. . PCK1 pri”) Fig. .PCK1 mRNA levels were robustly decreased by PCK1 shRNA , were individually transduced to PANC-1 cells and PATU-8988 cells. After selection by puromycin, stable cancer cells (PANC-1 and PATU-8988) bearing the PCK1 shRNA were established. The qRT-PCR assay results confirmed that RNA Fig. . PCK1 prRNA Fig. . PCK2 mRNAs Fig. .Fig. 2PCColony formation assay results, Fig. PCK1 mRNA silencing  led to robust ing Fig. . Functioing Fig. , viabiliing Fig. , migratiing Fig.  in primaWe next studied whether PCK1 silencing could provoke apoptosis activation in pancreatic cancer cells. In PANC-1 cells and PATU-8988 cells, following PCK1 silencing by targeted shRNAs see Fig. , the casPCK1 mRNA . As demonstrated RNA Fig.  and protRNA Fig.  expressiRNA Fig.  and inhiRNA Fig. . QuantifRNA Fig.  and “MatRNA Fig.  assays cFurther studies demonstrated that JC-1 green monomer intensity increase, reflecting mitochondrial depolarization, was detected in PCK1 KO cells Fig. . In addiWe next studied the possible underlying signaling mechanisms of PCK1-driven pancreatic cancer cell growth. High-throughput transcriptional profiling, or RNA-seq, was applied to analyze differentially expressed genes (DEGs) in PCK1-knockdown cells Fig. . As compPI3K-Akt-mTOR activation is an extremely important signaling pathway for cell growth . The abnTo further support the role of PCK1 in Akt-mTOR activation, a PCK1-S90A was stably transduced to PATU-8988 cells. As shown, PCK1-S90A largely inhibited PCK1 Ser-90 phosphorylation Fig. . ConsequTAS2R14, GAST4, FAM229A, C19orf73, and LY6G5C, was significantly corrected with better overall survival  was transduced to PANC-1 cells and PATU-8988 cells. Stable cell lines were established through selection with puromycin. As compared to control cells with empty vector (“Vec”), sed Fig. . PCK1 prsed Fig. , where Pged Fig. . Functioged Fig. . More imged Fig. .Fig. 6ExIn the primary pancreatic cancer cells, pPC1/ pPC2/pPC3, stable transfection of the OE-PCK1 construct led to robust PCK1 mRNA upregulation Fig. . In thesn = 5). Tumor growth curve results, Fig. To test whether PCK1 can exert similar tumor-promoting activity in vivo, we utilized a xenograft mouse model. The equal amount of PANC-1 cells, bearing PCK1 shRNA (“sh-PCK1-seq1”) or scramble control shRNA (“shC”), were subcutaneously (s.c.) injected into the flanks of the nude mice (five mice per group, PCK1 mRNA reduction in PCK1 shRNA-expressing PANC-1 xenograft tissues (Fig. Part of the tumor xenografts were lysed in fresh tumor tissue lysates. The qRT-PCR assay results confirmed robust ues Fig. . Resultsues Fig. .Emerging studies have revealed that metabolic reprogramming is a typical characteristic of cancers . The nutRecent studies have proposed a pivotal function of PCK1 in human cancer tumorigenesis progression , 29, 30.In this study, our results suggest that PCK1 could be an important gene for pancreatic cancer cell growth. PCK1 is overexpressed in pancreatic cancer tissues. PCK1 upregulation is also detected in established and primary human pancreatic cancer cells. Its expression is relatively low in pancreatic epithelial cells. In pancreatic cancer cells, shRNA-induced PCK1 silencing or CRISPR/Cas9-induced PCK1 KO robustly inhibited cell growth, viability, proliferation, migration and invasion, and provoked apoptosis activation. Conversely, ectopic overexpression of PCK1 in established and primary pancreatic cancer cells augmented cell proliferation and mobility. In vivo, the growth of PANC-1 xenografts in SCID mice was largely inhibited after PCK1 silencing.PCK1 kinase activity promoted SREBP1-dependent lipogenesis to promote HCC growth and tumorigenesis . PCK1 pSHere we found that PCK1 is important for Akt-mTOR activation in pancreatic cancer cells. RNA-seq analyzing DEGs in PCK1-silenced PANC-1 cells showed that DEGs are enriched in PI3K-Akt-mTOR cascades. Importantly, in PANC-1 and PATU-8988 cells, Akt-mTOR activation was largely inhibited by shRNA-induced silencing of PCK1 but was augmented after ectopic PCK1 overexpression. Furthermore, Akt-mTOR inactivation was detected in PCK1 shRNA-expressing PANC-1 xenograft tumor tissues. Moreover, the PCK1-S90A suppressed Akt-mTOR activation and inhibited pancreatic cancer migration and proliferation. These results implied a pivotal role of PCK1 in the activation of PI3K-Akt-mTOR cascade in pancreatic cancer cells. The underlying signaling mechanisms of PCK1-driven pancreatic cancer growth may warrant further characterizations. The pathological mechanisms of increased PCK1 expression and phosphorylation in pancreatic cancer require further investigations as well. Although our preliminary findings implied a possible role of microRNA dysregulation in the process.PCK1 is a potential therapeutic target for pancreatic cancer.Supplementary MethodsFigure S1


Age-Friendly communities are charged with fostering a social environment where social connections are available, accessible, and meaningful. Thematic content analysis of 67 interviews (representing 73 communities) conducted between 12/09/2019 and 01/24/2020) and 59 interviews (representing 71 communities) conducted between 12/14/2020 and 1/19/2021 with age-friendly leaders in rural Maine suggested the importance of expanding multi-sectoral collaborations and developing flexible strategies that allow older people to create and maintain social connections, even during COVID. Prior to the pandemic, common strategies were: (1) local partners and volunteers; (2) in-person activities; (3) accessibility; (4) reciprocity; and, (5) neighborhood-specific solutions. During the pandemic, there was an increased reliance on regional partners and funders to develop low or no-tech and tech-enabled social opportunities. Additional adaptive strategies included: (1) intergenerational ties; (2) emphasizing fun; and, (3) flexibility. The study has implications for understanding how rural age-friendly communities develop and grow multi-sectoral collaborations to enhance social connections.


Scientific Reportshttps://doi.org/10.1038/s41598-018-31106-0, published online 27 August 2018Correction to: The original version of this Article contained errors where the works of Giansanti (2015) and Sechi (2017) were incorrectly cited in the Materials and Methods section, under subheading ‘In situ hybridization and immunohistochemistry’. As a result, References 29 and 30 were omitted from the Reference list.et al., 2015; Sechi et al., 2017), mouse anti-dlg IgG ; 4F3), rabbit polyclonal anti-RFP IgG , mouse monoclonal anti-Lamin IgG  and mouse monoclonal anti-Peanut IgG .”“The following antibodies were used for immunostaining: rabbit polyclonal anti-anillin IgG  ; 4F3), rabbit polyclonal anti-RFP IgG , mouse monoclonal anti-Lamin IgG  and mouse monoclonal anti-Peanut IgG .”“The following antibodies were used for immunostaining: rabbit polyclonal anti-anillin IgG The original Article has been corrected.


KRAS mutant malignancies with a potential common cell of origin. PDAC ductal, but not NE, lineage traits have been associated with cell-intrinsic activation of interferon (IFN) pathways. The present studies demonstrate that the MUC1 C-terminal subunit (MUC1-C), which evolved to protect mammalian epithelia from loss of homeostasis, is aberrantly overexpressed in KRAS mutant PDAC tumors and cell lines. We show that MUC1-C is necessary for activation of the type I and II IFN pathways and for expression of the Yamanaka OCT4, SOX2, KLF4 and MYC (OSKM) pluripotency factors. Our results demonstrate that MUC1-C integrates IFN signaling and pluripotency with NE dedifferentiation by forming a complex with MYC and driving the (i) achaete-scute homolog 1 and BRN2/POU3F2 neural, and (ii) NOTCH1/2 stemness transcription factors. Of translational relevance, targeting MUC1-C genetically and pharmacologically in PDAC cells (i) suppresses OSKM, NE dedifferentiation and NOTCH1/2, and (ii) inhibits self-renewal capacity and tumorigenicity. In PDAC tumors, we show that MUC1 significantly associates with activation of IFN signaling, MYC and NOTCH, and that upregulation of the MUC1-C → MYC pathway confers a poor prognosis. These findings indicate that MUC1-C dictates PDAC NE lineage specification and is a potential target for the treatment of recalcitrant pancreatic carcinomas with NE dedifferentiation.Pancreatic ductal adenocarcinomas (PDAC) and poorly differentiated pancreatic neuroendocrine (NE) carcinomas are  Our results demonstrate that MUC1-C drives PDAC progression by integrating induction of neuroendocrine (NE) dedifferentiation with self-renewal capacity and tumorigenicity, in support of MUC1-C as a druggable target for the treatment of poorly differentiated pancreatic NE carcinomas. KRAS, TP53, SMAD4 and CDKN2A, among which KRAS mutations are the most frequent being found in up to 90% of tumors  is a highly aggressive malignancy with an increasing incidence . Curativf tumors ,7. Two dnt cells ,9. OtherMUC1 gene appeared in mammals to protect epithelia from the external environment (MUC1 encodes (i) an N-terminal subunit that includes glycosylated tandem repeats and is shed from the cell surface, and (ii) a C-terminal transmembrane subunit (MUC1-C) that is activated by loss of homeostasis and is associated with wound healing responses of inflammation, proliferation and remodeling  type I and II IFN pathways, (ii) Yamanaka pluripotency factors , (iii) the achaete-scute homolog 1 (ASCL1) and BRN2 NE lineage TFs and (iv) the NOTCH1/2 stemness TFs. Our results further demonstrate that MUC1-C integrates induction of NE dedifferentiation with self-renewal capacity and tumorigenicity in PDAC progression, in support of MUC1-C as a druggable target for the treatment of poorly differentiated pancreatic NE carcinomas.The present work demonstrates that MUC1 is overexpressed in KRAS cells  were cultured in Eagle’s Minimum Essential Medium (ATCC) supplemented with 10% fetal bovine serum. AsPC-1 mutant KRAS (ATCC), Panc-1 mutant KRAS (ATCC), MiaPaCa-2 mutant KRAS (ATCC) and BxPC-3 wild-type KRAS (ATCC) cells were cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum and 2 mM glutamine. Authentication of the cells was performed by short tandem repeat analysis every 4 months. Cells were monitored for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit  every 3 months.HPAF-II mutant MUC1shRNA , MYCshRNA  or a control scrambled shRNA  was inserted into the pLKO-tet-puro vector . The viral vectors were produced in 293T cells as described . Cells tTotal lysates prepared from subconfluent cells were subjected to immunoblot analysis. Immunoblotting was performed with anti-MUC1-C , Danvers, MA), anti-MYC , anti-OCT4 , anti-SOX2 , anti-KLF4 , anti-BRN2 , anti-ASCL1 , anti-SYP , anti-AURKA , anti-NOTCH1 , anti-NOTCH2  and anti-GAPDH .Total RNA from cells cultured separately in triplicates was isolated using Trizol reagent (Invitrogen) as described . TruSeq HPAF-II and AsPC-1 cells were fixed in 3.7% paraformaldehyde (Sigma) at room temperature for 15 min. These samples were washed three times with phosphate-buffered saline and then incubated with 0.3% Triton X-100 (Sigma) at room temperature for 10 min. The samples were blocked with 3% BSA and incubated with anti-MUC1-C  or anti-MYC  at 4°C overnight. The samples were then incubated with goat anti-Armenian hamster IgG H and L labeled with Alexa Fluor 488 (Abcam) and goat anti-rabbit IgG H and L labeled with Alexa Fluor 647 (Abcam) at room temperature for 1 h. DAPI (Sigma) was used for staining of nuclei. The cells were analyzed by confocal microscopy using an inverted Leica TCS SP5 microscope. Immunofluorescence intensities were calculated using ImageJ software.4) were seeded per well in 24-well ultra-low attachment culture plates  in DMEM/F12 50/50 medium  with 20 ng/ml epidermal growth factor (Millipore Sigma), 20 ng/ml bFGF  and 1% B27 supplement (Gibco). Growth factors were replenished every 3 days. Cells were treated with 0.1% DMSO as the vehicle control or 500 ng/ml DOX for 7 days. HPAF-II and AsPC-1 cells (1 × 104) were seeded per well in 24-well ultra-low attachment culture plates in tumorsphere culture medium in the absence or presence of 10 μM GO-203 for 72 h. Tumorspheres with a diameter >50 μm were counted under an inverted microscope in triplicate wells.HPAF-II/tet-MUC1shRNA and AsPC-1/tet-MUC1shRNA cells (1 × 10Cells were seeded at 1000 cells/well in 6-well plates and treated with (i) vehicle and 500 ng/ml DOX for 6 days, or (ii) 10 μM GO-203 for 4 days. The cells were stained with 0.5% crystal violet in 25% methanol on day 14 after treatment. Colonies >25 cells were counted in triplicate wells.6 tumor cells in 100 μl of a 1:1 solution of medium and Matrigel (BD Biosciences). When the mean tumor volume reached 100–150 mm3, mice were pair-matched into groups. In studies of HPAF-II/tet-MUC1shRNA and AsPC-1/tet-MUC1shRNA tumors, mice were fed without or with DOX . In studies of HPAF-II tumors, mice were treated intraperitoneally each day with phosphate-buffered saline or GO-203 at a dose of 12 μg/gm body weight. Tumor measurements and body weights were recorded every 3 days. Mice were sacrificed when tumors reached >1000 mm3 as calculated by the formula: (width)2 × length/2. These studies were conducted in accordance with ethical regulations required for approval by the Dana-Farber Cancer Institute Animal Care and Use Committee under protocol 03-029.Six-week-old female nude mice  were injected subcutaneously in the flank with 2–5 × 10U test was used to determine differences between means of groups. A P-value of <0.05 denoted by an asterisk (*) was considered statistically significant.Each experiment was performed at least three times. Data are expressed as the mean ± standard deviation (SD). The unpaired Mann–Whitney MUC1 is significantly overexpressed in KRAS mutant (n = 108), as compared to KRAS wild-type (n = 55), tumors (KRAS mutations (Carcinogenesis Online). MUC1 encodes the 158 aa MUC1-C subunit that includes a 58 aa extracellular domain, a 28 aa transmembrane domain and a 72 aa cytoplasmic domain or tail (Carcinogenesis Online). Analysis of pancreatic cancer cell lines demonstrated that MUC1-C is overexpressed in HPAF-II (KRAS G12D) and AsPC-1 (KRAS G12D) cells (Carcinogenesis Online). Treatment of the HPAF-II transfectants with DOX resulted in the suppression of MUC1-C in HPAF-II/tet-MUC1shRNA and not HPAF-II/tet-CshRNA cells (Carcinogenesis Online). As compared to that found for cells grown as monolayers, MUC1-C levels were clearly upregulated in association with the formation of tumorspheres (Carcinogenesis Online). Importantly, silencing MUC1-C significantly decreased the number of tumorspheres  , tumors , includiD) cells , which wD) cells . By contD) cells . To addrNA cells . As a seNA cells . HPAF-IINA cells ,22. We, rspheres  and F, dKRAS mutant PDAC cell-intrinsic activation of an IFN signature has been associated with a ductal-lineage specification  and that, like MUC1-C, the OSKM factors are induced in AsPC-1 and HPAF-II tumorspheres (Carcinogenesis Online), consistent with the involvement of pluripotency in driving stemness and self-renewal (Carcinogenesis Online) and AsPC-1 (Carcinogenesis Online) cells. SYP, SNAP25 and chromogranin A are vesicle markers of neuronal cells  pluripotency reprogramming factors confer lineage plasticity and dedifferentiation of fibroblasts . Among tgression . We foun-renewal . In conc-renewal , in supp-renewal . Confocaal cells , whereasal cells ,41. Surpal cells . MUC1-C al cells ,40. In tal cells . Along tal cells , in furtCarcinogenesis Online). Consistent with the effects of silencing MUC1-C genetically, treatment of HPAF-II and AsPC-1 cells with GO-203 resulted in the downregulation of MUC1-C and suppression of OCT4, SOX2, KLF4 and MYC (Carcinogenesis Online). In addition, we found that GO-203 suppresses ASCL1, BRN2 and AURKA in HPAF-II and AsPC-1 cells (Carcinogenesis Online). In further support of driving dedifferentiation, targeting MUC1-C with GO-203 decreased the capacity of HPAF-II and AsPC-1 CSCs to form tumorspheres (Carcinogenesis Online).Targeting MUC1-C with the GO-203 inhibitor, which blocks MUC1-C homodimerization and nuclear localization , phenocoNOTCH1 signaling has been linked to driving NE dedifferentiation of CSCs . AdditioIn determining whether MUC1-C drives tumorigenicity, we established HPAF-II/tet-MUC1shRNA tumor xenografts in nude mice and found that feeding DOX to suppress MUC1-C expression significantly decreased tumor growth . In addiCarcinogenesis Online) and the HALLMARK NOTCH SIGNALING (Carcinogenesis Online) and NOTCH2 (Carcinogenesis Online) expression. We found that MUC1-high/MYC-high, as compared to MUC1-low/MYC-high, tumors associate with significant decreases in patient survival  in a manner that is potentiated by p53 and RB suppression . We founStudies in castration-resistant prostate cancer cells have demonstrated that MUC1-C regulates lineage specification in the progression to NE prostate cancer . In thatbgab097_suppl_Supplementary_MaterialClick here for additional data file.


Aging is associated with accumulation of various tryptophan degradation products that may having either bone anabolic or catabolic effects. In epidemiologic studies, elevated levels of 3-hydroxyanthranilic acid (3-HAA) are associated with a higher bone mineral density (BMD). We have previously shown that the C57BL/6 mouse loses bone mass with age. Thus, we hypothesized that administering 3-HAA via a daily intraperitoneal (IP) injection would result in preserved or increased BMD. In an IACUC-approved protocol, we injected 26-month-old C57BL/6 mice with either a low dose (0.5 mg) or high dose (5 mg) of 3-HAA IP five days a week for eight weeks. At the end of this time mice were sacrificed and body composition and bone mineral density measured by DigiMus. BMD was significantly lower in the high dose 3-HAA group: 0.0570 + 0.004 vs 0.0473 + 0.006 vs 0.0432 + 0.0075 gm/cm2, . 3-HAA had no significant impact on body composition . Thus, 3-HAA did not prevent bone loss in older mice but instead significantly worsened bone loss. 3-HAA is known to have both pro- and anti- oxidant effects depending on the environment. These data would suggest that at the higher concentrations 3-HAA is acting predominantly as a pro-oxidant molecule accelerating age-related bone loss.


The use of virus-vectored platforms has increasingly gained attention in vaccine development as a means for delivering antigenic genes of interest into target hosts. Here, we describe a single-cycle influenza virus-based SARS-CoV-2 vaccine designated as scPR8-RBD-M2. The vaccine utilizes the chimeric gene encoding 2A peptide-based bicistronic protein cassette of the SARS-CoV-2 receptor-binding domain (RBD) and influenza matrix 2 (M2) protein. The C-terminus of the RBD was designed to link with the cytoplasmic domain of the influenza virus hemagglutinin (HA) to anchor the RBD on the surface of producing cells and virus envelope. The chimeric RBD-M2 gene was incorporated in place of the HA open-reading frame (ORF) between the 3′ and 5′ UTR of HA gene for the virus rescue in MDCK cells stably expressing HA. The virus was also constructed with the disrupted M2 ORF in segment seven to ensure that M2 from the RBD-M2 was utilized. The chimeric gene was intact and strongly expressed in infected cells upon several passages, suggesting that the antigen was stably maintained in the vaccine candidate. Mice inoculated with scPR8-RBD-M2 via two alternative prime-boost regimens  elicited robust mucosal and systemic humoral immune responses and cell-mediated immunity. Notably, we demonstrated that immunized mouse sera exhibited neutralizing activity against pseudotyped viruses bearing SARS-CoV-2 spikes from various variants, albeit with varying potency. Our study warrants further development of a replication-deficient influenza virus as a promising SARS-CoV-2 vaccine candidate. Severe acute respiratory syndrome coronavirus SARS-CoV-2), a newly emerged human coronavirus, has been identified as a causative agent of COVID-19 and has led to a global pandemic. Effective vaccines that elicit efficient adaptive immunity, including B and T cell responses against SARS-CoV-2, are essential for the fight against the spread of the virus and the return to pre-COVID-19 normalcy. While several COVID-19 vaccines [, a newlyWell-established reverse genetics techniques have shown to facilitate rapid influenza virus recovery . MoreoveA single-cycle influenza A virus (scIAV)-based vaccine was designed to allow the expression of foreign genes and served as a safe viral-vectored vaccine candidate  for COVIIn this study, we aimed to generate a scIAV-based SARS-CoV-2 vaccine to improve its safety and immunogenicity. The prototype influenza A/Puerto Rico/8/1934  strain was chosen as the viral backbone for the generation of a scIAV-based vaccine (scPR8) due to its attenuated phenotype in humans . The syn® medium  supplemented with 10% fetal bovine serum (FBS) and 1X antibiotic-antimycotic . Cells were grown at 37 °C in a 5% CO2 atmosphere.Human embryonic kidney  and Madin–Darby canine kidney  cells were maintained in Opti-MEMMDCK cells stably expressing the HA protein (MDCK-HA) derived from A/Puerto Rico/8/1934  have been previously described . BrieflyThe codon-optimized construct, tPA-RBD-HAcyt-2A-M2 (RBD-M2) comprising the signal peptide of human tissue plasminogen activator (tPA), the RBD of SARS-CoV-2 spike (Wuhan-Hu-1 (EPI_ISL_402125); amino acid 325–532), the transmembrane and the cytoplasmic tail of the PR8 HA (amino acid 517–565), -2A and the PR8 M2 was synthesized for high expression in human cells . To generate the plasmid expressing RBD-M2 for IAV reverse genetics, the pHW2000-PR8 HA plasmid was used as a template to remove the original HA, leaving the 3′ non-coding region (NCR) and 100 nucleotides (nt) of 3′ HA, and the 5′ NCR, and 180 nt of 5′ HA of linearized plasmid (pHW-ΔHA-PS). The pHW-ΔHA-PS was subsequently ligated with the RBD-M2 construct by In-Fusion™ cloning technique . In addition, the reporter gene, mCherry, was also cloned into the pHW-ΔHA-PS for making the pHW-ΔHA-mCherry as a plasmid control.pHW-ΔM2 plasmid was constructed as described previously . BrieflySARS-CoV-2 spikes , B.1.351 , and P.1 ) were codon-optimized and synthesized (Genscript). Each synthetic gene was cloned into pCAGGS expression plasmid. Sequences used for gene synthesis were derived from the GISAID database.scPR8-RBD-M2 viruses were recovered using the 9-plasmid reverse genetics rescue system as described previously . Briefly® containing 2 µg/mL of TPCK-treated trypsin at 37 °C. Cell supernatants were collected at indicated time points. For virus titration, MDCK-HA cells grown in flat-bottom 96-well plates were inoculated with serial 10-fold dilutions of scPR8-RBD-M2 and incubated at 37 °C for 72 h. scPR8-RBD-M2-infected MDCK-HA cells were observed microscopically with fluorescent conjugated secondary antibodies recognizing monoclonal antibodies (MAb) against influenza virus NP (FluA-NP 4F1) . Virus titers were calculated by Reed and Muench method (TCID50/mL) [To assess the growth of scPR8-RBD-M2 compared to the wild-type (WT) PR8, MDCK and MDCK-HA cells were infected with the viruses at an MOI of 0.01 and maintained in Opti-MEMID50/mL) .® 488  were used as secondary antibodies. After washing, cells were mounted with ProlongTM Gold Antifade Mountant with DAPI . The samples were observed by FluoviewTM FV1000 confocal microscopy .MDCK-HA cells were grown on coverslips in 6-well plates and infected with scPR8-RBD-M2 and scPR8-mCherry-M2 viruses for 48 h. After infection, cells were fixed with 4% paraformaldehyde at 4 °C for 20 min and blocked under non-permeabilized and permeabilized conditions with 5% FBS and 0.5% BSA at room temperature for 1 h. Cells were then probed with rabbit anti-RBD or anti-S antibodies . Goat anti-rabbit antibodies conjugated with Alexa FluorPR8, scPR8-RBD-M2 and scPR8-mCherry-M2 were concentrated by ultracentrifugation through 20% glycerol in PBS. Five microliters of each virus sample were negatively stained with 3% phosphotungstic acid solution (PTA) for 30 sec. The virus/PTA mixture was incubated on a carbon-coated copper grid (EMS) and incubated for 1 min. For immunogold labeling, the virus/PTA mixture was adsorbed onto a carbon-coated copper grid for 30 min followed by blocking with 1% bovine serum albumin (BSA) for 1 h. The grids were then incubated with rabbit anti-Spike RBD monoclonal antibodies  at 1:30 dilution in PBS for 1 h and rinsed six times with PBS, followed by incubation with goat anti-rabbit IgG antibodies (conjugated with 10-nm gold particles) at 1:100 dilution in PBS for 1 h. After washing, the samples were fixed for 5 min in 2% glutaraldehyde and negatively stained with 3% PTA. The grid was kept in a vacuum incubator before observing by transmission electron microscope  at 80 kV.Cells were collected and re-suspended in the Pierce™ mammalian cell lysis buffer . Protein samples were loaded onto polyacrylamide gel, and transferred to nitrocellulose membranes. Membranes were probed with rabbit anti-Spike RBD, anti-S , anti-influenza A NP (FluA NP 4F1) (Southern Biotech), Mouse anti-2A . Goat anti-rabbit IgG-HRP  and anti-mouse IgG-HRP antibodies were used as secondary antibodies for chemiluminescence detection by ChemiDoc™ XRS+ imager .n = 5/group) and received the vaccine using two alternative prime-boost  regimens including (i) intranasal-intranasal (IN-IN) and (ii) intranasal-intramuscular (IN-IM) routes. Mice were anesthetized with isoflurane before vaccination. For the first dose, mice were intranasally infected with scPR8-RBD-M2 or scPR8-mCherry-M2 . At 3 weeks after the first vaccination, mice were subsequently intranasally or intramuscularly boosted with the same dose of viruses. Throughout the immunization period, percentage of body weight loss and clinical signs of infection  were monitored. At 3 weeks after boosting, mice were sacrificed for sample collection . All mouse experiments were conducted following the guidelines of the Institutional Animal Care and Use Committee (IACUCs), National Center for Genetic Engineering and Biotechnology .To determine vaccine immunogenicity in the mouse model, 8-week-old female BALB/c mice were purchased from Nomura Siam International Co., Ltd. . Mice were divided into 5 groups  was pre-coated with 5 µg/mL SARS-CoV-2 spike protein purified from HEK293T cells transfected with pCAGGS expressing soluble SARS-CoV-2 spike (a kind gift from Dr. Florian Krammer distributed via BEI resources). After blocking with 1% BSA , serially diluted mouse sera or trachea/lung supernatants were added into each well and incubated at room temperature for 1 h. Next, the plates were washed with PBST (0.1% Tween-20) and incubated with HRP-conjugated goat antibodies against mouse IgG (KPL) or mouse IgA (Abcam) for 1 h. After washing, the substrate 3,3′,5,5′-tetramethylbenzidine  was added to the plates and the reaction was stopped by adding 1N H2SO4. The absorbance was measured by Synergy, HTX multi-mode reader at 450 nm . The flat-bottom 96-well ELISA plate coated with purified PR8 HA  was used to measure IgG specific to PR8 HA in mouse sera.The trachea and lung were homogenized in 400 µL PBS and centrifuged at 10,000× ® mouse IFN-γ ELISPOT kit . Briefly, 96-well ELISPOT plates were activated by adding 15 µL of 70% ethanol per well and washed 3 times with 1 x PBS. The plates were then coated with anti-murine IFN-γ antibodies diluted in murine IFN-γ capture solution at 4 °C overnight. Next, single-cell suspensions prepared from the spleens of vaccinated mice were added to the wells (3 × 105 cells/well) and incubated with pools of overlapping 15-mer peptides spanning the entire length of SARS-CoV-2 spike protein  or NP peptide 147–158 [2 for 24 h. After washing the plates with PBS and 0.05% Tween-PBS, biotinylated-labeled anti-mouse IFN-γ mAbs were added to each well and incubated at room temperature for 2 h. After additional washes, streptavidin-conjugated alkaline phosphatase was added and further incubated at room temperature for 30 min. Lastly, 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP) solution was added to measure IFN-γ-producing T cell spots by an automated ELISPOT reader system and ImmunoSpot 3 software . Results were expressed as the number of spot-forming cells (SFC) per 106 input cells.According to the manufacturer’s protocol, the assay was performed using an ImmunoSpot 147–158  (JPT Pep4 relative light unit (RLU/mL) PV at 37 °C for 1 h and diluted in DMEM supplemented with 10% FBS before being transferred to each well of a tissue culture treated, white opaque 96-well microplate. HEK 293T cells expressing ACE2 and TMPRSS2 cells (1 × 104 cells) were suspended in 50 µL DMEM supplemented with 10% FBS and then added into each well. The plate was incubated at 37 °C, 5% CO2 for 48 h. Supernatants were removed from each well before adding 25 µL of Bright-GloTM luciferase substrate . The luciferase signals (RLU/mL) correlating to the PV titers were measured using Synergy, HTX multi-mode reader  and normalized with the signal gained from no-serum control following the guideline described previously [Pseudotyped viruses (PV) with the firefly luciferase reporter displaying the full-length spike protein of various SARS-CoV-2 variants of concern (VOC), including Alpha, Beta, and Gamma were generated and titrated using methods as described previously . Mouse seviously . The halp values < 0.05 were considered statistically significant. GraphPad Prism 9.0  was used for statistical analyses.All data were expressed as means ± standard error of means (SEM). The differences in mean values of between groups were analyzed by two-way ANOVA. In order to generate the recombinant influenza gene that encodes the membrane-anchored receptor-binding domain (RBD) of SARS-CoV-2 spike presented on the influenza virus envelope, the RBD region fused with the transmembrane domain and cytoplasmic tail of PR8 HA (RBD-HAcyt) was constructed to replace the HA ORF of PR8 HA segment. The human tissue plasminogen activator signal sequence (tPA) was also included upstream of RBD-HAcyt to increase RBD-HAcyt protein expression and immunogenicity of the vaccine . In factTo maintain the stability of the inserted gene, a bi-cistronic protein expression cassette of RBD-HAcyt and M2 was generated by introducing 2A self-cleavage site between the RBD-HAcyt (or mCherry as the control) and M2 protein A. The chImmunofluorescence assay was also performed to confirm that an ectopic expression of HA was necessary for scPR8-RBD-M2 propagation. Wild-type MDCK and MDCK-HA cells were infected with scPR8-RBD-M2 at MOI of 0.1 for 24 h and probed with anti-NP and anti-RBD antibodies. The results showed that while MDCK and MDCK-HA are permissive to scPR8-RBD-M2 infection, only MDCK-HA could support the multi-cycle replication of scPR8-RBD-M2 A. The grWe next sought to determine whether cells infected with scPR8-RBD-M2 expressed RBD on their surface. To this end, scPR8-RBD-M2-infected MDCK-HA cells were fixed and probed with anti-spike RBD antibodies at 48 hpi. RBD expressions were found localized on the surface of infected cells A. These 5 TCID50 of scPR8-RBD-M2, scPR8-mCherry-M2 or PBS  were primed and boosted intranasally (IN-IN) or intranasally/intramuscularly (IN-IM) at a 3-week interval with 1 × 102 or PBS A. Mice w2 or PBS B. After 2 or PBS C. To tes 147–158 . The pre+ T cells in both IN-IN and IN-IM vaccination regimens, demonstrating that scPR8-RBD-M2 could induce strong SARS-CoV-2 RBD specific T cell responses.We next investigated whether scPR8-RBD-M2 could induce specific immune responses to the spike protein through different prime-boost strategies in mice. Serum ELISA shows that scPR8-RBD-M2 administered by both IN-IN and IN-IM induced robust anti-RBD antibody titers A. Intere50 values described  neutralization assay A. Both Iescribed . Notablyescribed B. This r50 value shown , B.1.351 (Beta variant), and P.1 (Gamma variant). To assess whether RBD-specific antibodies obtained from scPR8-RBD-M2 immunized mice conferred neutralizing activity against new SARS-CoV-2 variants, pseudotyped viruses with Alpha, Beta or Gamma spike variant were generated. It is also noteworthy that spike-RBD specific antibodies induced by scPR8-RBD-M2 exhibit a cross-neutralization activity among spike proteins derived from distinct SARS-CoV-2 variants, despite a decreased of ICue shown . While Pue shown , we coulThe application of viral vector systems for vaccine development against many infectious diseases has recently gained popularity. Influenza A viruses are the known causative agent of a highly contagious respiratory disease. Their preferred portal of entry through the respiratory mucosal epithelium makes the influenza A virus an attractive viral vector for the SARS-CoV-2 candidate vaccine development. A single cycle infectious influenza A virus (scIAV) as a vaccine platform for COVID-19 vaccine development was established in the present study. We report encouraging results of scIAV-vectored SARS-CoV-2 vaccine candidate as a safe vaccine and induce robust immune responses against the spike RBD of SARS-CoV-2. This advantage would potentially allow the use of single-cycle infectious influenza virus as a dual vaccine platform targeting both the seasonal flu as well as SARS-CoV-2.The membrane-anchored form of the RBD domain of SARS-CoV-2 spike protein (Wuhan strain) was inserted into the HA gene segment of a laboratory-adapted influenza A/PR/8/34 A. Due to+ T cells are associated with control of primary SARS-CoV-2 infection in humans [An ideal goal of vaccination is to induce humoral immune responses and cell-mediated immunity, leading to protective immunity upon subsequent exposure to relevant pathogens. While protective immunity against SARS-CoV-2 depends mainly on virus-specific antibody quantity and quality, recent studies in transgenic mouse models provided evidence that T-cell mediated clearance of the virus is also known to play essential roles in the complete resolution of the infection . A crossn humans . Anothern humans  recapituBy profiting from the already established cell culture-based system in influenza vaccine production, it is feasible to use a replication-deficient IAV harboring the highly immunogenic spike RBD gene as a candidate vaccine against two major respiratory viruses: IAV and SARS-CoV-2. Despite its potential as a promising dual-specific vaccine candidate, certain caveats still need to be addressed. Most importantly, while we validated that vaccinated mice display both the induction of spike-specific IFN-γ producing T cells and the neutralizing activity, we did not directly test if immune responses elicited by scPR8-RBD-M2 are protective against SARS-CoV-2. Additionally, as observed in the use of other vectored vaccine platforms ,29,30, t


The ketogenic diet (KD)has been considered an effective treatment for epilepsy, whereas its underlying mechanisms remain obscure. We have previously reported that the KD feeding increased Neuregulin 1 (NRG1) expression in the hippocampus; disruption of NRG1 signaling by genetically deleting its receptor-ErbB4 abolished KDs effects on inhibitory synaptic activity and seizures. However, it is still unclear about the mechanisms underlying the effect of KD on NRG1 expression and whether the effects of KD require ErbB4 kinase activity.Nrg1 promoter locus was examined using the chromatin immunoprecipitation technique. Kainic acid (KA)-induced acute seizure model was utilized to examine the effects of KD and histone deacetylase inhibitor-TSA on seizures. Synaptic activities in the hippocampus were recorded with the technique of electrophysiology. The obligatory role of ErbB4 kinase activity in KDs effects on seizures and inhibitory synaptic activity was evaluated by using ErbB kinase antagonist and transgenic mouse-T796G.The effects of the KD on NRG1 expression were assessed via western blotting and real-time PCR. Acetylation level at the Nrg1 promoter locus of KD-fed mice. Treatment of TSA dramatically elevates NRG1 expression and diminishes the difference between the effects of the control diet (CD) and KD. These data indicate that KD increases NRG1 expression via up-regulating histone acetylation. Moreover, both pharmacological and genetic inhibitions of ErbB4 kinase activity significantly block the KDs effects on inhibitory synaptic activity and seizure, suggesting an essential role of ErbB4 kinase activity.We report that KD specifically increases Type I NRG1 expression in the hippocampus. Using the chromatin immunoprecipitation technique, we observe increased acetylated-histone occupancy at the These results strengthen our understanding of the role of NRG1/ErbB4 signaling in KD and shed light on novel therapeutic interventions for epilepsy.The online version contains supplementary material available at 10.1186/s13578-021-00611-7. Epilepsy is one of the most common neurological disorders that affects over 1% of people worldwide , which eThe ketogenic diet (KD) is a low-carbohydrate, moderate-protein, and high-fat diet known to induce a sustained ketotic state by producing heightened ketone bodies. Since the 1920s, the KD has been utilized to treat refractory epilepsy in the clinic . HoweverNrg1 gene produces more than 30 different isoforms comprising six types of protein: I-VI, in which the I, II, and III are the primary types [ErbB4 diminished the KDs effects on GABAergic synaptic transmission and seizures [Neuregulin 1 (NRG1) belongs to a trophic-factor family and contains an epidermal growth factor (EGF) domain . Due to ry types . A distiry types , 8. Locary types . By stimry types . In our seizures . These dBesides, evidence from in-vitro experiments demonstrated that both full-length and kinase-dead mutant of ErbB4 construct promoted presynaptic maturation , and typNrg1 promoter locus of KD-fed mice. Treatment of TSA, a broad-spectrum histone deacetylase (HDAC) inhibitor, dramatically elevates NRG1 expression in the hippocampus and diminishes the difference between the control diet (CD) and KD groups, indicating that KD increases NRG1 expression via up-regulating histone acetylation. Furthermore, both pharmacological and genetic inhibitions of ErbB4 kinase activity significantly block the KDs effects on inhibitory synaptic activity and seizure, suggesting an essential role of ErbB4 kinase activity. These results strengthen our understanding of the role of NRG1/ErbB4 signaling in KD.In the present study, we find that KD feeding selectively increases type I NRG1 expression in the hippocampus. Using the chromatin immunoprecipitation (CHIP) technique, we observe increased acetylated-histone occupancy at the The adult C57/B6 mice were treated with KD or its control diet (CD). Three weeks later, we found that the serum level of -hydroxybutyrate (a major form of ketone) was dramatically increased in KD-fed mice , a broad-spectrum HDAC inhibitor . TSA treTSA Fig. F. FurtheTSA Fig. G, H. In Previous studies have indicated that the GABA level was increased in the brain of KD-fed rodent animals , 23. OurMoreover, we tested whether TSA affects KDs anti-seizure effects. After C57/B6 mice were treated with CD or KD for 3 weeks, KA was then infused into the amygdala to induce seizures the next day after TSA injection  ; Mouse anti-ErbB4 (Santa Cruz Biotechnology) ; Rabbit anti-P-ErbB4  ; Mouse anti-GAPDH (Santa Cruz Biotechnology) ; Rabbit anti-H3 (Active motive) ; Rabbit anti-H3k18ac (Active motive) ; Rabbit anti-H3k27ac (Active motive) ; Rabbit anti-H4 (Active motive) ; Rabbit anti-H4k8ac (Active motive) ; Rabbit anti-H4k12ac (Active motive) ;); Rabbit anti-acetylated H4 (abcam) . Rabbit anti-acetylated H3 (Sigma) .Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich. DL-AP5 (0105), CNQX (0190) were purchased from Tocris Bioscience. 1NM-PP1 (HY-13942) and ITSA-1 (HY-100508) were from MedChemExpress.T796G, 5- CTT AGC AAT CTG GAC ACA CCAG-3 and 5- CCT ATT GGG AGT GTG TCT GAG TCC CAC TAT CCA GGT TAC G-3 and 5- CCC ACT ATC CAG TTG GTT GGC-3. In all experiments, significant efforts were made to minimize the total number of animals used while maintaining statistically valid group numbers. Animal housing conditions were maintained at a temperature of 221 C, at>30% humidity and a standard 12 h light/dark cycle. All animal experimental protocols were approved by the Animal Ethics Committee of Guangzhou Medical University.Eight-to twelve-week-old male mice were used for experiments. C57/B6 mice were purchased from the Laboratory Animal Center of Guangdong Province. The primers for genotyping were listed as below: As we reported previously , SD  according to the manufacturers instructions. Briefly, after sterilization with 70% ethanol, the tail tips of mice under tests were cut using a clean scissor, and a drop of blood was collected. Using the test strip (Abbott), levels of -hydroxybutyrate or glucose were determined.Brain tissue homogenates were prepared in RIPA Buffer containing (in mM): 50 TrisHCl, pH 7.4, 150 NaCl, 2 EDTA, 1 PMSF, 50 sodium fluoride, 1 sodium vanadate, 1 DTT with 1% sodium deoxycholate, 1% SDS and 1% protease inhibitors cocktails. Samples were resolved on SDS/PAGE and transferred to nitrocellulose membranes, which were incubated in TBS buffer containing 0.1% Tween-20 and 5% milk for 1 h at room temperature before incubating with primary antibodies (overnight at 4 C). After washing, the membranes were incubated with HRP-conjugated secondary antibodies (Absin ImmunoResearch) in the same TBS buffer for 1 h at room temperature. Immunoreactive complex bands were visualized using enhanced chemiluminescence (Pierce) and captured using the Genesys imaging system . Band densities of interested proteins were normalized with by the loading control.Nrg1 were normalized to those of GAPDH. Primer sequences were: Type I Nrg1, 5-AAG GGC AAG AAG AAG GAC CG-3 and 5-AAT CTG GGA GGC AAT GCT GG-3; Type II Nrg1, 5-TGC AAG CGG TGC GCA TT-3 and 5-TAC GGT TCA GCT CAT TCC CG-3; Type III Nrg1, 5- GCT GTC TGC TTT TCC TCC CTT-3 and 5-TGT TTG TGG CTG AGT TCC TGA-3; GAPDH, 5- GGT TGT CTC CTG CGA CTT CA-3 and 5-CCA CCA CCC TGT TGC TGT AG-3.qRT-PCR was performed as described previously . BrieflyNrg1 (CAAGAGTAGCCCCGAGACAC and AAAAGTTTGTCCCGGGAGGG). Each experiment was conducted at least three times.The CHIP assay was performed as directed by the manufacturer (Millipore). Briefly, samples were crosslinked with 1% formaldehyde at 37 C for 15 min and then subject to sonication into 2001000 bp fragments. The chromatin was immunoprecipitated with anti-acetylated H3 or H4 primary antibodies. The crosslinking was reversed, and the DNA was isolated on the columns provided by the kit. Real-time PCR was conducted with primers targeted to the promoter of type I 2, 0.5 CaCl2, 1.25 NaH2PO4, 25 NaHCO3, and 10 glucose. Coronal hippocampal slices (300 m) were sectioned in ice-cold modified ACSF using a VT-1000S vibratome  and transferred to a storage chamber containing regular ACSF (in mM)  at 32 C for 30 min and at room temperature (241 C) for additional 1 h before recording. All solutions were saturated with 95% O2 / 5% CO2 (vol/vol).Adult male mice were anesthetized with isoflurane. Brains were quickly removed and chilled in ice-cold modified artificial cerebrospinal fluid (ACSF) containing (in mM): 120 Choline-Cl, 2.5 KCl, 7 MgClSlices were placed in the recording chamber superfused (2 ml/min) with ACSF. Whole-cell patch-clamp recording from CA1 pyramidal neurons was visualized with infrared optics using an upright microscope equipped with an infrared-sensitive CCD camera . Pipettes were pulled by a micropipette puller  with a resistance of 35 M. Recordings were made with MultiClamp 700B amplifier and 1440A digitizer (Molecular Device).2, 4 Mg-ATP, 0.3 Na-GTP, 10 phosphocreatine and 5 QX314 . To measure evoked inhibitory postsynaptic currents (eIPSCs), stimulation electrode was positioned on the Schaffer Collaterals (SC)-CA1 pathway,~100 m away from the recording pipette.To record spontaneous inhibitory postsynaptic current (sIPSC), pyramidal neurons were held at -70 mV in the presence of 20 M CNQX and 50 M AP-5, with the pipette solution containing (in mM): 140 CsCl, 10 Hepes, 0.2 EGTA, 1 MgClIn all experiments, series resistance was maintained below 20 M and not compensated. Cells would be rejected if membrane potentials were positive more than -60 mV; or if series resistance fluctuated more than 20% of initial values. Data were filtered at 1 kHz and sampled at 10 kHz.Adult male mice were maintained anesthetized with isoflurane (23%) and head-fixed in a stereotaxic device (RWD Life Science.Inc). After an incision was made in the scalp, a small hole was drilled into the skull, and a guide cannula  was implanted inside the right amygdala  or the left lateral ventricle , and cemented onto the skull with dental cement. Mice were then returned to their homecages for at least 1 week.As previously reported , mice wiP<0.05 was considered to be statistically significant.Adult male mice (2 months) were used in the present study. Animal or replicate numbers for each experiment and results of the statistical analyses, including degrees of freedom and exact p-values were mentioned in the figure legends. Statistical analyses were performed using GraphPad Prism (GraphPad Software). Students t-test was used to compare data from two groups. One-way ANOVA with sidaks post multiple comparison test was used to compare data from multiple groups. Regular two-way ANOVA with sidaks post multiple comparison test was used in the studies that analyze more than two parameters. All tests were two-sided. All data represent meanSEM, unless otherwise stated. Additional file 1. Supplementary figures.


Microbial biofilms are represented by sessile microbial communities with modified gene expression and phenotype, adhered to a surface and embedded in a matrix of self-produced extracellular polymeric substances (EPS). Microbial biofilms can develop on both prosthetic devices and tissues, generating chronic and persistent infections that cannot be eradicated with classical organic-based antimicrobials, because of their increased tolerance to antimicrobials and the host immune system. Several complexes based mostly on 3D ions have shown promising potential for fighting biofilm-associated infections, due to their large spectrum antimicrobial and anti-biofilm activity. The literature usually reports species containing Mn(II), Ni(II), Co(II), Cu(II) or Zn(II) and a large variety of multidentate ligands with chelating properties such as antibiotics, Schiff bases, biguanides, N-based macrocyclic and fused rings derivatives. This review presents the progress in the development of such species and their anti-biofilm activity, as well as the contribution of biomaterials science to incorporate these complexes in composite platforms for reducing the negative impact of medical biofilms. Infectious diseases remain at the top of the list of mortality and morbidity causes, resulting from many converging factors such as global emergence and the spread of genetically encoded resistance to all currently used antibiotics, the delays in the discovery of novel antimicrobials, and complications associated with biofilm development on tissues and prosthetic devices ,2. MicroCandida–streptococcal associations in the case of oral diseases SO4·2H2O (1) (Hcip: ciprofloxacin-a fluoroquinolone antibiotic) was studied as anti-biofilm species able to provide a high concentration of Hcip in the lungs. ·2C2H5OH}n (2)  showed both strong inhibition of C. albicans clinical isolates biofilm formation at subinhibitory concentration and the ability to reduce its adherence to human non-small cell lung cancer A549 cells in vitro. Moreover, this compound inhibits pyocyanin production and biofilm formation in P. aeruginosa, results that recommend its further examination in the mixed Candida-P. aeruginosa infections [Complex {[ZnClfections .2O)6]0.5[Mn(smx)3] (3) (smx: sulfamethoxazole–an antibiotic from the second generation of sulfonamides) was fully characterized by single crystal X-ray diffraction and proved to be an inhibitor of both the planktonic and biofilm embedded Staphylococcus aureus strain  (4) (Ph: phenyl) species for all tested strains pyrimidine scaffold aroused the pharmaceutical interest due to their resemblance with purine bases. As result, several complexes of Co(II), Ni(II), Cu(II), Zn(II) with such ligands were developed and tested on as anti-biofilm species. Among these, the series of complexes of type [MpmtpX2]  and Gram positive  bacterial strains, as well as the C. albicans fungal strain. A broad spectrum of anti-biofilm activity was demonstrated by all Cu(II) species [Cu2(pmtp)2Cl4(OH2)2] (5), [Cu(pmtp)(CH3COO)2]·0.5H2O (6) and [Cu(pmtp)(OH2)3](ClO4)·3H2O (7) from this series at subinhibitory concentrations, including MRSA and other clinical isolates pyrimidine (dmtp) [Co(dmtp)2Cl2] (8) also exhibited a broad spectrum of anti-biofilm activity, being tested on the same strains [2(OH2)](ClO4)2·dmtp (9/10) proved to exhibit a stronger antimicrobial and anti-biofilm effect against the Gram-positive strains, including MRSA. In addition, compounds display an antiproliferative effect on murine melanoma (B16 cells); low toxicity on normal (BJ) cells, do not affect the membrane integrity and behave as metallonucleases Cl2·2H2O (11) (Him: imidazole) inhibits the E. coli biofilm [2(acr)4(Hbzim/Me2bzim)2] (12/13)  exhibited anti-biofilm activity on a wide range of bacteria  as well as C. albicans, activity complemented for (12) by an antiproliferative effect on the colon adenocarcinoma (HT29) cell line  (14) (tcpp: 1-thiocarbamoyl-5-(4-chlorophenyl/)-3-phenyl-4,5-dihydro-1H-pyrazole; tbpp: 1-thiocarbamoyl-5-(4-bromophenyl)-3-phenyl-4,5-dihydro-1H-pyrazole) species on C. glabrata biofilm. Moreover, these complexes do not exhibit mutagenic potential or cytotoxicity against cervical cancer  cell lines and Vero non-tumor cells [Good activity was reported for [Co(tcpp/tbpp)or cells .2O)2(NO3)2] (15/16)  are able to inhibit QS by successful modulation of signalling molecules production. Aside from the inhibitory activity of complexes on the acyl homoserine and 2-alkyl-4-quinolones (AHQs) level, they are also potent inhibitors of biofilm formation in P. aeruginosa PAO1 [Some Cu(II) complexes of type [Cu(pym/quz)(Hosa PAO1 .3)2X] (17)  with β-carboline (cbl) at sub-MIC concentration interfered significantly with the QS regulated functions in Chromobacterium violaceum (violacein), P. aeruginosa  and S. marcescens (prodigiosin). Aside from the inhibitory effect on the EPS production and swarming motility, these complexes also demonstrated potent broad-spectrum inhibition of biofilm formed by P. aeruginosa, E. coli, C. violaceum, S. marcescens, K. pneumoniae and L. monocytogenes n (19) ethane, H2muco: trans, trans-muconic acid) inhibited S. aureus biofilm formation. Furthermore, the inflammatory response of the spine surgery incision was supressed by this species (ClO4)2 (20)  proved to be a very potent antibacterial agent against both susceptible or resistant Gram-positive (S. aureus) and Gram negative  strains in planktonic or biofilm growth states. The minimum biofilm eradication concentration (MBEC) values followed a similar trend with that of MIC recorded for most of the tested strains [2(pmtp)](ClO4)2 (21/22)  showed antibacterial potential against several bacterial strains, including MRSA, extended-spectrum beta-lactamase (ESBL) producing E. coli and multi-drug-resistant P. aeruginosa, both in planktonic and biofilm growth states. In addition, both compounds exhibited superoxide scavenging ability, intercalative DNA properties, metallonuclease activity and an antiproliferative effect on B16 cells Xivatives .P. aeruginosa biofilm activities were exhibited by the complex [Ag(mmphi)2]NO3 (24) (mmphi: (E)-7-(4-methoxybenzylidene)-3-(4-methoxyphenyl)-2-pyridyl-3,3a,4,5,6,7-hexahydro-2H-indazole) at MIC concentrations in a dose-dependent manner for P. aeruginosa, whereas a biphasic response was obtained for MRSA showing that the sub-MIC doses enhanced biofilm formation while its reduction was recorded at higher concentration  (25)  in P. aeruginosa biofilm eradication. The structure-activity relationship analysis evidenced an enhanced activity for pyridine derived ligands for hydroxyl as a substituent and nitrate as a counterion. In addition, complexes are non-toxic on primary human fibroblasts, exhibit catalase like activity and the ability to easily reach at Mn(III), associated with the compound’s ability to interact with biological target involved in biofilm destruction through redox processes [Good activity was demonstrated for a series of complexes of type [Mn(snh)rocesses .2]ClO4 (26) and [Cu(phendione)3](ClO4)2·4H2O (27)  were tested on several carbapenemase-producing Acinetobacter baumannii strains, a microorganism often exhibiting a multidrug-, extended drug- and even pan-drug-resistance profile. Both compounds affected the biofilm formation and disrupted the mature biofilm sub-MIC concentration in a typically dose-dependent manner, reducing biomass and viability parameters, with Cu(II) species again being more potent [Compounds [Ag(phendione)e potent .2(bmic)2(tet)2]·3DMF (28)  was proved as a good inhibitor of S. aureus biofilm formation including in a bacterial infection model confirmed by counting colony forming unit (CFU) numbers in the skin following experimental infection in vivo [The complex with mixed ligands [Zn in vivo .2]BF4 (29) (Hphtt: (E)-2-(2-(pyridin-2-ylmethylene)hydrazinyl)-4-(p-tolyl)thiazole) was reported as P. aeruginosa anti-biofilm species with a mechanism involving the transcriptional activator protein complex 3-oxo-C12-HSL-dependent QS system (LasI/LasR system) [2] (30) pyridine), where Co(II) adopts an unusual distorted square pyramidal geometry according with single-crystal X-ray diffraction data. The complex was screened for antibacterial activities against Gram-positive (B. substilis) and Gram-negative  bacterial strains. The effects of these complexes on QS-regulated behaviours of bacteria such as swarming and biofilm formation were also examined [A complex [Co(phtt) system) . As a QSexamined .31)  against S. aureus and P. aeruginosa together with a strong antioxidant activity [Good anti-biofilm activity was demonstrated by complex [Cu(dfx)(py)] , Ni(II), Cu(II), Zn(II)) with the biological activity of N-heterocycles , several valuable anti-biofilm species were developed. One of the potential mechanisms of action revealed by different studies is ROS generation.The antimicrobial activity is enhanced when two or more isolated or fused N-heterocycle rings are present in different ligand molecules. Moreover, complexes bearing mixed ligands, both from this class of derivatives, proved to be more active compared with those having only one type of N-heterocycle in their structure.E. coli and multi-drug-resistant P. aeruginosa.It is worthy of mentioning the wide spectrum of most of Cu(II) complexes as well as the good activity against resistant strains such as MRSA, ESBL In recent years, researchers have drawn significant attention toward Schiff bases and their metal complexes considering their numerous applications in the biological field, such as their antiviral, antimicrobial, antimalarial, and antitumor properties. Furthermore, some of complexes exhibit a good anti-biofilm activity besides the antimicrobial one against planktonic bacteria.2O (32) -methyl phenol, X: Cl ·nHl, X: Cl , CH3COO Cl  (33) (M: Co(II), Ni(II), Cu(II), HBS2: Schiff bases derived from cefotaxime/ceftazidime and salicylaldehyde, n = 2, 0) were studied as anti-biofilm species against E. coli, K. pneumoniae, S. aureus, and B. subtilis, the most active being Cu(II) compounds against E. coli and P. aeruginosa biofilms at sub-MIC concentrations [2)4]Cl2·4H2O (34) and [Cu{Ca(BS3)(OH2)2}2]Cl4·H2O (35) (HBS3: 2-hydroxy-8-methyl-tricyclo[7.3.1.02.7]tridec-13-N-4′(benzo-15-crown-5-ether)-imine) exhibited superior anti-biofilm activity compared to that of the ligand against several bacterial strains and C. albicans  (36/37) -hydrazones, alchil: CH3, C2H5, n-C3H7, i-C3H7). All complexes also exhibited a relatively high toxicity on human immortalized keratinocyte (HaCaT) cells at high concentrations  (neration .40) (H2BS7: 3-methoxy-2-oxidobenzylidenebenzohydrazide) were characterised by single crystal X-ray diffraction, and it was found that it repressed both P. aeruginosa and S. aureus biofilm formation. Moreover, the compound exhibited the ability to intercalate into the DNA strands [Compounds with the mixed ligands [Cu(BS7)(phen)]  complexes with multidentate Schiff bases having N,O or N,O,O donor sets and bulky substituents like phenyl, pyridine, triazole or pyrazole exhibit a very good anti-biofilm activity on a wide range of Gram-positive and Gram-negative bacteria. The activity is enhanced for the species bearing besides Schiff base another chelate ligand such as 1,10-phenantroline. However, the specific mechanisms of action for this type of complex remains to be elucidated in future research.From the perspective of anti-biofilm activity, the complexes with biguanide derivatives have also shown promising potential. The biguanides are valuable ligands that can coordinate in neutral, anionic or cationic form. Due to their chelate coordination through the imide groups they form stable complexes with transition metal ions as neutral or anionic species .N,N′-dimethylbiguanide, Hdmbg) compound used for type II diabetes treatment by decreasing the glucose release from the hepatic tissue is also the best known of these derivatives as complex formatters Cl·0.5H2O (42) [2]Cl2·H2O (43) Cl2 (44) (Htbg: 1-(o-tolyl)biguanide) [2](ClO4)2 (45) exhibited excellent antibacterial properties against biofilm embedded Gram-negative  and Gram-positive  bacteria, more intense in comparison with ampicillin. The best efficacy was noticed against E. cloacae and L. monocytogenes with MBEC of 1.95 µg/mL. One of the possible mechanisms of antimicrobial activity is represented by ROS generation. The complex was not cytotoxic on L929 fibroblasts, and the in silico analysis confirmed its drug-likeness and safety profile Cl2 (46)  proved to have good efficiency against the biofilm embedded S. aureus, B. subtilis and E. coli cells at sub-MIC values. The most efficient compounds showing the largest spectrum of anti-biofilm activity were Pd(II) and Pt(II) complexes. Moreover, the Pt(II) compound exhibited the most significant antiproliferative activity on the human cervical cancer (SiHa) cell line, inducing a cell cycle arrest in the G2/M phase X  ; R1= biph) proved to be active against a large spectrum of microbial strains, including MRSA, being able to also disrupt and eradicate the bacterial mature biofilm  ( biofilm .49)  for applications in the treatment of infections produced by pathogenic microorganisms, including those complicated by biofilm development. A broad anti-biofilm spectrum on P. aeruginosa, E. faecium, E. faecalis, C. albicans and C. parapsilosis was registered for Ni(II) complex with macrocycle resulted in ammonia system [Recent studies indicated that the biguanide incorporation into a macrocycle ligand generated promising complexes [M(dmbgMc)] , Zn(II)) and non-essential (Ni(II), Pd(II), Pt(II), Ir(III)) metal ions. The anti-biofilm activity is enhanced for perchlorate species and biguanides bearing bulky substituents as a result of the favourable balance between their water solubility and lipophilicity. The ROS generation was reported as one of the potential mechanisms of action for Cu(II) species.2]·nH2O (50/51) -methanone; mc2: -bisphenylen-1,3,6,8,10,13-hexaazacyclotetradecan-bis(pyrid-3-yl)methanone) synthesized by the template condensation were screened for anti-biofilm activity on both susceptible and resistant strains of P. aeruginosa, E. coli, K. pneumoniae, S. aureus, B. subtilis and C. albicans, and proved to be strong inhibitors in most cases at sub-MIC concentrations, especially the Cu(II) species. These species also exhibited antiproliferative activity on HEp 2 cells by inducing the cellular cycle arrest in the G2/M phase Cl by MRSA  while thomplexes .2(bmc1)](CH3COO)4 nH2O (53) -benzene) were designed as antimicrobials and Zn(II) complex exhibit ability to inhibit the S. aureus, B. subtilis, E. faecalis, K. pneumoniae, E. coli, E. cloacae, P. aeruginosa and C. krusei adherence on inert substratum ·nH2O (56) -benzene) exhibited good anti-biofilm activity, especially against S. aureus and E. coli strains, the most active being the copper(II) complex. In addition, all complexes disrupt the membrane integrity of human ileocecal adenocarcinoma (HCT 8) tumour cells ·nH2O (57),  complex displayed good potential ·H2O (58) (Hapa: 2-aminopyridine-4-carboxylic acid) based on an oxalate linker associated with a lysozyme activity of the compound [3[Fe(C2O4)3].3H2O (mbzim: 5-methylbenzimidazole) disrupted the biofilm only in case of C. albicans DMF·H2O}n (59)  was prepared and structurally characterized. The results of the violet crystal staining experiment showed that the new compound significantly inhibited the formation of the S. aureus biofilm in vitro [The metal-organic framework (MOF) {[Coin vitro .2] (60) (cur: curcumin) inhibited biofilm formation in the case of S. aureus and significantly repressed the expression of lasI and lasR genes, demonstrating its QS inhibitory effect ·2bza (61)  indicated moderate to very good anti-biofilm activity [The assays performed on activity .S. epidermidis [24(H2O)2] (62/63)  exhibited anti-biofilm activity on a clinical S. aureus isolate similarly or even better than doxycycline used as positive control (NO strains .2 (65)  is very active on the old biofilms of S. aureus, as indicated in the studies performed by confocal laser scanning microscopy which revealed its bactericidal activities, possibly by membrane alterations, as demonstrated by the propidium iodide (PI) uptake ) uptake .2] (66/67) (bedtcm: N-(benzyl)-(ethyl)-dithio carbamate, imdtcm: N-(4-isopropyl-benzyl)-(4-methoxy-benzyl)-dithiocarbamate) exhibited anti-biofilm activity against both methicillin susceptible and resistant S. aureus [2NO3] (68) (aptes: 3-aminopropyltriethoxysilane) showed pronounced antibacterial effects against P. mirabilis isolated from patients with urinary tract infections, and exhibited a clear decrease in the ability of this bacteria to form biofilms at MIC concentration Cl3 (71) into the same polymeric matrix exhibited the same ability to generate nitric oxide (NO) from endogenous nitrite. This material exerted anti-biofilm activity against Bacillus sp., in addition to nitrifying bacteria biofilm dispersion  and Ag(I). The composites were tested for their antibacterial potential and were found to effectively resist nalogues .L-lysine functionalized mesoporous silica SBA-15 for the selective coordination of Ag(I). This nano-species (72) exhibited inhibitory effect on E. coli, S. aureus, M. tuberculosis and C. albicans. Besides killing the C. albicans cells, this system inhibited biofilm formation and eliminated preformed biofilms, with no development of resistance during continuous serial passaging. The antifungal activity is related to the disruption of bacterial cell membranes and increased levels of intracellular ROS. In mouse models of multidrug-resistant C. albicans infection, nano-species exhibited an efficient in vivo fungicidal efficacy superior to the antifungal drugs, amphotericin B and fluconazole. Moreover, treatment induced negligible toxicity against normal tissues (NO3)2 (73)  was prepared by electrospinning PLA with a suspension of polyvinylpyrrolidone-stabilized of (73). MOF particles formed aggregates that, after being electrospun, became completely embedded inside of polymeric fibres, which inhibited t S. aureus biofilm [A composite material based on polylactic acid (PLA) fibres containing cobalt-based MOF [Co(mcim) biofilm .3[(Cu4Cl)3(BTTri)8] benzene) blended with chitosan demonstrated the ability to reduce by 85% the P. aeruginosa adhesion in the first 6 h, and maintained the inhibitory effect up to 24 h. Moreover, the system elicited the same inhibitory effect after a second round of experiments, suggesting reusability of the materials [Another material based on water-stable MOF Haterials .The development of an effective treatment for MRSA infections is complicated by the fact that antibiotics can be degraded by β-lactamases, and the antibiotics cannot penetrate the full depth of biofilms. Considering the nanoparticle-based carriers’ ability to deliver antibiotics with better biofilm penetration, a platform for β-lactam antibiotics and β-lactamase inhibitors co-delivery based on metalcarbenicillin framework-coated mesoporous silica nanoparticles (MSN) was developed. Carbenicillin, a β-lactam antibiotic, was used as a ligand for Fe(III) in order to generate a metalcarbenicillin framework able to block the pores of the MSN. The study evidenced that this system achieved a better penetration in the depth of biofilms and exhibited an inhibitory effect on the MRSA biofilm both in vitro and in vivo .Despite the current tendency to use drug delivery systems (DDSs) based on biocompatible and biodegradable matrices, the studies concerning the use of DDS for anti-biofilm species are rather few. The available studies are reporting Cu(II) or Ag(I) coordination to dendrimers or natural or synthetic polymers providing N as donor atoms or the incorporation of some complexes into polymeric (linear or branched) or silica matrices, or even in organic-inorganic composites.Biofilm development on viable tissues and prosthetic devices represents an important challenge for the medical field, due to their involvement in biofilm-associated infections, which are persistent and hard or often impossible to treat. Therefore, finding efficient agents capable of surpassing the numerous mechanisms of biofilms tolerance to high doses of antimicrobials represents one of the hot fields of research for both microbiologists and chemists. Metal complexes offer promising leads for the development of biofilm disrupting agents due to their multi-target, complex mechanism of action. The current data reveal the efficiency of metal complexes against biofilms formed by epidemiologically important resistant strains, such as MRSA. From the point of view of ligands, it was observed that nitrogen-based ligands mostly involved in chelation lead to an enhanced anti-biofilm activity, and Cu(II) complexes with these species exhibit the most promising activity associated with biofilm disruption. However, most studies in the field are focused only on assessing the in vitro anti-biofilm activity, and very few address the elucidation of the intimate mechanisms of action; the current studies identified the QS inhibition or ROS/NOS generation as some of the main mechanisms involved in biofilm disruption by metal complexes. From these data it is obvious that this represents an open field, and there are many aspects that must be elucidated by further studies.One of the most promising leads for the design of new complexes with anti-biofilm activity are the redox active metal ions such Cu(II), Fe(III) and Mn(II) but the less-studied ones such VO(IV) and Ru(II) should be also considered. All of these ions have ROS or NOS generation as a common mechanism of action. The best anti-biofilm activity is achieved then these ions are combined with multidentate ligands, especially bearing N as donor atoms, assuring enhanced stability. Furthermore, the perchlorate anion that easily generates single crystals seems to enhance the anti-biofilm activity in complexes bearing neutral organic ligands. The most active compounds show an improved activity after incorporation in organic, inorganic or composite matrices. The majority of the current literature refers to the in vitro study of the anti-biofilm activity of complexes, this explaining the paucity of novel anti-biofilm agents in medical practice. Thus, there is an urgent need for additional in vivo studies in this field in order to elucidate the safety, efficacy and toxicity of these species in order to develop new valuable drugs for the treatment of biofilm-associated infections.


Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C+ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C+ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C+ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2Cwt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2Cdel/del) and heterozygous (NKG2Cwt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C+ cells. We therefore analyzed the PUUV-mediated NKG2C+ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients.Infections with the + NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C+ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2Cdel and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2Cwt/del allele was significantly overrepresented, compared to the NKG2Cwt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2Cwt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2Cwt/wt NK cells.NKG2C+ NK cell response, as reflected by NKG2Cwt/del variant, may be associated with a higher risk for a severe hantavirus infections.Our results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C Orthohantaviruses are an emerging worldwide public health concern. Infections with the Puumala Orthohantavirus (PUUV) may cause hemorrhagic fever with renal syndrome (HFRS), termed nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. The knowledge about potentially protective immune responses against PUUV in the human host is still limited. The present study contributes to the understanding of NK cell based human immune responses against PUUV. Based on in vitro NK cell proliferation and activation experiments as well as on analysis of genetic risk factors in NE patients, we provide evidence that the NKG2C+ NK cell mediated immune responses are an important factor in the prevention of severe PUUV-infections. These findings do not only contribute to the deeper understanding of the PUUV-specific NK cell responses, but also identify a potential protective immune mechanism, which may also contribute to strategies for PUUV vaccine development.Infections with  Orthohantavirus are an emerging worldwide public health concern, affecting approximately 200.000 people annually in the Americas, Asia and Europe. In severe cases, hantavirus infections may lead to hantavirus hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) [Puumala orthohantavirus (PUUV) is the most common hantavirus in Europe. It is transmitted by bank voles (Myodes glareolus) [in vitro, PUUV-infections of endothelial cells are associated with the induction of an antiviral state, which involves on one hand a strong intrinsic immune response and on the other hand the upregulation of classical and non-classical HLA-molecules [Infections with e (HCPS) . Puumalaareolus) . In the areolus) , 5. Altholecules , 7.+ natural killer (NK) cells subset [+ NK cells are characterized by the absence of inhibitory NKG2A/CD94 and high expression levels of activating NKG2C/CD94 receptors [+ NK cells is mediated by the interaction of NKG2C with its cellular ligand HLA-E and by complex formation with the leader sequence of MHC-1, such as HLA-G [+ NK cells provide a potent antiviral phenotype, characterized by a high cytotoxic potential and the high-level secretion of pro-inflammatory cytokines, such as IFNγ and TNFα [A recent study in patients with acute NE showed that PUUV infection may lead to the expansion of the NKG2Cs subset . This was subset . NKG2C+ as HLA-G  or the Has HLA-G . NKG2C+ and TNFα .+ NK cells in HCMV-infected individuals [The presence of distinct HLA-E and NKG2C alleles may however influence the peptide presentation and NK cell responses in NE patients. Heterozygous or homozygous deletion of NKG2C decreases the expression of NKG2C, leading to an impaired expansion of NKG2Cividuals . HLA-E*0ividuals .+ NK cell responses and the relationship between NKG2C and HLA-E alleles in NE patients.This study elucidates the functional basis of PUUV-specific NKG2CThe study was approved by the institutional review board of the Medical University of Vienna (EK No. 1389/2019). Written/oral consent was not obtained due to the retrospective study design. All data were analyzed anonymously.in vitro testing.In this study, we included 130 patients with NE, as defined previously  and whicHCMV-specific IgG were measured in the plasma of NE patients, controls, and blood donors by ELISA (Euroimmune).2O. HLA-E*0101/0103 genotypes were determined by a Taqman assay and NKG2Cwt/del variants were determined by touchdown PCR as described before [Genomic DNA was isolated from plasma of NE patients, matched controls and hantavirus naïve blood donors using NucliSens EasyMag extractor (BioMérieux). DNA was eluted in 50μl of nuclease-free Hd before , 16.Briefly, Vero E6 cells (ATCC) were maintained in E-MEM with EBSS (BioWhittaker), and 10% FBS and infected at 0.01 MOI with the PUUV strain Sotkamo. After 14 days of infection, cell-free virus was harvested and stored at -80°C.4 cells were seeded in 96-well plates and incubated at 37°C with 50μL of 1:10 serially diluted viral concentrations for three days. Subsequently, cells were fixed with 80% acetone for 10 min and stained with mouse anti-hantavirus mAb A1C5  and AF488 conjugated goat anti-mouse mAb . Cell nuclei were counterstained with DAPI and counted using LAS X software (Leica Microsystems). Viral titers were calculated as infected cells per μL virus containing supernatant.Pooled HUVECs (Lonza) were cultured using the EGM-2 BulletKit (Lonza) according to the manufacturer’s instructions. Prior to infection, the cells were cultured without hydrocortisone. For viral titration, 1.5x105 HUVEC cells were seeded in 24-well plates and infected with PUUV laboratory strain Sotkamo (European Virus Archive) and a MOI of 1.5 or mock infected with UV-inactivated virus. After incubation for indicated time-points, HUVEC were harvested and either stained for the HLA-E or HLA-G surface expression by flow-cytometry. HLA-E or HLA-G expression levels were defined in comparison with unstained HUVEC.2.5x10HLA-G knockout in HUVEC cells was performed using HLA-G CRISPR/Cas9 KO and HLA-G HDR Plasmid (Santa Cruz) according to the manufacturer’s instruction. Successful HLA-G knockout was confirmed by HLA-G surface expression by flow-cytometry and qPCR, using a commercially available assay .+/CD3- NK cells were isolated by magnetic labeling of undepleted PBMCs  followed by MACS column filtration (Miltenyi Biotec). Subsequently, 5x106 cells were stored in aliquots containing FBS (Gibco) and 5% DMSO at—80°C until further analyses. Cells were thawed and placed overnight in RPMI 1640 medium supplemented with 10% FBS, 10 ng/mL IL-12 (PeproTech), and 100 ng/mL IL-18 (Biozym Scientific). RMA-S/LFA3/HLA-E cells  were maintained as previously described [PBMCs were isolated from the whole blood of naïve hantavirus donors (Austrian Red Cross) using Ficoll-Paque centrifugation . CD56escribed .6 RMA-S/LFA3/HLA-E cells were incubated with 300 μM of indicated peptides (peptides&elephants) in 1 mL serum-free Opti-MEM (Thermo Fisher) at 37°C with 5% CO2 for 16h. Peptide-pulsed RMA-S/LFA3/HLA-E cells and CFSE (Thermo Fisher) stained NK cells (E:T: 2:1) were co-cultivated with 100 ng/ml IL-18 and 10 ng/ml IL-12 for 20h, followed by cultivation in RPMI 1640 medium with or without 10 ng/mL IL-15 for seven days. New 2x106 peptide-pulsed RMA-S/LFA3/HLA-E cells were added each three days of co-culture. After co-culture all cells were harvested and analyzed by flow-cytometry.HLA-E surface stabilization was performed as previously described . 2x106 R5 cells were seeded in 24-well plates and incubated at 37°C over-night in a humidified incubator. Cells were infected with a MOI of 1.5 or treated with UV-inactivated virus for 72h hours. The viral suspension was afterwards removed and CFSE (Thermo Fisher) stained NK cells (E:T: 2:1) were co-cultivated with 100 ng/ml IL-18 and 10 ng/ml IL-12 for 20h, followed by cultivation in RPMI 1640 medium with or without 10 ng/mL IL-15 for seven days. After three days, NK cells were transferred to new PUUV-infected cells. After co-culture NK cells were harvested and analyzed by flow-cytometry.For NK cell proliferation assays, pooled HUVECs (Lonza) were cultured using the EGM-2 BulletKit (Lonza) according to the manufacturer’s instructions. Prior to infection, HUVECs were cultured without hydrocortisone. 2.5x105 cells were seeded in 24-well plates and incubated at 37°C over-night in a humidified incubator. Cells were infected with a multiplicity of MOI of 1.5 or treated with UV-inactivated virus for 72h hours. The viral suspension was afterwards removed and NK cells were added in a 2:1 (E:T) ratio in RPMI 1640 medium. In addition, 5μL of mouse-anti human APC-H7-CD107a mAB (BD Biosciences) was added. After one hour of co-culture, GolgiStop (BD Biosciences) was added according to the manufacturer’s instructions. After additional 6h of co-culture, NK cells were harvested and analyzed by flow-cytometry.For NK cell cytotoxicity assays, pooled HUVECs (Lonza) were cultured using the EGM-2 BulletKit (Lonza) without hydrocortisone. 2.5x10Cells were fixed and permeabilized for flow cytometry using the FIX & PERM Cell Permeabilization Kit (Thermo Fisher). The following mouse anti-human conjugated antibodies were used: APC-Cy7-CD16, PE-Cy7-CD57, PE-CD94, APC-HLA-G, APC-HLA-E (BioLegend), BV421-CD56, BV510-NKG2A, PE-TNF, PE-Cy7-IFNγ and AlexaFluor647-NKG2C (R&D Systems). The mouse anti-hantavirus mAB A1C5 was coupled with a secondary AlexaFlour488 antibody using a commercially available labeling kit . Live cells were identified using 7-AAD (BD Biosciences) or a LIVE/DEAD cell viability assay (Thermo Fisher). FACS analysis was performed using the FACSCanto2 platform, FACSDiva version 6.1.2, and Flowjo version 7.2.5 (BD Biosciences).2 test. The patients’ ages were compared using the Mann-Whitney U test. The proportions of proliferating NKG2C+ and NKG2A+ NK cells were analyzed using a paired t-test. ANOVA and Dunn’s post-test were used to analyze HLA-G- and HLA-E-expressing HUVECs at multiple time points. Baseline NKG2C+ were compared with the NK cell proliferation of distinct cell subsets using the coefficient of determination and the nonparametric Spearman test. P-values < 0.05 were considered significant. Statistical analyses were performed using IBM SPSS Statistics v24.0.The distribution of patients’ gender and genetic variants was compared using χ+ NK cell expansion, we performed an in vitro stimulation of CFSE labeled CD56+ NK cells with PUUV-infected and non-infected human umbilical vein endothelial cells (HUVECs). In response to PUUV-infected cells and IL-15, an expansion of CD56+ , for HCMV-seropositive donors: mean: 8% (1% - 15–1%), and for HCMV-seronegative donors: mean 2% (0.5%– 3.2%). The correlation between the pre-existing NKG2C+ NK cell subset and the proliferation of NKG2C+ in response to PUUV-infected cells was for all NK cell donors: r2 = 0.31 p = 0.08, for HCMV-seropositive NK cell donors: r2 = 0.29 p = 0.24, and for HCMV seronegative NK cell donors: r2 = 0.19 p = 0.59. Thus the pre-existing HCMV-serostatus had only a limited effect on the in vitro NKG2C+ NK cell proliferation in response to PUUV-infected cells.We further correlated the baseline NKG2C+ NK cell proliferation [The peptide-dependent stabilization of HLA-E is essential for NKG2C/CD94-mediated NKG2Cferation . Therefo+ NK cell expansion is dependent on the presentation of a virus-derived UL40 peptide by HLA-E [+ NK cell proliferation. We screened the PUUV-encoded peptides homologous to HCMV-UL40 peptides  and selected the most similar peptide (SRKPRNFLL). We also predicted the binding affinity of PUUV-encoded peptides to HLA-E using NetMHCpan and selected three candidates with the highest binding affinity to HLA-E . We then performed an NK cell proliferation assay using RMA-S/HLA-E/LFA-E cells to study the impact of the four selected peptides on NK cell proliferation. However, NK cell proliferation was not observed  from the same cells. A recently published study found that endothelial cells induce the release of sHLA-E in response to TNFα, IFN-β and IFNγ , all whiwt/del patients, may favour the development of severe PUUV-infections.In conclusion, the data provide evidence that the NKG2C/HLA-E axis has a substantial impact on the PUUV-specific NK cell response via the cellular stress response. Furthermore, it appears that an impaired NKG2C response, as described for NKG2CS1 FigOne representative example of 18 independent replicates is shown.(TIFF)Click here for additional data file.S2 Fig+ and NKG2A+ NK cells obtained from 18 different healthy PUUV-naive human donors in response to PUUV-infected HUVEC in the presence of IL-15. The data of the 18 samples are shown as median ± min/max. Paired T-Test was used for statistical comparison. MFI: mean fluorescence intensityExpression level of (A) CD16, (B) CD57, and (C) CD94 measured in proliferating NKG2C(TIFF)Click here for additional data file.S3 Fig+ cells, (B) NKG2A+ cells or (C) CD56+ NK cells obtained from 18 different healthy PUUV-naive human donors measured in response to NK cells cultured either without (-PUUV-infected HUVEC) or with PUUV-infected HUVEC (+PUUV-infected HUVEC) in the presence or absence of IL-15. The median ± min/max of the results of the 18 samples is shown. ANOVA and Dunn’s post test were used to compare the percentage of NK cells between mock-infected and PUUV-infected HUVEC. (D) Proliferation of NKG2C+ and NKG2A+ NK cells obtained from 6 different healthy PUUV-naive human donors  measured in response to NK cells in the presence of IL-15 and the presence or absence of either a α-NKG2C or α-NKG2A blocking antibody. The median ± min/max of the results of the 6 samples is shown. Paired T-Test was used to compare the percentage of proliferating NK cells between the presence or absence of respective blocking antibodies. mAB: monoclonal antibody, PUUV: Puumala-Virus.(A-C) Proliferation of (A) NKG2C(TIFF)Click here for additional data file.S4 Fig+ (B) NKG2C+ and (C) NKG2A+ NK cells between HCMV-seropositive (N = 10) and HCMV-seronegative (N = 8) donors in the presence of IL-15. Data are shown as median of 18 different donors ± min/max. Mann-Whitney t-test was used to for statistical comparison between HCMV-seropositive and HCMV-seronegative donors. HCMV: Human Cytomegalovirus.NK cell proliferation was measured in samples obtained from 18 different healthy PUUV-naive human donors, including HCMV-seropositive (N = 10) and HCMV-seronegative (N = 8) donors. Comparison of NK cell proliferation of (A) CD56(TIFF)Click here for additional data file.S5 FigOne representative example of 3 independent replicates is shown.(TIFF)Click here for additional data file.S6 FigPUUV: Puumala orthohantavirus.Kinetics of the PUUV-infection of HUVEC measured using flow cytometry. Percentage of PUUV-infected HUVEC, evaluated after 1, 2, 3, 4, 8 and 15 days of PUUV or mock infection. Results are shown as means of three independent technical replicates ± SD. Unpaired t-test was used to compare the percentage of PUUV-antigen positive and PUUV-antigen negative HUVEC between mock-infected and PUUV-infected HUVEC for each time point. (TIFF)Click here for additional data file.S7 Fighttp://www.cbs.dtu.dk/services/NetMHCpan/) and were identified by BLAST (SRKPRNFLL). The HLA-G leader peptide (VMAPRTLFL) served as a positive control. NK cells stimulated alone or with only RMA-S/LFA3/HLA-E cells served as a negative control. One representative example of three independent technical replicates is shown.AIPPAAYSL, VLPDSLFSF, LLPDKNLRM peptides were selected according to their %rank by NetMHCpan ((TIFF)Click here for additional data file.