The blood–testis barrier (BTB) is essential to the microenvironment of spermatogenesis, and Sertoli cells provide the cellular basis for BTB construction. Numerous nuclear transcription factors have been identified to be vital for the proper functioning of Sertoli cells. PA1 has been reported to play important roles during diverse biological processes, yet its potential function in male reproduction is still unknown.Pa1 knockout resulted in an azoospermia-like phenotype in mice. The knockout of this gene led to multiple defects in spermatogenesis, such as the disorganization of the cytoskeleton during basal and apical ectoplasmic specialization and the disruption of the BTB. Further transcriptomic analysis, together with Cut-Tag results of PA1 in Sertoli cells, revealed that PA1 could affect the expression of a subset of genes that are essential for the normal function of Sertoli cells, including those genes associated with actin organization and cellular junctions such as Connexin43 (Cx43). We further demonstrated that the expression of Cx43 depended on the interaction between JUN, one of the AP-1 complex transcription factors, and PA1.Here, we show that PA1 was highly expressed in human and mouse testis and predominantly localized in the nuclei of Sertoli cells. Sertoli cell-specific Overall, our findings reveal that PA1 is essential for the maintenance of BTB integrity in Sertoli cells and regulates BTB construction-related gene expression via transcription factors. Thus, this newly discovered mechanism in Sertoli cells provides a potential diagnostic or even therapeutic target for some individuals with azoospermia.The online version contains supplementary material available at 10.1186/s13578-022-00773-y. The differentiation and maintenance of some tissues require special niches that need to exclude macromolecules from these tissues, and all these processes are dependent on the blood–tissue barrier. General examples for barriers are the blood–brain-, the blood–retina-, the blood–placenta- and the blood–testis barrier (BTB) –4. AmongOccludin or Claudin11 in mice has been shown to lead to the disruption of seminiferous tubules and the progressive germ cell loss . I. I37]. IJUN Fig. d. Next, JUN Fig. e, furtheCx43 promoter [Cx43 in Amh-pa1−/− Sertoli cells, as the RT-qPCR results confirmed . Pa1F/F mice were bred with Amh-cre mice to generate Amh-cre, Pa1F/+ (F1). Amh-cre, Pa1F/+ were mated with Pa1F/F mice to generate Amh-cre, Pa1F/F (Amh-pa1−/−) mice (F2). All mice are kept in a specific pathogen-free (SPF) environment. The primers used for genotyping were listed in Additional file Pa1 and Jun amplified from mouse testis cDNA were cloned into the pRK-flag and pCS2-myc vector, respectively, according the protocol of Clon Express Kit . Rabbit anti-PA1 antibody (ABE1863) was used for western blot and Cut-Tag experiments, rabbit anti-PA1 (NBP1-89792) was used for immunofluorescence and immunohistochemistry. Rabbit anti-SYCP3 (ab15093) and mouse anti-γH2AX (05–636) were used for immunofluorescence. Rabbit anti-DDX4 antibody (ab13840), mouse anti-PCNA (ab29) and goat anti-PLZF (AF2944) were used for immunofluorescence. Rabbit anti-WT1 (ab89901) and mouse anti-WT1 (NB110-60011) were used for immunofluorescence. Rabbit anti-CLAUDIN11 antibody (ab53041) was used for immunofluorescence and western blot. Rabbit anti-OCCLUDIN (711500) antibody, rabbit anti-CTTNB1 (712700) antibody, and rabbit anti-ZO-1 antibody (402200) from Invitrogen was used for immunofluorescence and western blot. Rabbit anti-GJA1 (26980-1-AP) and rabbit anti-GJA1 (A11752) were used for immunofluorescence and western blot experiments. Rabbit anti-JUN (A0246) was used for immunofluorescence experiments. Rabbit anti-MYC (BE2011), mouse anti-MYC (M20002L), and mouse anti-Flag (M20008L) were used for immunoprecipitation and immunoblot experiments. Mouse anti-GAPDH (AC002) antibody and mouse anti-β-TUBULIN (AC021) antibody were purchased from ABclonal. Rabbit IgG (B30011), purchased from Abmart, was used for Cut-Tag experiments. FITC-labeled Phalloidin (40735ES75) purchased from Yeasen was used for immunofluorescence . TRITC- or FITC-labeled goat anti-mouse, TRITC- or FITC-labeled goat anti-rabbit, and HRP-conjugated goat anti-rabbit secondary antibodies  were used for immunohistochemistry and immunofluorescence experiments. Alexa Fluor 680-labeled goat anti-mouse and Alexa Fluor 800-labeled goat anti-rabbit secondary antibodies purchased from Invitrogen were used for western blot.Mouse Fertility experiments were conducted as previously described . 8-week-g for 3 min at room temperature. The cells were further digested with a mixture of 2 mg/ml hyaluronidase, 2 mg/ml collagenase IV and 0.5 mg/ml DNase I in 1 mg/ml trypsin  and incubated in 2 ml Eppendorf tubes for 15 min at 37 °C. The digestion was halted using equal volume of F12-DMEM  including 15% fetal bovine serum . The products were filtered through a 200-mesh filter , and washed twice with PBS centrifuged at 600×g for 3 min at room temperature. The separated cells were resuspended with F12-DMEM , 10 μg/ml insulin, 2.5 ng/ml EGF, and 5 μg/ml transferrin and cultured in 35 mm plastic dishes  at 34 °C and 5% CO2. After 8 h, the dishes were gently washed with PBS twice to remove the unattached germ cells.The isolation of primary Sertoli cell was performed as previous description with some modifications . In brie2. When the cell density reached 60–80% in 6-well plates , the cells were further transfected with the constructed plasmid DNA (2 ng) with lippomax  for 8 h followed the protocol. Cells were harvested 2 days post transfection for further experiments.Mouse TM4 Sertoli cell line from the American Type Culture Collection  were cultured in 35 mm plastic dishes  with F12-DMEM containing 15% fetal bovine serum  at 37 °C and 5% COSeparation of germ cells was performed using a STA-PUT method with minor modification as previously described  with detTestes and caudal epididymides for each genotype of mice were fixed in Bouin’s solution or 4% (m/v) paraformaldehyde (PFA) for 24–48 h, embedded in paraffin, and cut into 5 μm sections. After deparaffinization, the tissue sections were further stained with hematoxylin and eosin (H&E). For Periodic acid-Schiff staining, deparaffinized slides were stained with PAS and hematoxylin followed the protocol in the PAS kit .2HPO4 and 25 mM NaH2PO4) for 3 times, the tissues were immersed in 1% osmium acid for 2 h. Through the acetone gradient dehydration and immersion of acetone: epoxy resin (1:1), the tissues were embedded, and placed at 60 °C for 24 h. The sections (70 nm) were sliced via the Leica EM UC7 ultra-thin microtome, and stained with uranyl acetate and lead citrate. Images were captured and analyzed with JEM-1400 electron microscope and Gatan digital camera.After the mice were anesthetized, 50 ml of fixative solution  was perfused into the heart within 1 h, and the testes were then removed and fixed in the fixative solution for 4 h. After washed with 0.1 M PB buffer , separated via SDS-PAGE and electrotransferred to a nitrocellulose membrane. The membrane was further blocked with 5% milk and incubated in corresponding primary antibody. In the next day, the membrane was further incubated with Alexa Fluor 800 or 680-labeled goat anti-rabbit or Alexa Fluor 800 or 680-labeled goat anti-mouse secondary antibodies. Finally, the ODYSSEY System  was used to scan the membrane.3VO4, 0.1% NP-40, 50 mM β-glycerolphosphate) with the addition of protein inhibitor cocktail, and further incubated with relevant primary antibody at 4 ℃ overnight. Protein A-Sepharose beads  were prepared and added to the proteins at 4 ℃ for 2 h. Immunoprecipitated product were washed 3 times with TAP buffer with centrifuged at 845×g for 5 min at 4 ℃, and the pellets were eluted with loading buffer containing 1% SDS, heated for 10 min at 95 ℃ and examined by immunoblotting.For Immunoprecipitation, the transfected cells proteins were extracted with the TAP buffer .Cut-Tag experiments and data analysis were performed followed the published articles , 60 withGja1 promoter (− 2000 to + 36) was cloned into the pGL3-basic plasmid . TM4 cells seeded in 24-well-plate were transfected using lippomax  with 20 ng plasmids containing Renilla luciferase, 200 ng of plasmids encoding firefly luciferase reporters and empty pCS2 vector, pCS2-pa1, pCS2-jun or pCS2-pa1 with pCS2-jun plasmids. The luciferase activity was measured using the Dual-Lumi™ Luciferase Reporter Gene Assay Kit (RG088S).The https://metascape.org).Total RNA of Sertoli cells from 20-day-old mice testes was extracted with Trizol (Invitrogen). NEBNext® Ultra™ RNA Library Prep Kit from Illumina®  was used to construct the libraries. Sequencing was carried out with Illumina HiSeq 2000. The genome index was built with Bowtie2 v2.2.3, and the Clean Data was aligned to the reference genome, Mus_musculus.GRCm38.90.chr, with HISAT2 v2.1.0. HTSeq v0.6.0 was used to count the reads number of each gene in each sample. FPKM (Fragments Per Kilobase Millon Mapped Reads) was calculated to estimate the expression level of genes in each sample. Differential gene expression was analyzed with with DEGseq v1.18.0 between the samples in biological replicates. The genes with |log2 Fold change|≥ 1 and q (padj) < 0.05 were determined as differentially expressed genes. GO analyses were performed using DAVID27. Upstream analysis was done using Metascape , 0.01(**), 0.001(***) and 0.0001(****).Additional file 1. The primers used in this study including Table S1 and Table S2.Additional file 2. The detailed supplementary methods and materials mentioned in the article.Additional file 3: Fig. S1. The expression pattern of PA1 in human and mouse. a) PA1 protein levels gradually increased during the development of testis. The expression pattern of PA1 in different developmental stages of murine testes  using Western Blot. Statistical analysis of the expression level of PA1 during the development of testis was showed in the lower panel. b) Semi RT-PCR results showed Pa1 was ubiquitously expressed in different mouse organs but especially enriched in the testis. β-actin was used as the loading control. c) Semi RT-PCR results showed Pa1 levels gradually increased in mouse testis with the development of testis. β-actin was used as the loading control. d) PA1 is localized in Sertoli cells in mouse testis during the whole spermatogenic cycle while the expression level of PA1 in different stages varies. Testes sections from adult wild-type mice (8-week-old) were stained for PA1 using immunohistochemistry as indicated. Arrows indicate positive cells for PA1 expression. e) PA1 was expressed in different testicular cells but especially abundant in Sertoli cells. Immunoblotting of PA1 was performed in Sertoli cells, SC; spermatogonium, SPG; spermatocytes, SPC; round spermatids, rST; elongated and elongating spermatids, eST.Additional file 4: Fig. S2. Generation of Amh-pa1−/− mice. a) Schematic diagram for the generation of Amh-pa1−/− mice. Exon 1 and exon 2 (blue) of Pa1 were flanked by loxp sites (yellow) in Pa1F/F mice. These exons were specifically deleted in the Sertoli cells in Amh-pa1−/− mice. b) Amh-pa1−/−mice genotyping. The 250 bp band indicated floxed Pa1, the 200 bp band indicated Pa1, and the 400 bp band indicated Amh-cre. c) PA1 was specifically knocked out in testicular Sertoli cells of 5-week-old mice. Seminiferous tubule sections of Pa1F/F and Amh-pa1−/− testes were co-stained for WT1(green) and PA1 (red). The nuclei were stained with DAPI (blue). Solid boxes showed localization of the enlarged images. Dashed circle indicated basal membrane of seminiferous tubules. Arrows indicated the nuclei of Sertoli cells. d) Body weights of 8-week-old Pa1F/F mice and Amh-pa1−/− male mice (n = 5 independent experiments) were similar. Data are presented as mean ± SEM. e) Testis weights of Amh-pa1−/− mice were significantly lower than those of Pa1F/F mice (n = 5 independent experiments). Data are presented as mean ± SEM. ****p < 0.0001.Additional file 5: Fig. S3. Disorganized basal actin cytoskeleton is detected in Amh-pa1−/− mice testis. F-actin at the basal compartment of seminiferous tubules was observed to be perturbed and disorganized in adult Amh-pa1−/− testes. Arrows indicate F-actin bundles. Immunofluorescence using FITC-labeled phalloidin (green) was performed on the frozen testis sections of Pa1F/F mice (upper panels) and Amh-pa1−/− mice (lower panels). Nuclei were stained with DAPI (blue). Dashed boxes showed the localization of the enlarged images. Dashed curve indicated basal membrane of seminiferous tubules.Additional file 6: Fig. S4.Amh-pa1−/− mice show massive germ cell loss. a) Most of the residual cells in the empty tubules of Amh-pa1−/− testis were identified as Sertoli cells. Immunofluorescence analysis using anti-WT1 (green) was conducted on the sections of testis from Pa1F/F mice (upper panels) and Amh-pa1−/− mice (lower panels). Nuclei were stained with DAPI (blue). Dashed curve indicated basal membrane of seminiferous tubules. b) PCNA immunofluorescence analysis of seminiferous tubules showed that 8-week-old Amh-pa1−/− mice had decreased numbers of spermatocytes and spermatogonia compared with 8-week-old Pa1F/F mice. Seminiferous tubule sections of Pa1F/F and Amh-pa1−/− testes were stained for PCNA (red), and the nuclei was stained with DAPI (blue). Dashed boxes showed the localization of the enlarged images. Dashed curve indicated basal membrane of seminiferous tubules. Data are presented as mean ± SEM. ***p < 0.001. c) DDX4 immunofluorescence analysis of germ cells showed that 8-week-old Amh-pa1-/- mice had decreased numbers of germ cells compared with 8-week-old Pa1F/F mice. Seminiferous tubule sections of Pa1F/F and Amh-pa1−/− testes were stained for DDX4 (red), and the nuclei was stained with DAPI (blue). Dashed boxes showed the localization of the enlarged images. Dashed curve indicated basal membrane of seminiferous tubules. Data are presented as mean ± SEM. **p < 0.01. d) PLZF immunofluorescence analysis of germ cells showed that 8-week-old Amh-pa1−/− mice had decreased numbers of undifferentiated spermatogonium compared with 8-week-old Pa1F/F mice. Seminiferous tubule sections of Pa1F/F and Amh-pa1−/− testes were stained for PLZF (red), and the nuclei was stained with DAPI (blue). Dashed boxes showed the localization of the enlarged images. Dashed curve indicated basal membrane of seminiferous tubules. Arrows indicated PLZF positive cells. e) PCNA positive cell number per tubule was significantly decreased Amh-pa1−/− (27.34 ± 1.319) mice testes compared with Pa1F/F mice (62.72 ± 3.167). (n = 3 independent experiments). Data are presented as mean ± SEM. ***p < 0.001. f) DDX4 positive cell number per tubule was significantly decreased in Amh-pa1−/− (50.39 ± 2.342) mice compared with Pa1F/F mice (122.9 ± 12.20). (n = 3 independent experiments). Data are presented as mean ± SEM. **p < 0.01. g) PLZF positive cell number per tubule was significantly decreased Amh-pa1−/− (0.5789 ± 0.1919) mice testes compared with Pa1F/F mice (2.763 ± 0.2321). (n = 3 independent experiments). Data are presented as mean ± SEM. ***p < 0.001. h) Homologous chromosome synapsis, pairing, or segregation were normal undergoing in Amh-pa1−/− spermatocytes. Spermatocytes of Pa1F/F and Amh-pa1−/− mice were stained with SYCP3 (green) and γH2AX (red) antibodies.Additional file 7: Fig. S5. 5 GO analysis of downregulated genes in Amh-pa1−/− Sertoli cells. a) GO-Molecular function analysis of 1045 downregulated genes in Amh-pa1−/− Sertoli cells. Molecular function of protein binding, actin filament binding and actin binding were labeled by asterisks. b) GO-Cellular component analysis of 1045 downregulated genes in Amh-pa1−/− Sertoli cells. Cellular component of membrane, cell junction and actin cytoskeleton were labeled with asterisks. c) KEGG pathway analysis of 1045 downregulated genes in Amh-pa1−/− Sertoli cells. KEGG pathways of phagosome, cell adhesion molecules, regulation of actin cytoskeleton and focal adhesion were labeled with asterisks.Additional file 8: Fig. S6. GO analysis of upregulated genes in Amh-pa1−/− Sertoli cells. a) GO-Cellular component analysis of 899 upregulated genes in Amh-pa1−/− Sertoli cells. Cellular component of chromosome was labeled with asterisks. b) KEGG pathway analysis of 899 upregulated genes in Amh-pa1−/− Sertoli cells. KEGG pathways of cell cycle was labeled with asterisks. c) GO-Biological process analysis of 899 upregulated genes in Amh-pa1−/− Sertoli cells. Biological process of cell cycle was labeled with asterisks. d) GO-Molecular function analysis of 899 upregulated genes in Amh-pa1−/− Sertoli cells. Molecular function of calcium ion binding, and actin binding were labeled by asterisks.Additional file 9: Fig. S7. PA1 together with JUN regulates downstream gene expression. a) GO-Biological process analysis of the 184 overlapped genes identified in both downregulated genes of Amh-pa1−/− Sertoli cells and PA1 bound genes. Biological process of actin cytoskeleton organization, cell adhesion and transforming growth factor beta receptor signaling pathway were labeled with asterisks. b) Upstream transcription factor analysis of downregulated genes in Pa1-knockout MEFs. The identified top 1 transcription factor JUN was labeled with asterisk. c) The endogenous co-immunoprecipitation results of PA1 and JUN in TM4 cells. The proteins of TM4 cells were collected and immunoprecipitated using anti-PA1 antibody and the products were further used for immunoblot analysis with anti-PA1 and anti-JUN antibodies. The bands for PA1 and JUN were labeled with the arrows. d) Venn diagram showing the overlap of PA1 bound genes in Sertoli cells with JUN bound genes in MCF-7 cells [-7 cells . e) GO-M-7 cells . Molecul-7 cells . Biologi


PdgfrβCre + ;FAKWT/WT, PdgfrβCre + ;FAKY397F/Y397F and PdgfrβCre + ;FAKY861F/Y861F mice, our data demonstrate that Lewis lung carcinoma tumour growth, tumour blood vessel density, blood vessel perfusion and pericyte coverage were affected only in late stage tumours in PdgfrβCre + ;FAKY861F/Y861F but not PdgfrβCre + ;FAKY397F/Y397F mice. Further examination indicates a dual role for pericyte FAK-Y861 phosphorylation in the regulation of tumour vessel regression and also in the control of pericyte derived signals that influence apoptosis in cancer cells. Overall this study identifies the role of pericyte FAK-Y861 in the regulation of tumour vessel regression and tumour growth control and that non-phosphorylatable FAK-Y861F in pericytes reduces tumour growth and blood vessel density.Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is overexpressed in many cancer types and in vivo studies have shown that vascular endothelial cell FAK expression and FAK-phosphorylation at tyrosine (Y) 397, and subsequently FAK-Y861, are important in tumour angiogenesis. Pericytes also play a vital role in regulating tumour blood vessel stabilisation, but the specific involvement of pericyte FAK-Y397 and FAK-Y861 phosphorylation in tumour blood vessels is unknown. Using The online version contains supplementary material available at 10.1007/s10456-021-09776-8. Angiogenesis is a complex biological process that involves multiple cell types, crosstalk between these cells and responses to different stimuli –3. InterY397F/Y397F and FAKY861F/Y861F mice.It is well documented that focal adhesion kinase (FAK) plays a central role in different aspects of tumour growth and is overexpressed in many types of cancer –11. One In this study, we show that in a subcutaneous Lewis Lung Carcinoma (LLC) tumour model, both angiogenesis and tumour growth are reduced only in mice which have the pericyte-specific FAK-Y861F mutation. This correlates with a significant increase in vessel regression. Furthermore, examination of the secretome and protein expression in FAK-Y861F pericytes highlight an altered pericyte signature involving cytokines and protein secretion that are involved in cancer cell apoptosis. Thus, pericyte FAK-Y861 plays a role in the control of tumour growth.PdgfrβCre;FAKfl/fl mice to generate mutant FAK-knockin and endogenous FAK-knockout in pericytes under Cre control . These mice show no defects in Mendelian ratios, gender distribution or weights and had no obvious adverse phenotype . We also confirmed reduced pY397 in FAK-Y397F pericytes and reduced pY861 in FAK-Y861F pericytes  targeted to the Rosa26 (R26) locus. These mice were bred with PC-specific PdgfrβCre + ;FAKWT/WT control mice, PdgfrβCre + ;FAKY861F/Y861F and PdgfrβCre + ;FAKY397F/Y397F mice were injected subcutaneously with Lewis Lung Carcinoma cells (LLC). In vivo tumour growth was reduced in PdgfrβCre + ;FAKY861F/Y861F, but not PdgfrβCre + ;FAKY397F/Y397F mice. Furthermore, the PdgfrβCre + ;FAKY861F/Y861F but not PdgfrβCre + ;FAKY397F/Y397F mice had significantly reduced blood vessel density  compared with PdgfrβCre + ;FAKWT/WT control mice  from PdgfrβCre + ;FAKWT/WT, PdgfrβCre + ;FAKY397F/Y397F and PdgfrβCre + ;FAKY861F/Y861F mice showed a significant increase in endomucin-negative / collagen IV-positive blood vessel sleeves in tumours from PdgfrβCre + ;FAKY861F/Y861F compared with tumours from PdgfrβCre + ;FAKWT/WT, PdgfrβCre + ;FAKY397F/Y397F mice  and pericyte coverage  were both reduced in PdgfrβCre + ;FAKWT/WT and PdgfrβCre + ;FAKY861F/Y861F mice . VEGF treatment significantly increased angiogenic sprouting in both genotypes to the same extent suggesting that mutation of FAK-Y861F in pericytes is not sufficient to directly affect angiogenic sprouting in a tumour-free environment .Examination of early stage tumours was undertaken to determine at what stage, during growth of the tumour, angiogenesis was being affected. In early stage tumours (day 12 post tumour cell inoculation) LLC tumour size, blood vessel density and blood vessel perfusion were similar between ice Fig. a-c, suggPdgfrβCre + ;FAKY861F/Y861F mice had a significant increase in tumour necrosis compared with PdgfrβCre + ;FAKWT/WT control mice  mice. This pro-apoptotic tumour cell phenotype was confirmed, in vivo, where tumours from PdgfrβCre + ;FAKY861F/Y861F mice displayed significantly increased levels of apoptosis compared with tumours from either PdgfrβCre + ;FAKWT/WT or PdgfrβCre + ;FAKY397F/Y397F mice  domain to bind to FAK, including Src. FAK-Src complexing are thought to potentiate further FAK phosphorylation at other FAK domains including Y861. Since phosphorylated Src was not reduced in Y861F pericytes, we wanted to determine if the phenotype observed in Y861F pericytes was dependent on Src. To address this, Src levels were depleted in FAK-WT, FAK-Y397F and FAK-Y861F pericytes followed by examination of the effect of CM from these cells on LLC cell death. CM from Src-depleted FAK-WT and FAK-Y397F pericytes significantly increased LLC apoptosis. Conversely, CM from FAK-Y861 Src-depleted pericytes , PdgfrβCre;FAKY397F/Y397F (Y397F) mice and PdgfrβCre;FAKY861F/Y861F (Y861F) mice [To examine the effect of pericyte-specific FAK mutations in vivo, we developed 1F) mice . All exp6 Lewis lung carcinoma cells  into the flanks. Tumour dimensions were measured over time and tumour growth was determined using the formula: length x width2 × 0.52. When tumours reached the maximum legal size allowed, mice were killed, tumour sizes measured and tumour samples were either snap-frozen, fixed in 4% paraformaldehyde (PFA) or fixed in 4% PFA/sucrose for histological analysis. For early tumour growth studies, tumours were harvested at day 12 post tumour cell injection.Male and female mice aged 12–16 weeks were injected subcutaneously with 0.5 × 102. For examination of blood vessel regression, tumours were fixed with 4% PFA/sucrose then frozen in OCT.Five µm frozen tumour sections were air-dried for 10 min, washed once in PBS, fixed in acetone for 10 min at -20 °C, washed in PBS three times and then blocked with 5% normal goat serum for 30 min at room temperature. After blocking, sections were incubated with primary antibodies overnight at 4 °C. Primary antibodies used were directed against endomucin . Sections were then washed with PBS and incubated with Alexa-Fluor®-conjugated secondary antibody  for 45 min at room temperature before mounting the slides with Prolong® Gold anti-fade reagent . Tumour blood vessels were counted across entire midline sections, and the numbers were expressed as vessels/mmFor analysis of the % of functional tumour vessels, 100 μl PE-PECAM antibody  was injected via the tail vein 10 min prior to culling mice. Tumours were dissected immediately, snap-frozen and sectioned. Frozen sections were then immunostained for endomucin, as described above. To calculate the % number of functional vessels, the number of PE-PECAM-positive blood vessels was divided by the total number of endomucin-positive blood vessels.Frozen tumour sections were double immunostained as described in “Frozen tumour sections were air-dried for 30 min at room temperature, permeabilised for 3 min with PBS + 0.5% TritonX-10 then blocked with 5% BSA/PBS for 1 h at room temperature. Sections were then incubated overnight at 4 °C with Collagen IV  and endomucin  antibodies. The following day, sections were washed three times with PBS and incubated for 1 h at room temperature with AlexaFluor conjugated secondary antibodies (1:100). Finally, sections were washed twice in PBS and once in distilled water then mounted (Prolong Gold with DAPI) with a glass slide and images were acquired using a confocal spinning disk microscope and sCMOS confocal camera (Nikon). Image analysis was performed using ImageJ software by making maximum intensity projections of z stacks and the numbers of vessels counted manually.Early stage LLC tumours were fixed in 4% formaldehyde and bisected. Tumour sections were H&E stained, scanned using a Panoramic scanner and the area of necrosis (as identified by acellular regions of tumour tissue) quantified with ImageJ software.Tumour cell apoptosis was examined in frozen tumour sections using the In Situ Cell Death Detection Kit, TMR red  following the manufacturer’s instructions. Stained sections were scanned using a Panoramic scanner and the TUNEL-positive area was calculated as the percentage of TUNEL-positive area/ tumour area.4 cells in DMEM + 10% FCS. At the same time WT and 861F pericytes were grown to 50–60% confluency and conditioned medium (CM) removed. CM was centrifuged to remove cell debris and added to LLCs after removal of DMEM and two PBS washes. Recombinant mouse MCP-1  was added to the CM at either 10 or 50 ng/ml. Cells were incubated with CM for 24 or 48 h after which cells were stained to detect DNA fragmentation in apoptosis using the BrdU-Red DNA (TUNEL) kit  following the manufacturer’s instructions. Within 3 h of staining the cells were analysed for BrdU using a Zeiss microscope and Axiovision software. The percentage of TUNEL-positive cells was calculated by counting the total number of cell nuclei and the number of nuclei that were TUNEL positive. For Src siRNA experiments, WT, Y397F and Y861F pericytes were transfected for 48 hr with SMARTpool Src siRNA or non-targeted siRNA  as a control. After 48 hr cells were lysed for protein extraction to measure Src levels by Western blot analysis. Conditioned medium was also removed and added to LLCs as described above, prior to TUNEL staining.LLC were plated on coverslips at a density 5 × 10Primary mouse brain pericytes were isolated from the mice, cultured and characterised as previously described , 54. BriAortic rings were isolated from all mouse genotypes as previously described .Pericyte angiogenesis and cytokine profiles were compared using the angiogenesis array  and cytokine array . Briefly, cell lysates were prepared as follows: sample buffer was added to the cell culture, the cells were scraped and transferred into a 1.5 ml Eppendorf tube. After sonication, samples were adjusted to the array conditions and mixed with a Detection Antibody Cocktail as indicated by the manufacturer’s instructions. Lysates were incubated overnight at 4 °C on dot-blot membranes. Membranes were washed, incubated with streptavidin-HRP for 30 min at RT, washed again and ECL was applied to the membrane to reveal the dots. Quantification of dot intensity was performed using ImageJ™ software.Primary brain pericytes were grown to 70–80% confluency then lysed in RIPA buffer. 15–30 μg protein was run on 8% polyacrylamide gels then transferred to nitrocellulose membranes. Membranes were probed with primary antibody overnight at 4 °C. Myc-tag , total FAK , phospho-397 FAK , phospho-861 FAK , PDGFRβ , endomucin , Src , pSrc . The anti-HSC70  or GAPDH  antibody, for loading controls, were used at 1:5000 dilution. Densitometric readings of band intensities were obtained using the ImageJ™ software.P < 0.05 was considered statistically significant, unless otherwise indicated. For tumour growth statistics, non-parametric two-sided Mann–Whitney U rank sum test was performed to compare tumour volumes each day. One-way ANOVA was performed for the in vitro TUNEL and aortic ring assay, two-way ANOVA for the blood vessel regression and in vivo TUNEL study. Wilcoxon test was performed for tumour necrosis.Statistical significance was calculated using Prism 8 software and Supplementary file1 (PDF 391 KB)Supplementary file2 (PDF 55 KB)Supplementary file3 (PDF 568 KB)Below is the link to the electronic supplementary material.


Intravenous fluid therapy is a vital and life-saving therapeutic in veterinary medicine. In the absence of heart or lung disease, trauma or sepsis there is limited evidence that fluid therapy will have a detrimental effect on lung function. In healthy dogs there is a reasonable level of experimental evidence that supraphysiologic rates of fluid are required before signs of fluid overload are made evident. In cats, however, this may not be the case. There are higher rates of asymptomatic myocardial disease, but even in the absence of that it seems that some cats may be susceptible to fluid overload. Where systemic inflammation already exists the careful homeostatic and protective mechanisms within the lung are deranged and increases in hydrostatic pressure are more likely to result in fluid movement into the lung tissues. Strategies including restricting the use of intravenous crystalloid fluid administration and using blood products for management of severe hemorrhage are of increasing importance in human trauma and seem to be associated with fewer pulmonary complications, and lower mortality. Managing dogs and cats with sepsis and acute respiratory distress syndrome is already challenging, but ensuring adequate vascular expansion needs to be balanced with avoiding excessive volume administration which may negatively impact pulmonary function. While fluids remain crucial to management of these conditions, there will be an ongoing requirement to balance need without providing excess. The use of point of care ultrasound may provide clinicians with a non-invasive and accessible way to do this. Fluid therapy is a vital part of the management of the acutely or critically ill veterinary patient. Despite being considered a relatively benign therapy in veterinary practice, emerging evidence in human patients has identified that there is a need for careful fluid balance to avoid excessive morbidity or mortality . This efFluid dynamics within tissues are complex. In recent years the endothelial glycocalyx has been identified as an important consideration in the formation of extravascular fluid, as well as the forces described by Starling , 4. StarIn healthy lung tissue there are several additional protective measures that limit the formation of extravascular lung water (EVLW) . CapillaWhen there is pulmonary inflammation or damage to the glycocalyx, fluid therapy affects these delicate relationships by increasing capillary hydrostatic pressure. In these circumstances the result can be overwhelming, resulting in alveolar flooding and pulmonary edema. The impact of fluid therapy and specifically that of the volume, or dose administered, is therefore of most importance in animals where there is endothelial damage or inflammation. There is limited data that supports the formation of non-cardiogenic pulmonary edema in veterinary species as a result of appropriate doses of intravenous fluid in the absence of lung injury or damage. Experimental data in healthy dehydrated dogs examining the dose response effect of lactated Ringer's solution identified that high rates and doses were associated with clinical signs of fluid overload. However, the doses that resulted in side effects associated with fluid overload were between 270 and 360 mL/kg/h administered for an hour, which would not be used in clinical circumstances . Further2  or arterial blood gas analysis. Assessment for pulmonary edema can be more challenging. Techniques that measure EVLW are not available in clinical veterinary practice and are invasive. Thoracic imaging, including radiography and ultrasound are easily available and generally non-invasive. Assessing animals for the development of pulmonary edema during clinical care has been improved by the advent of point-of-care ultrasound. Several studies evaluating the use of focussed protocols for lung ultrasound have been published in veterinary medicine –22. ThisThe detrimental effects of intravenous fluid therapy on lung contusions have been known for over 60 years . An assoExperimental studies in pigs in a model of blunt lung injury have confirmed a relationship between increased lung water and administration of crystalloid or artificial colloid solutions, with variable clinical effects –31. In tIn an older animal model of lung contusion and hemorrhage the infusion rate of Ringer's lactate solution used for resuscitation was directly related to the degree of pulmonary insufficiency identified . ClinicaLarge multi-center clinical studies in people have also identified an association between severity of hypoxemia and the volume of crystalloid administered, particularly if administered within the first 6 h following arrival at the hospital . FurtherApplying this evidence to veterinary patients is challenging. Our patient population generally has less significant injury, or potentially our capability to manage severe injury is less than in human medicine. Availability of large volumes of blood products is also more limited in all but the largest hospitals. However, current evidence supports the use of restricted volumes of crystalloid at rates of <30 mL/kg/h to limit pulmonary effects, while ensuring adequate resuscitation using physical examination markers of perfusion, lactate concentrations and other markers of perfusion. When there is severe hemorrhage requiring fluids for resuscitation, the use of blood products instead of non-sanguineous fluids is justified where possible.Fluid therapy in human sepsis is a very controversial topic despite decades of research. Following the publication of improved outcomes with early goal directed therapy large volumes of fluid were recommended to target specific blood pressure and central venous pressure end-points . HoweverIn an experimental pig model of sepsis resuscitation with large volumes of fluid in early sepsis did not result in increased EVLW . SimilarOnce ARDS is present, the management of fluid therapy becomes complex. As seen previously, many patients with ARDS have sepsis, and management of sepsis typically requires administration of intravenous fluids to maintain homeostasis. Assessing fluid requirements, responsiveness and balance is of particular challenge in these patients and an area of active research. Given that ARDS is a form of non-cardiogenic pulmonary edema associated with alterations to pulmonary vascular permeability, it makes biological sense to limit increases in hydrostatic pressure to minimize further worsening of pulmonary function. Restrictive fluid therapy strategies in ARDS are supported by several retrospective studies –47. A laIntravenous fluid therapy is a vital and life-saving therapeutic in veterinary medicine. The evidence supporting a detrimental impact of fluid therapy on lung function is limited to disease states. The effects of excessive fluid therapy in animals with known cardiac disease is well-known. In addition to this, animals that have suffered trauma, have sepsis or inflammatory lung disease should be considered at risk of deteriorating lung function as a result of overzealous fluid therapy after initial resuscitation.The effects of fluid overload in healthy animals usually only occur in supraphysiological doses, however due to their smaller blood volume and increased incidence of undiagnosed or asymptomatic myocardial disease, extra caution is required in cats.In sick animals the impact of the underlying condition should be considered as this may impact on the fluid therapy plan and influence monitoring.There is clear evidence in the literature that limiting fluid rates in people with trauma is important to prevent respiratory complications. In patients with sepsis the data is more difficult to interpret. Restoration of perfusion is of critical importance, and in patients that are fluid deplete administration of intravenous fluid therapy is important, but overload must be avoided. Early use of vasopressors is increasingly promoted in people with sepsis to avoid fluid overload. The importance of this in veterinary species is unknown, and clinical experience suggests that fluid deficits are common in these patients. Assessing whether fluid deficit is present is potentially challenging, but the use of point-of-care ultrasound using for example, measurements of the caudal vena cava is allowing more careful and individualized patient care. In addition, the use of monitoring tools such as body weight, serial physical examination, urine output and specific gravity allows individualized patient care and may improve prescription of fluid therapy.While management of these animals will remain challenging when formulating individualized and flexible fluid therapy plans, using phased approaches, e.g., ROSE approach  will allow veterinary clincians to limit any potentially detrimentaleffects.The author confirms being the sole contributor of this work and has approved it for publication.SA is employed by Linnaeus Veterinary Limited which is a part of Mars Veterinary Health.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The crystalloid amount was a risk factor for prolonged POI . In the context of RARC, increased intravenous fluids are associated with prolonged POI and longer LOS.This study aimed to investigate the effect of intraoperative fluid volume on the postoperative ileus (POI) recovery period. A retrospective review of the Korean robot-assisted radical cystectomy database identified 718 patients who underwent robot-assisted radical cystectomy (RARC). Regression analyses were performed to identify the associations between the amount of intraoperative fluid administration , POI period (time to flatus/bowel movements), and length of hospital stay (LOS) after adjusting for covariates. In addition, we analyzed the risk factors for gastrointestinal complications and prolonged POI using a logistic regression model. An increasing volume of the administered crystalloid/total fluid was associated with prolonged POI (crystalloid R Recent studies have shown that RARC improves perioperative morbidity and facilitates early recovery4.Since its introduction in 2003, robot-assisted radical cystectomy (RARC) is a minimally invasive technique that has been increasingly used worldwide. It shows comparable results in terms of oncologic and perioperative outcomes5. Weak and ineffective peristalsis associated with POI can lead to accumulation of gastrointestinal secretion, causing abdominal distension and vomiting; prolonged POI even requires parenteral nutrition6. POI is traditionally considered inevitable after major abdominal surgery, leading to increased patient morbidity, hospital costs8, and 30-day readmission rates9.Gastrointestinal complications are the primary complications of both RARC and open surgical approach. Postoperative ileus (POI), which occurs in approximately 20% of patients, is commonly implicated for delayed recovery and increased risk of postoperative complications13. Several studies have reported that hypervolemic management may result in bowel edema, prolong the recovery of bowel function, disrupt tissue oxygenation, and adversely affect the healing of anastomotic sites17. On the other hand, fluid restriction has been shown to accelerate the recovery of bowel function and facilitate the early intake of oral diet; but owing to the heterogeneity of these studies and the lack of a precise definition of “restricted,” the real impact of fluid restriction on the POI period is yet to be established19. In an attempt to overcome this limitation, we attempted to determine the independent effect of the intraoperative volume on the POI period and length of hospital stay (LOS) through regression analysis, after adjusting for other covariates.A complex interplay between neurogenic, inflammatory, fluid and electrolyte, and pharmacologic components plays a role in the development of POI. Among these, fluid and electrolyte imbalances can be managed with intraoperative care. However, proper perioperative fluid management strategies for patients undergoing elective surgery have not yet been establishedThe Korean Robot-Assisted Radical Cystectomy (KORARC) study group has a quality assurance database comprising > 750 RARC patients. In this study, the KORARC database was queried for the oncologic and functional outcomes of a large series of patients who underwent RARC. Using this database, we tested the hypothesis that unrestricted fluid management in patients during operation could be associated with prolonged bowel function convalescence.22. However, we adopted a slightly modified ERAS program based on special circumstances in Korea. As per anesthesiologists’ requirements, all patients fasted from 12 AM of the day preceding the surgery; a policy that applies to almost all hospitals in South Korea. Further, we could not administer alvimopan, a μ-receptor opioid antagonist, that has been reported to be effective in bowel function recovery in several studies, because it has not been introduced in Korea yet. Most of the other guidelines were followed, such as the avoidance of mechanical bowel preparation, avoidance of nasogastric tube insertion or its early removal, early ambulation, early feeding (within 24 h post-surgery), and use of multimodal analgesia to minimize opioid use.The prospectively collected KORARC database comprises over 750 patients from 11 tertiary referral institutions (21 surgeons) who were treated with RARC between April 2007 and October 2019. Study participants included patients with recurrent/multiple or high-grade superficial or muscle-invasive bladder cancer without evidence of metastatic disease on preoperative imaging. Further, eligible patients medically cleared for radical cystectomy with pelvic lymph node dissection (PLND), were aged ≥ 18 years, and had clinical stage Ta–T4/N0–N3/M0. We excluded study participants with evidence of metastasis who had previously undergone abdominal surgery or pelvic radiation therapy. The KORARC database is a web-based electronic database and is a brain-child of the Korean Society of Endourology and Robotics. All 718 patients enrolled in this study received perioperative patient care according to the previously established enhanced recovery after surgery (ERAS) program23.Based on a systematic review, the definition of POI was proposed as follows: “POI” is defined as the bowel dysfunction that occurs immediately after surgery, and recovery of POI refers to a case in which flatus or stool is passed and oral diet is tolerated; “prolonged POI” can be defined as a case in which one or more of following conditions are applicable on or before postoperative day 7 without POI resolution as per the above-mentioned criteria: (1) nausea or vomiting, (2) inability to tolerate oral diet for the preceding 24 h; (3) no flatus or stool passage for the preceding 24 h (4) abdominal distension, and/or (5) radiologic bowel distension sign with no mechanical obstructionThe primary endpoint was the correlation between the intravenous fluid volume , POI period, and LOS, after controlling for confounders. Regression analyses were performed to identify the combination of crystalloid volume, colloid volume, total fluid volume, and LOS associated with prolonged POI. The secondary endpoint was identification of gastrointestinal complications and prolonged POI. Multivariate logistic regression was used to assess the following potential predictors: age, sex, body surface area, body mass index, corporeal method , urinary diversion type , crystalloid/colloid injection amount, operation time, American Society of Anesthesiologists (ASA) score, PLND extent, and tumor-node-metastasis (TNM) stage.Continuous variables are expressed as median (interquartile range [IQR]) or as a number (percentage) of cases. Categorical variables are reported as numbers and percentages. Pearson’s correlation coefficients were used for statistical comparisons of continuous variables. Logistic regression analysis was performed to analyze the predictors of complications. Simple and multiple linear regression analyses were performed. All analyses were performed using the Statistical Package for the Social Sciences (SPSS) version 20.0  and SigmaPlot . Results were considered statistically significant if the P-value was less than 0.05.This study conformed to the standards of the Declaration of Helsinki and the current ethical guidelines. The study protocol was reviewed and approved by the Institutional Review Board of Korea University Hospital (No. 2019AN0102). Written informed consent was obtained from all the study participants prior to their enrolment.The average age of patients was 65 years , of which 615 (85.0%) were men; 57 (7.9%) had an ASA score > 3  of surgery. The incidence of major and minor complications was 18% (129 patients) and 38% (273 patients), respectively. Among all categories, the gastrointestinal complication rate was 24% (173 patients), with 31% (47 patients) experiencing high-grade complications. The most common gastrointestinal complications encountered were prolonged POI , small bowel obstruction , nausea/vomiting , and diarrhea   and total intravenous fluid volumes , and not associated with colloid volumes. LOS was positively associated with crystalloid , colloid , and total intravenous fluid volumes .On multivariate linear regression analyses, the total intraoperative intravenous volume and crystalloid volume were significantly positively associated with the time to flatus , while the fluid volume was not statistically significant. As for prolonged POI, the crystalloid amount  was statistically significant .The main strength of this study was that it specifically evaluated the independent impact of intraoperative fluid volume on the recovery of bowel function in the context of complex radical surgery, limiting the risk of bias due to several intraoperative confounding factors. On regression analyses, our results confirmed that the administration of less fluid produced small, but significant postoperative recovery advantages 25. Clinically, bowel edema, which results in ileus, is the most common symptom occurring secondary to excessive fluid therapy during RARC, and tends to be more prominent under the care of inexperienced anesthesiologists. RARC is a relatively complex operation that takes approximately 6 h on average , and pneumoperitoneum and head-down positions make fluid therapy indices difficult to interpret. Several studies have found that perioperative fluid overload is associated with increased morbidity26, and it has been demonstrated that avoiding fluid overload leads to improved outcomes not only in urology, but also in major gastrointestinal surgery27. In the Multicenter Danish study, when fluid was restricted during surgery, postoperative complications were reduced by almost half regardless of the volume of fluid infusion before and after surgery10. Lobo et al.28 reported that fluid overload during elective surgery was related to increased time until the first passage of flatus or stool, increased gastric emptying time, and increased time to tolerance of solid food.The goal of intraoperative fluid management is to create a “zero” fluid balance at the end of the surgery without complications. For physiological maintenance, enough fluid should be given to maintain the patient's weight prior to surgery, and for replacing fluid loss through urine, sweat, and other routes30. In their animal model, a distinct submucosal pale band was found in the hypervolemia group, and a narrow and dense submucosal layer was found in the volume-restriction group. In addition, the volume of crystalloid infusion during surgery was reported to have a significant effect on the structural and functional stability of the intestinal anastomosis in the early postoperative period.The reconstructive portion of the RARC for bowel resection and anastomosis is closely associated with POI. Marjanovic et al. demonstrated that hypervolemia increases intravascular hydrostatic pressure and causes damage to the endothelial glycocalyx, a mediator that induces fluid retention in the interstitial space32. Crystalloid have a hemodilution effect, while colloids have the ability to maintain osmotic pressure in the vasculature; hence, even with the same volume, colloids are expected to induce lesser bowel edema.However, there are a few issues in our findings that need to be addressed. First, in our results, colloids, unlike crystalloids, were not a risk factor for POI. Some studies have found a greater influence of colloids on the fluid resuscitation of critically ill patients compared with crystalloids; but studying how colloids might impact POI is largely a novel area of research33. Our study showed significantly better results for ICUD compared to ECUD in terms of the overall and gastrointestinal complication rates, which is consistent with the International Robotic Cystectomy Consortium data34. The duration for which the peritoneum is exposed to the external environment is proportional to intestinal inflammation and oxidative stress response, and these factors could cause the delayed restoration of bowel function35. However, in our study, the corporeal type did not significantly affect the occurrence of gastrointestinal complications (P = 0.407) or prolonged POI (P = 0.745). This is attributed to the following reasons: The anesthesiologist of each operation was different, which resulted in differing strategies of fluid therapy administration. Moreover, our data did not reflect the clinical experience of surgeons, which means that a few outcomes of surgeons who had not overcome the learning curve yet were included.We previously investigated whether intracorporeal urinary diversion (ICUD) has more clinical benefits than extracorporeal urinary diversion (ECUD)Our study has several limitations. First, further fluid management-related measures, such as urine output, were not measured. However, owing to the surgical principles of RARC that include bilateral ureter clamping in the early stage of surgery, the impact might be insignificant and can be a confounding factor considering that it is difficult to accurately account for. Certainly, it should be acknowledged that various surgical situations warrant varying interventions and different surgeons/anesthesiologists, potentially limiting the generalizability of these results in centers with different principles of perioperative care.In conclusion, intravenous fluid administration during surgery is an easily modifiable part of RARC, and intense collaboration between urologists and anesthesiologists is necessary to encourage optimal gastrointestinal recovery. Within its limitations, this study shows that an increased volume of fluid received during RARC may impair return to normal bowel function. To maximize the advantage of RARC as a minimally invasive operation, careful monitoring is required to control the amount of fluid infusion during surgery and to avoid fluid overload.


Cefiderocol (CFDC) is a novel siderophore-conjugated cephalosporin with broad activity against Gram-negative (GN) bacteria, including carbapenem-resistant isolates, and non-fermentative organisms. CFDC is approved by the FDA for complicated urinary tract infection (cUTI), hospital-acquired bacterial pneumonia, and ventilator-associated bacterial pneumonia. In this study, we analyzed the susceptibility of CFDC and comparators against aerobic nonfastidious GN isolates collected from US patients hospitalized with pneumonia (PHP) in 2020 as a part of the SENTRY Antimicrobial Surveillance Program. Enterobacterales  and extensively drug resistant  phenotype isolates were analyzed.A total of 1,877 Gram-negative isolates were consecutively collected from PHP in 27 US hospitals during 2020. Susceptibility (S) testing was performed using the CLSI broth microdilution method. CFDC was tested in iron-depleted Mueller-Hinton broth. CLSI or FDA (2021) breakpoints were used. Both CLSI and FDA (2021) interpretations are shown in the table. Carbapenem-resistant Pseudomonas aeruginosa , followed by Klebsiella pneumoniae (n=239). The %S and MIC50/90 values of CFDC for both CLSI and FDA breakpoints and comparators are shown in the table for all organisms and resistant subsets. For Enterobacterales, all tested drugs had >99%S. The 18 CRE isolates had 94.4%S to CFDC and ceftazidime-avibactam. CFDC was the most active antimicrobial tested against PSA  and XDR PSA (94.6/93.2%). CFDC had the highest %S against Acinetobacter baumannii-calcoaceticus species complex , XDR ABC (94.6/93.2%), and against Stenotrophomonas maltophilia . The most common GN organism isolated from PHP was in vitro results suggest that CFDC may be an important option for the treatment of PHP caused by GN organisms, particularly for pathogens which have few treatment options. CFDC was highly active against US GN isolates from PHP, including CRE, XDR PSA and ABC, as well as SM. These Dee Shortridge, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support) Jennifer M. Streit, BS, GlaxoSmithKline, LLC (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Leonard R. Duncan, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Basilea Pharmaceutica International, Ltd. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)Department of Health and Human Services Shionogi (Research Grant or Support) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support


MoS2 was incorporated into the HPMC matrix as a strengthening additive. The mechanical properties, bonding, and water vapor permeability of the composites were analyzed. The mechanical and vapor barrier properties of the HPMC films were significantly enhanced. The ultimate tensile strength and Young’s modulus of the composite films increased with the addition of up to 1 wt% MoS2. The water vapor permeability of HPMC films reduced when additives were incorporated. The wear test proves that the MoS2 additives can improve the tribological performance of the HPMC composite while reducing the friction coefficient. The main reason for enhanced tribological performance is the improvement in load capacity of the composite coating by the MoS2 additive. This MoS2/HPMC biocomposite can be used in food packaging.Synthetic polymers are the most commonly used polymers in daily life. Therefore, it is necessary to develop environmentally friendly polymers. Hydroxypropyl methylcellulose (HPMC) is a potential candidate for a biopolymer, owing to its unique properties. However, HPMC biopolymers have some disadvantages compared to synthetic polymers. In this study, the mechanical properties and tribological performance of MoS When food is exposed directly to air, it is not contaminated by dust easily but becomes more likely to breed bacteria and other microorganisms, which may cause diseases. Food packaging can block direct contact with air, moisture, and light and can prevent contamination during transportation and sale, thereby extending shelf life and making the food safer and more hygienic. Since the development of food packaging in response to urban development and the needs of people’s lives, it has also become necessary to consider the convenience and efficiency of food management in a large number of transport, handling, storage, sales, and eating cases. The advantages of traditional plastic packaging are its low price, low density, toughness, ease of production, and low water vapor permeability. However, the development of new food packaging materials is necessary due to ecological demands. Moreover, a food packaging material must ensure freshness, safety, and environmental protection. Some studies have employed nanotechnology and decomposable materials for food packaging ,2. The bChemically modified polymers have been extensively investigated for the development of new biomaterials with excellent physicochemical properties. Cellulose is the most abundant polysaccharide found in nature. It is regular and linear and has a rigid structure because of its configuration and intramolecular hydrogen bonds. The intermolecular hydrogen bonds between the hydroxyl groups result in aggregates or crystalline forms of cellulose. This association between the molecules leads to the formation of highly ordered crystalline regions, which makes cellulose only slightly soluble in pure water .2) additives on mechanical properties and tribological behavior.Synthetic polymers are the most used polymers in daily life. However, they are non-biodegradable, difficult to recycle, and based on non-renewable materials. Therefore, the development of environmentally friendly and biodegradable polymers of natural origin that can replace synthetic polymers is necessary for environmental and resource conservation. Hydroxypropyl methylcellulose (HPMC) is an odorless and tasteless edible plant derivative with properties such as transparency, oil resistance, and the ability to form a gel upon heating. Considerable work has been conducted on the characteristics of HPMC, which possesses unique tribological properties , self-he2 has a low friction coefficient. When added to polymers, it forms composites with improved strength and reduced friction. Previous studies have demonstrated the use of MoS2 additives in applications, such as lubrication, anti-wear, anticorrosion [MoSorrosion , and bioorrosion .2 can enhance the tribological performance of HPMC, and the mechanism responsible for this is well-understood; MoS2 and HPMC will peel off from the coating surface to form a transfer layer [The addition of appropriate quantities of MoSer layer . However2 addition, and their mechanisms. In addition, the factors that influence the water vapor permeability (WVP) of the composite films are discussed. The findings of this study can serve as a useful reference for the diversification of HPMC film applications. HPMC has potential for applications in food packaging ,19, pedi2 particles with an average size of 2 μm were obtained from Sigma Aldrich . The material properties of HPMC and MoS2 are listed in Hydroxypropyl methylcellulose  of three grades based on viscosity and molecular weight as shown in 2/HPMC composite films followed a four-step process. Initially, water and ethanol were mixed in a ratio of 1:4 in a clean flask and then heated to 60 °C. Then, 3 g of all grades of HPMC powder was poured into the solvent of 100 g and magnetically stirred at 500 rotations per minute for 12 h. Then, MoS2 additives (listed in 2/HPMC solution was then poured into a petri dish for film preparation and placed inside a controlled-environment chamber at 40 ± 10% relative humidity (RH) and at 30 ± 10 °C to dry the film. After 6 h, the petri dish was removed from the chamber, placed at a temperature of 25 °C with a relative humidity of 60%, and dried for 24 h. After drying, the films were removed from the Petri dish and stored under 30% RH at 25 °C. Both film thickness and surface roughness (Ra) were measured using a 3D laser scanning microscope , controlled at 200 ± 10 µm and 2 ± 0.5 µm. A scanning electron microscope  was used to observe the surface morphology of the coating.The preparation of MoSThe cast films were removed and cut into a bone shape using an ASTM D-638-V “dog bone” punch. Films with cracks, nicked sides, or bubbles were discarded. The mechanical properties of the cast films were determined using a micro/nano tensile testing machine  according to the ASTM D882 standard. 1 and p2 are the partial pressures of water vapor on the lower and upper side of the film, respectively.The crystallization parameters were collected by a Bruker D2 Phaser diffractometer, using CuKα  at an operating voltage and current of 30 kV and 10 mA, respectively. The blank scan was carried out using a clean zero background plate with no applied sample. The blank scan was subtracted from the sample scan during the analysis. The intermolecular force was determined using attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy . The WVP of films was measured according to the ASTM 1290-93 standard . A uniquWater vapor transmission rate (WVTR) was in turn obtained as follows:The tribological performance was evaluated using a ball-on-disk tribometer  under a load of 2 N and disk speed of 0.03 m/s. A chrome steel ball  with a diameter of 6.31 mm was employed as the upper ball, and the composite films were employed as the lower disk test piece. The wear test was performed in an environment at 25 °C and a RH of 70%. The friction coefficient of the coating was monitored and recorded in real time, and the wear volume was measured using a 3D laser scanning microscope.The third-body theory describes the interaction of the counterpart of the materials involved in the abrasion dry friction conditions ,30. The 2 1 wt%/HPMC composite is presented in 2 1 wt%/HPMC composite. The composite material is black in color, which means that the addition of MoS2 turns the originally transparent HPMC into black and can absorb a large amount of light, achieving the purpose of blocking light. The tribological behavior of MoS2 is immensely sensitive to environmental conditions. In extremely humid environments, sulfides are prone to combination with the moisture in air and form oxides, resulting in a decrease in tribological performance [2 affect its abrasive performance [2 additives in composite materials following the preparation process. The above samples were analyzed via X-ray diffraction (XRD) analysis, and the results are shown in 2/HPMC composite are shown in 2/HPMC composite clearly displays diffraction peaks of MoS2, which can be indexed to the (002), (004), (100), (103), (006), (105), and (110) planes of 2H-MoS2 (JCPDS card No. 37-1492). This result indicates that the preparation process does not damage the 2H-MoS2 additives. Meanwhile, the highly intense (002) peak indicates that the layered structure of MoS2 is parallel to the composite surface, which helps to discern its tribological properties.The SEM image showing the surface pattern of the MoSformance . Meanwhiformance . Therefo2 additives enhanced the ultimate tensile strength (UTS) of the film but reduced its ductility. Furthermore, as the amount of MoS2 increased, UTS and ductility of the film decreased. This may be because an appropriate amount of additive could form better local bonding with the HPMC matrix. This hypothesis can be verified with the experimental results of FTIR.The tensile strength, elastic modulus, and percentage elongation of the films were measured to evaluate the improvements in the mechanical properties of composite films compared to those of pure HPMC. The stress–strain curves of the composite films with three different molecular masses (molecular chain lengths) are shown in 2 provided the best result in terms of UTS, that is, an increase of up to 20%. A further increase in MoS2 additives led to a significant decrease in UTS. As shown in 2/HPMC composite considerably improved when the amount of additive was less than 3%, suggesting that the elastic behavior of the films was strongly affected by a small amount of additives. The elastic moduli of the films containing 1–3% MoS2 were dramatically more significant than those of the pure HPMC matrix from 1500 to 30%. Therefore, the addition of MoS2 within this range improved the matrix bonding. Based on the elongation (EL) analysis shown in 2 embrittled HPMC films and changed these films from ductile to brittle materials. Shi et al. [2 resulted in optimal tribological properties. This is due to the fact that the primary mechanical property in this study is tensile strength, whereas wear and lubrication occur under compressive conditions. As the addition of MoS2 particles embrittled an HPMC film, the material broke down when subjected to stress, allowing the MoS2 particles to directly contact the wear component. Hence, the attainment of optimal wear resistance and friction coefficients at higher additive ratios (5–10%) was caused by the combination of material breakage with various accommodation mode tribological mechanisms of MoS2 [2 synergizes with HPMC to form the transfer layers. However, the breakdown of the material also accelerated film depletion. This is consistent with previous findings, which have reported that the addition of MoS2 cannot extend the service life of a film [i et al.  found th of MoS2 ,34 and tf a film .2 particles modulate bonding between the additive and matrix before and after particle addition. Angles of incidence greater than the critical angle result in continuous total reflectance of an infrared beam between the sample surface and the ATR crystal. This internal reflectance creates an evanescent wave that the sample may absorb. The infrared beam spectrum, in which the energy is attenuated after multiple reflections, may then be used to analyze the molecular vibrations on the sample surface. The FTIR spectra of the HPMC and MoS2/HPMC composite films are shown in 2 additive, minor changes were observed in the intermolecular H bonding and OH bonding (3600–3200 cm−1) of pure HPMC, which indicated that the addition of MoS2 particles did not reduce the number of molecular bonds. A new band was observed at 690 cm−1 (indicated by a dashed line) after the addition of MoS2 to the matrix owing to the interaction of S2− with H+ in HPMC. MoS2 bonded with HPMC and formed molybdenum oxides. The peak at 690 cm−1 indicates the formation of MoO2 (MoS2 oxidized by water) bonding between HPMC and MoS2. Based on the change in molecular bonding, an appropriate addition of MoS2 particles (1 wt%) increases the bonding between additive particles and the HPMC matrix, thus enhancing the mechanical properties of the composite films. The MoS2 additives used in this study have large particle sizes (at the μm level), and longer-chain HPMC (HPMC 615) may establish more bonding with additives, resulting in higher tensile strength, as demonstrated in The properties of composite materials greatly affect the intermolecular interactions between various components ,36. The 2 is hydrophobic [2 particles effectively reduces WVP. However, without the addition of a dispersant, WVP eventually reached a limit (approximately 1.9 ± 10% g·mm/kPa·h·m2 in this study). A small amount of MoS2 additives can succeed in blocking water vapor from entering the composite material due to the intermolecular bonding between the additive and matrix. It is also helpful to reduce the frictional resistance caused by water vapor.The significant index  for industrial food packaging is the WVP index ,40. As Mrophobic , we pred2/HPMC 615 composite film offered the best mechanical properties and was used for subsequent wear tests. As shown in 2 additives effectively reduced the friction coefficient by 70%. However, the wear volume hardly reduced. This is in agreement with the study conducted by Shi [2 additive can facilitate the formation of a transfer layer, thereby reducing the coefficient of friction. MoS2 with a layered structure caused delamination during abrasion and detached itself from the bonded matrix, resulting in wear. A small amount of wear reduction occurred when MoS2 was bonded to the matrix, which led to better load resistance. The deformation of the film surface reduced, and the wear contact area and abrasion volume were lessened. However, the effect of MoS2 additives in reducing the wear of the composite film was not apparent.The MoSd by Shi . The MoS2 results in a transfer layer during abrasion, which can effectively reduce the coefficient of friction [2 additive. The third-body theory can explain this behavior adequately. With the addition of 1 wt% MoS2 additives, UTS increased by 20% and the elastic modulus increased by 1500%, indicating that MoS2/HPMC composites are very strong and rigid. When the composite material was subjected to a force, its deformation resistance increased and it provided a better S5M1  third-body velocity accommodation mechanism, and a better coefficient of friction was obtained. The main reason for transfer layer formation is the delamination of MoS2 [2 in the composite can provide transfer layer formation. In addition, this behavior explains why the coefficient of friction was practically constant with the addition of MoS2 in composites. The additives simultaneously increased the load resistance of the composite material and the embrittlement of the coating. Embrittlement coating was more likely to abrade out of the system by a third-body flow during abrasion, resulting in wear [A previous study had reported that the addition of MoSfriction . However of MoS2 ; the pre in wear . These t2/HPMC composite films were successfully prepared using the solvent evaporation method. The mechanical properties, such as the UTS and Young’s modulus, were enhanced dramatically with 1 wt% additive. The addition of MoS2 to HPMC strengthened the mechanical and vapor barrier behavior of the HPMC composite because of high interfacial bonding between MoS2 and the HPMC matrix. A matrix with a large molecular weight provided more sites for bonding with additives, thus improving the mechanical properties and load resistance. However, embrittlement of the composite film promoted the separation of MoS2/HPMC composite material from the film surface, which formed an effective transfer/protection layer and resulted in excellent tribological properties. The properties of HPMC were enhanced when the additives were incorporated. Although FTIR spectra illustrate that intermolecular forces transpire between additive and matrix materials, results from other analytical instruments is essential. The principal components analysis-assisted ATR–FTIR and thermogravimetric analysis can provide additional evidence to make the findings more significant. The application of environmentally friendly materials with improved mechanical properties would be beneficial for the food packaging industry.MoS


Enterobacterales (ENT) and CRE isolates, but have limited activity against metallo-β-lactamase (MBL)-producing ENT. We evaluated the activity of QPX7728 (QPX), a novel BLI with intravenous (IV) and oral availability, in combination with BL agents. We tested ENT isolates carrying the most common BL genes such as blaCTX-M, transferable AmpCs, oxacillinases, MBLs, and serine carbapenemases.The β-lactam (BL)/ β-lactamase inhibitor (BLI) combinations approved in the last 10 years are active against most ESBL-producing A total of 1,027 ENT isolates were susceptibility (S) tested by reference broth microdilution against aztreonam (ATM), cefepime (FEP), cefdinir (CDR), ceftibuten (CTB), ceftolozane (CT) and piperacillin (PT) with fixed 4 mg/L of tazobactam, biapenem (BPM), meropenem (MER), and tebipenem (TEB) combined with QPX at fixed 4 and 8 mg/L. All isolates were genetically characterized using whole genome sequencing and included 520 ESBL-producers and 507 CRE with 168 producing MBLs.90, ≥32 mg/L); however, MIC90 values decreased ≥32-fold with the addition of QPX at the highest concentration tested (Table). Oral agents, CTB,CDR and TEB were tested with QPX at a fixed 4 mg/L and showed a 32- to 128-fold increase in potency . ATM and FEP were tested with QPX at a fixed 4 and 8 mg/L and displayed MIC90 values ranging from 0.12-0.5 mg/L. ATM and FEP, tested with 8 mg/L of QPX, inhibited 99.8% of isolates at the breakpoint for the BL agent alone. BLI inhibitor combinations PT and CT displayed MIC90 values of 2 and 4 mg/L with the addition of 8 mg/L QPX. MER with QPX at a fixed 4 mg/L and 8 mg/L inhibited 99.8% and 100% of isolates, respectively. BL agents tested alone had limited activity against this challenge set of isolates ContraFect Corporation (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support) Yahse Edah, AS, Qpex (Research Grant or Support) Olga Lomovskaya, PhD, Qpex Biopharma (Employee) Mariana Castanheira, PhD, AbbVie (formerly Allergan) (Research Grant or Support)Bravos Biosciences (Research Grant or Support)Cidara Therapeutics, Inc. (Research Grant or Support)Cipla Therapeutics (Research Grant or Support)Cipla USA Inc. (Research Grant or Support)GlaxoSmithKline (Research Grant or Support)Melinta Therapeutics, Inc. (Research Grant or Support)Melinta Therapeutics, LLC (Research Grant or Support)Pfizer, Inc. (Research Grant or Support)Qpex Biopharma (Research Grant or Support)Shionogi (Research Grant or Support)Spero Therapeutics (Research Grant or Support) Mariana Castanheira, PhD, Affinity Biosensors  Involved: Self): Research Grant or Support; Allergan  Involved: Self): Research Grant or Support; Amicrobe, Inc  Involved: Self): Research Grant or Support; Amplyx Pharma  Involved: Self): Research Grant or Support; Artugen Therapeutics USA, Inc.  Involved: Self): Research Grant or Support; Astellas  Involved: Self): Research Grant or Support; Basilea  Involved: Self): Research Grant or Support; Beth Israel Deaconess Medical Center  Involved: Self): Research Grant or Support; BIDMC  Involved: Self): Research Grant or Support; bioMerieux Inc.  Involved: Self): Research Grant or Support; BioVersys Ag  Involved: Self): Research Grant or Support; Bugworks  Involved: Self): Research Grant or Support; Cidara  Involved: Self): Research Grant or Support; Cipla  Involved: Self): Research Grant or Support; Contrafect  Involved: Self): Research Grant or Support; Cormedix  Involved: Self): Research Grant or Support; Crestone, Inc.  Involved: Self): Research Grant or Support; Curza  Involved: Self): Research Grant or Support; CXC7  Involved: Self): Research Grant or Support; Entasis  Involved: Self): Research Grant or Support; Fedora Pharmaceutical  Involved: Self): Research Grant or Support; Fimbrion Therapeutics  Involved: Self): Research Grant or Support; Fox Chase  Involved: Self): Research Grant or Support; GlaxoSmithKline  Involved: Self): Research Grant or Support; Guardian Therapeutics  Involved: Self): Research Grant or Support; Hardy Diagnostics  Involved: Self): Research Grant or Support; IHMA  Involved: Self): Research Grant or Support; Janssen Research & Development  Involved: Self): Research Grant or Support; Johnson & Johnson  Involved: Self): Research Grant or Support; Kaleido Biosceinces  Involved: Self): Research Grant or Support; KBP Biosciences  Involved: Self): Research Grant or Support; Luminex  Involved: Self): Research Grant or Support; Matrivax  Involved: Self): Research Grant or Support; Mayo Clinic  Involved: Self): Research Grant or Support; Medpace  Involved: Self): Research Grant or Support; Meiji Seika Pharma Co., Ltd.  Involved: Self): Research Grant or Support; Melinta  Involved: Self): Research Grant or Support; Menarini  Involved: Self): Research Grant or Support; Merck  Involved: Self): Research Grant or Support; Meridian Bioscience Inc.  Involved: Self): Research Grant or Support; Micromyx  Involved: Self): Research Grant or Support; MicuRx  Involved: Self): Research Grant or Support; N8 Medical  Involved: Self): Research Grant or Support; Nabriva  Involved: Self): Research Grant or Support; National Institutes of Health  Involved: Self): Research Grant or Support; National University of Singapore  Involved: Self): Research Grant or Support; North Bristol NHS Trust  Involved: Self): Research Grant or Support; Novome Biotechnologies  Involved: Self): Research Grant or Support; Paratek  Involved: Self): Research Grant or Support; Pfizer  Involved: Self): Research Grant or Support; Prokaryotics Inc.  Involved: Self): Research Grant or Support; QPEX Biopharma  Involved: Self): Research Grant or Support; Rhode Island Hospital  Involved: Self): Research Grant or Support; RIHML  Involved: Self): Research Grant or Support; Roche  Involved: Self): Research Grant or Support; Roivant  Involved: Self): Research Grant or Support; Salvat  Involved: Self): Research Grant or Support; Scynexis  Involved: Self): Research Grant or Support; SeLux Diagnostics  Involved: Self): Research Grant or Support; Shionogi  Involved: Self): Research Grant or Support; Specific Diagnostics  Involved: Self): Research Grant or Support; Spero  Involved: Self): Research Grant or Support; SuperTrans Medical LT  Involved: Self): Research Grant or Support; T2 Biosystems  Involved: Self): Research Grant or Support; The University of Queensland  Involved: Self): Research Grant or Support; Thermo Fisher Scientific  Involved: Self): Research Grant or Support; Tufts Medical Center  Involved: Self): Research Grant or Support; Universite de Sherbrooke  Involved: Self): Research Grant or Support; University of Iowa  Involved: Self): Research Grant or Support; University of Iowa Hospitals and Clinics  Involved: Self): Research Grant or Support; University of Wisconsin  Involved: Self): Research Grant or Support; UNT System College of Pharmacy  Involved: Self): Research Grant or Support; URMC  Involved: Self): Research Grant or Support; UT Southwestern  Involved: Self): Research Grant or Support; VenatoRx  Involved: Self): Research Grant or Support; Viosera Therapeutics  Involved: Self): Research Grant or Support; Wayne State University  Involved: Self): Research Grant or Support


We find a positive/facilitating interaction. We discuss the implication of these results to theories about the joint effect of fluid intelligence and non-cognitive characteristics on crystalized intelligence.Most of the studies of the effects of fluid intelligence and non-cognitive characteristics on crystalized intelligence examined additive effects. The results of the few studies that examined interactive effects are inconsistent. Some find a positive (facilitating) interaction and some find a negative (compensatory) interaction. We improve on these previous studies by examining non-cognitive characteristics that were not studied before and by using a very large representative sample ( Cattell  distinguAlthough the original formulation of Cattell's theory had a clear interactive aspect to it, in that the extent to which fluid intelligence is invested in the formation of crystalized intelligence was assumed to depend on the levels of the investment traits, most of the models of the joint effects of fluid intelligence and investment traits on crystalized intelligence have been additive. For example, the PPIK  effects that investment has on fluid intelligence in the acquisition of knowledge; that is, with the idea that, with regard to the development of crystalized intelligence, fluid intelligence may be futile if it is not invested in the acquisition of knowledge. Alternatively, this interaction may be viewed as the result of a facilitation effect of fluid intelligence leading to a more efficient channeling of investment resources; that is with the idea that investment may be futile if fluid intelligence is missing. In this respect, the effects of intelligence and investment on the acquisition of knowledge is similar to the effects of ability and motivation on performance, effects which are associated with the idea that people will not act if their actions do not serve relevant goals , conducted with a probability sample of 12,686 Americans  born between 1957 and 1964 . The mean age of the participants when they took the cognitive ability tests in 1980 was 18.66 (std = 2.25). Because of missing values, the number of subjects in the regression analyses was 11,448. The data are available at Armed Services Vocational Aptitude Battery (ASVAB), which included 10 tests: coding speed, numerical operations, general science, arithmetic reasoning, mathematical knowledge, word knowledge, paragraph comprehension, auto and shop information, mechanical comprehension, and electronics information. ASVAB scores were based on item response theory statistics, with higher scores indicating better performance. To facilitate interpretation, ASVAB scores were standardized prior to the analyses.Cognitive abilities were measured by the Since the measures available in this data set did not allow for deriving a pure measure of fluid intelligence, we relied on the idea that fluid intelligence is linked to processing speed ; parents' education was the mean of the highest grade completed by the father and mother . Sex was coded as 1 for males and 0 for females.To examine our interaction hypothesis, we regressed each of the eight crystalized abilities on fluid intelligence, our focal investment trait variables and the interaction between fluid intelligence and each of the three investment traits. In addition, our regressions also include the quadratic terms of the independent variables to account for a possible overlap between interaction and quadratic terms. To facilitate interpretability of the main effects, all independent variables were mean centered.The results of these eight regressions are presented in It is clear that although the signs of all the significant interactions were in the hypothesized directions, there were quite a few interactions that were not significant. Although all the 14 significant interactions were consistent with our hypothesis, the remaining 10 interactions were not significant, neither supporting nor opposing the investment as interaction hypothesis.ical abilities as measures of crystalized intelligence and parents' education and educational aspirations as investment traits. Since these two traits are associated with socioeconomic background, these non-significant interactions, together with the significant interactions involving these two traits when the crystalized abilities are stochastic abilities, suggest that socioeconomic factors are associated primarily with a motivation for scholastic, rather than technical, achievement.The non-significant interactions are also of interest. Six of them are associated with verbal abilities, which is consistent with the idea that the investment of fluid intelligence is less important in the development of verbal abilities than the development of mathematical and science abilities. Three of the non-significant interactions involve technIn addition to our focal interaction effect, an interesting pattern that emerges from our results is related to the quadratic effects of fluid intelligence, which was significantly positive for the two mathematical abilities and significantly negative for the other abilities. This pattern suggests an increasing marginal effect of fluid intelligence on mathematical abilities, but a diminishing marginal effect on the other abilities.Finally, we note that the implications of the significant interactions are not negligible. This is particularly the case for the interactions between fluid intelligence and sex, where a standard deviation increase in fluid intelligence is associated with additional increases of up to 0.200 standard deviations in test scores for males vs. females. These additional increases for males are smaller for the scholastic tests , and larger for the technical tests . As an example, The implications of the significant interactions involving parents' education and educational aspirations are smaller. A one standard deviation increase in in fluid intelligence is associated with an additional increase of up to 0.084 standard deviation in test scores for a participant with high educational expectations (1std above the mean) vs. participants with low educational expectations (1std below the mean). For parents' education the increase is up to 0.026 standard deviations. It is also evident from In the 1941 APA conference, Reymond Cattell (simultaneously with Donald Hebb, see Brown, Building on Cattell's work, and on the work of earlier scholars who suggested that non-cognitive traits are involved in the development of intelligence (Thorndike et al., Since an interaction model  is a mosTo solve this problem, we suggest relying on large representative databases that include information about fluid and crystalized intelligence, and using antecedents of interest that were not considered so far as moderators for the effect of fluid intelligence on crystalized intelligence. This suggestion is supported by the current results, but it is also supported by the fact that the few previous successful demonstrations of interactive relationships associated with fluid and crystalized intelligence were documented by analyzing large database, estimating the interaction between fluid intelligence and interest (Lechner et al., The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ariel. The patients/participants provided their written informed consent to participate in this study.YG initiated the study and analyzed the data, conceptualized the design, and wrote the paper.This work was supported by University grant.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.