Journal: Scientific Reports

Article Title: Second-trimester urine nephrin:creatinine ratio versus soluble fms-like tyrosine kinase-1:placental growth factor ratio for prediction of preeclampsia among asymptomatic women

doi: 10.1038/srep37442

Figure Lengend Snippet: FPR, serum soluble (sFlit-1): placental growth factor (PlGF) ratio; urine NCR, spot-urine nephrin:creatinine ratio; urine PCR, spot-urine protein:creatinine ratio. Totals of 239 and 15 women with neither SPIP nor hypertension were examined at GW 25 (22–27) and 24 (23–25), respectively; the former 239 women remained normotensive throughout pregnancy and the latter 15 women later developed PE at GW 35.7 (27.6–38.6). Eleven and 22 urine specimens from PE and normotensive women, respectively, were used previously , but nephrin concentrations in the 33 urines were measured again together with the remaining 221 samples for this study. All variable levels except PlGF differed significantly between the two groups. The median (range)/mean ± SD values in the 239 vs. 15 women were 49.6 (0.15–812)/69.6 ± 88.1 vs. 154 (52.2–1512)/261 ± 362 ng/mg Cr for NCR, 0.044 (0.001–0.215)/0.048 ± 0.031 vs. 0.062 (0.018–0.210)/0.073 ± 0.054 mg/mg Cr for PCR, 813 (192–1648)/1097 ± 1545 vs. 1414 (385–1969)/3440 ± 5027 pg/mL for sFlit-1, 497 (87–3034)/575 ± 397 vs. 336 (24–982)/474 ± 327 pg/mL for PlGF, and 1.65 (0.26–1334)/3.31 ± 10.3 vs. 2.32 (0.51–831)/64.6 ± 212 for FPR, respectively.

Article Snippet: The range of the standard curve is 0.8–200 ng/mL according to the protocol of the human nephrin ELISA kit (listed as #1035 in the catalog of Funakoshi Co. Ltd.).

Techniques:

Journal: Scientific Reports

Article Title: Second-trimester urine nephrin:creatinine ratio versus soluble fms-like tyrosine kinase-1:placental growth factor ratio for prediction of preeclampsia among asymptomatic women

doi: 10.1038/srep37442

Figure Lengend Snippet: Number of days until assay after urine collection varied from 80 to 662 in the 239 urines from 239 women who remained normotensive throughout pregnancy. There was no significant correlation between NCR levels and number of days after sampling until nephrin assay (Fig. 2, left). The median/mean NCR values were 57.2 (0.151–812)/79.9 ± 105, 40.0 (0.499–318)/55.5 ± 56.7, and 52.4 (0.239–671)/73.9 ± 96.0, for the 1 st , 2 nd , and 3 rd tertile groups divided by number of days until assay after sampling, respectively (Fig. 2, right). The GW at specimen sampling was 24.9 (23.4–27.4), 24.7 (22.0–26.7), and 24.9 (22.0–26.7) for the 1 st , 2 nd , and 3 rd tertile groups, respectively. Similar investigations on sFlit-1 and PlGF were performed (data not shown). There was no significant correlation of number of days after specimen sampling until assay with either sFlit-1 or PlGF level. The median/mean sFlit-1 values were 721 (250–16476)/1098 ± 1838, 813 (192–3268)/919 ± 622, and 882 (237–15986)/1277 ± 1846 pg/mL for the 1 st , 2 nd , and 3 rd tertile groups, respectively ( P = 0.1776 with Kruskal-Wallis test). However, there was a significant difference in the median/mean values of PlGF with unknown reasons; 528 (123–3034)/656 ± 474, 348 (86.9–1405)/407 ± 264, and 638 (123–2244)/663 ± 373 pg/mL for the 1 st , 2 nd , and 3 rd tertile groups, respectively ( P < 0.0001 with Kruskal-Wallis test).

Article Snippet: The range of the standard curve is 0.8–200 ng/mL according to the protocol of the human nephrin ELISA kit (listed as #1035 in the catalog of Funakoshi Co. Ltd.).

Techniques: Sampling

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: The BMP scavenger ERFE is upregulated in cachexia through STAT3 activation (A) Schematic representation of the C26 cancer cachexia model where mice injected with C26 cells develop severe cachexia within 11 days. (B) Immunoblot of phosphorylated Smad1/5/8 (pSmad1/5/8) and vinculin in the quadriceps of control and C26 tumor-bearing mice (n = 5/6). (C) Densiometric quantification of the immunoblot of pSmad1/5/8 and Smad1 in the quadriceps of control and C26 tumor-bearing mice (n = 5/6). (D) mRNA levels of ERFE relative to GAPDH in the gastrocnemii of C26 tumor-bearing mice and relative control mice 11 days post cancer cells inoculation (n = 4/5). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) ERFE protein concentration detected by ELISA in the serum of C26 and control mice (n = 4). (F) Expression levels of ERFE relative to beta-actin in human skeletal muscle biopsies from pre-cachectic and cachectic colorectal and pancreatic cancer patients compared to control individuals (control, n = 41; pre-cachectic, n = 77; cachectic, n = 63). Values are reported as fold change over control group (control vs. pre-cachexia q value = 0.0021; control vs. cachexia q value = 0.0007). (G) Expression levels of Erfe in C2C12 myotubes treated for 48h with 20% C26 CM (n = 6) and representative immunofluorescence of atrophic C2C12 myotubes treated with C26 CM and stained for myoglobin. (H) Experimental design of the ChIP-qPCR assay performed on C2C12 myotubes treated with C26 CM or atrophic skeletal muscles of C26 tumor-bearing mice at day 11. (I) STAT3 predicted binding sites on Erfe promoter. (J) ChIP-qPCR of pSTAT3 binding Erfe promoter on TSS (+629/+639 bp) in C2C12 myotubes treated with C26 CM compared with control cells (n = 2). (K) In vivo ChIP-qPCR of pSTAT3 binding Erfe promoter on TSS (+629/+639 bp). ChIP-qPCR assay was performed by pooling 4 quadriceps per condition for control or C26 tumor-bearing mice (n = 2). (L) Expression levels of Erfe on C2C12 myotubes treated with Stattic 5 μM and/or 50% C26 CM for 6 h (n = 3). (M) Luminescence (firefly/renilla) of NIH/3T3 transfected with Erfe promoter-Luc reporter vector and knocked down for STAT3 (siSTAT3). Cells were treated overnight with 50% C26 CM prior to performing the assay (n = 2). See also Figures S1 and . Data information: Statistical significance was tested with unpaired two-tailed Student’s t -test in (C)–(E) and (G); with non-parametric Krustal-Wallis test followed by Benjamini, Krieger, and Yekutieli multiple comparison tests in (F) and with two-way ANOVA followed by Sidak’s multiple comparison test in (J) and (K) and with one-way ANOVA followed by Sidak’s multiple comparison test in (L) and (M).

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Activation Assay, Injection, Western Blot, Control, Protein Concentration, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining, ChIP-qPCR, Muscles, Binding Assay, In Vivo, Transfection, Plasmid Preparation, Two Tailed Test, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: Muscle-specific knockdown of BMP inhibitors curbs muscle wasting in C26 tumor-bearing mice (A) Schematic representation of the pathway. The BMP scavenger ERFE is upregulated in cachexia and with the intracellular inhibitor FKBP12 contributes to the downregulation of pSmad1/5/8 in atrophic muscles. (B) Quantification of myotubes diameters after 24 h treatment with recombinant Fc-ERFE (1 μg/mL), inactive globular Fc-gERFE (1 μg/mL), and the relative Fc-CTR (1 μg/mL) (n = 3/4). (C) Myotubes diameters transfected with siRNA for Fkbp12 or scramble sequence (siCtr) for 48 h and treated for 24 h with 10% C26 CM (n = 3/4). (D) Expression levels in C2C12 myotubes knocked-down for FKBP12 with siRNA (n = 4/6). (E) Experimental design of muscle-specific AAV9 delivery in the skeletal muscle of C26 tumor-bearing mice. AAV9-shRNA (10 11 vp) viral particles were injected in the skeletal muscles of BALB/c mice, using the contralateral limb as control (AAV9-shScr). One month after AAV injection, BALB/c mice were subcutaneously inoculated with 750,000 C26 cancer cells. The mice were euthanized 11 days after C26 injection. (F) mRNA levels of ERFE in the quadriceps of AAV9-shERFE-injected mice vs. the contralateral AAV9-shScr limb (n = 7). (G) Quadriceps weight normalized for tibial length compared with the contralateral limb (n = 7). (H) Atrogin1 expression in the quadriceps analyzed in Figures 2 F and 2G (n = 7). (I) CSA distribution of AAV9-shERFE-injected mice (n = 3). (J) mRNA levels of FKBP12 in the quadriceps of AAV9-shFKBP12-injected mice vs. the contralateral AAV9-shScramble limb (n = 7). (K) Quadriceps weight normalized for tibial length compared with the contralateral limb (n = 7). (L) Atrogin1 expression in the quadriceps analyzed in Figures 2 J and 2K. (M) CSA distribution of AAV9-shFKBP12-injected mice (n = 3). See also Figure S3 . Data information: Statistical significance was tested with unpaired two-tailed Student’s t -test in (D); with one-way ANOVA followed by Sidak’s multiple comparison test in (B) and (C). One-sample t -test was performed in (F)–(H) and (J)–(L), while two-way ANOVA followed by Sidak’s multiple comparison test in (I) and (M).

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Knockdown, Muscles, Recombinant, Transfection, Sequencing, Expressing, shRNA, Injection, Control, Two Tailed Test, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: FK506 bypasses BMP pathway inhibition by activating pSmad1/5/8 through FKBP12 binding (A) Representation of the proposed approach to bypass the "BMP resistance." FK506 binds FKBP12 displacing it from the BMPR-I and restoring pSmad1/5/8 signaling. (B and C) Diameters quantification of C2C12 myotubes treated for 24 h with FK506 (1 μg/mL) and/or 10% C26 CM (B) (n = 5) or treated with recombinant Fc-ERFE (1 μg/mL) and FK506 in Fc-CTR (1 μg/mL) (C) (n = 4). (D) Immunoblot of C2C12 myotubes serum starved for 2 h and treated with FK506 (1 μg/mL) for 1 h (n = 4). (E) Dose-response and immunoblot of pSmad1/5/8 in C2C12 myotubes serum starved for 2 h and treated with increasing doses of murine BMP6 (from 0.01 to 3 ng/mL) with or without FK506 (1 μg/mL) for 1 h (n = 4). (F) Luminescence signal detected in C2C12 transfected with pGL3-BMP responsive element (BRE)-Luciferase and TK-Renilla for 24 h, switched to low-serum DMEM 2% fetal bovine serum for 4 h and treated with FK506 (10 μg/mL) and BMP6 (30 ng/mL) for 5 h (n = 6). (G) Immunoblot of pSmad1/5 and Smad1 of C2C12 myotubes knocked-down for ALK2 or ALK3 (siAlk2 or siAlk3) for 48 h and treated with FK506 (1 μg/mL) for 1 h upon 2 h of serum starvation (n = 2). (H) mRNA levels of ALK2 and ALK3 in C2C12 myotubes knocked-down for ALK2 or ALK3 with siRNA (n = 2/3). (I) Diameters of C2C12 myotubes treated with DMH1 (500 nM) in combination with FK506 (1 μg/mL) and/or 10% C26 CM for 24 h in differentiation medium (n = 3/4). (J and K) In vitro SuNSET assay and relative quantifications performed with C2C12 myotubes treated with FK506 (1 μg/mL), 10% C26 CM (J) (n = 4) or Fc-CTR (1 μg/mL), Fc-ERFE (1 μg/mL), and inactive globular gERFE (1 μg/mL). (K) (n = 3) For 24 h in differentiation medium. For the last 4 h of treatment, puromycin (1 μM) was added to the cells. (L) Myotubes diameters imaged at 24 h post FK506 (1 μg/mL) and/or Torin1 (100 μM) treatment (n = 3). (M and N) mRNA levels of Atrogin1 (M) (n = 3/5) and MuRF1 (N) (n = 3) in C2C12 myotubes treated for 48 h with FK506 (1 μg/mL) and/or 20% C26 CM. See also Figure S4 . Data information: All graphs’ statistical significance was tested with one-way ANOVA followed by Sidak’s multiple comparison test.

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Inhibition, Binding Assay, Recombinant, Western Blot, Transfection, Luciferase, In Vitro, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet: FK506 prevents the degeneration of NMJs morphology and preserves muscle strength (A) Final grip strength as percentage of initial grip strength at day 0 in FK506 or vehicle-treated C26 tumor-bearing and control mice at day 11 post-C26 injection (n = 5/7). (B and C) Volume of the NMJs quantified in the EDL muscle and stained with α-bungarotoxin-Alexa 488 (B) and relative representative picture of the experimental groups (C) (n = 3, 28–30 NMJs quantified per mouse). (D) Expression levels of Chrne gene in the gastrocnemii of FK506 or vehicle treated C26 and control mice at day 11 post-C26 injection (n = 3/4). (E) Graphical representation of the molecular mechanism. In cancer cachexia, systemic inflammation induces catabolism by hyperactivating TGFβ-Smad2/3 axis and by increasing the expression of BMP signaling inhibitors. Activated pSTAT3 induces the upregulation of the BMP scavenger ERFE, which in combination with the intracellular inhibitor FKBP12, contributes to the downregulation of the BMP-pSmad1/5/8 axis and therefore reducing protein synthesis in atrophic muscles. Our therapeutic approach proposes the use of low-dose FK506, that by binding the immunophilin FKBP12 induces its displacement from the BMPR-I, rescuing pSmad1/5/8 signaling and protecting from muscle atrophy in cancer cachexia. See also Figure S6 . Data information: Statistical significance was tested with one-way ANOVA followed by Sidak’s multiple comparison test.

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: Control, Injection, Staining, Expressing, Muscles, Binding Assay, Comparison

Journal: Cell Reports Medicine

Article Title: FK506 bypasses the effect of erythroferrone in cancer cachexia skeletal muscle atrophy

doi: 10.1016/j.xcrm.2023.101306

Figure Lengend Snippet:

Article Snippet: Serum protein levels of erythroferrone were measured with Intrinsic Lifesciences mouse ERFE ELISA kit (ERF-200) according to manufacturer’s instructions.

Techniques: FLAG-tag, Virus, Recombinant, Reporter Assay, BIA-KA, Enzyme-linked Immunosorbent Assay, Purification, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software

Journal: International Journal of Nanomedicine

Article Title: Design of Scaffolds Based on Zinc-Modified Marine Collagen and Bilberry Leaves Extract-Loaded Silica Nanoparticles as Wound Dressings

doi: 10.2147/IJN.S466905

Figure Lengend Snippet: ( A ) Cytocompatibility of Vaccinium myrtillus extract and encapsulated extracts in human keratinocytes normal cells after 24 and 72 h incubation; Secretion of IL-1β ( B ) and TNF-α ( C ) pro-inflammatory cytokine in pretreated HaCaT cells with different concentrations of bilberry extracts, determined by ELISA assay at 24 h cultivation. # p < 0.05 compared to positive control; * p < 0.05 compared to WB.

Article Snippet: Human normal keratinocytes HaCaT (ATCC - American Type Culture Collection, Manassas, VA, USA) were cultivated in RPMI 1640 medium with high glucose content supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-neomycin antibiotic mixture (PSN) at 37°C and 5% CO 2 .

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Positive Control

Journal:

Article Title: Utility of the Focus Technologies West Nile Virus Immunoglobulin M Capture Enzyme-Linked Immunosorbent Assay for Testing Cerebrospinal Fluid

doi: 10.1128/JCM.42.1.12-15.2004

Figure Lengend Snippet: CSF specimens evaluated

Article Snippet: The second specificity panel consisted of 60 CSF specimens positive for another virus by PCR testing performed by the Focus Technologies reference laboratory Molecular Diagnostics section; samples were frozen (−70C) after PCR testing until utilized for the present study. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Panel Description No. of specimens Sensitivity Positive using in-house WNV IgM ELISA (native antigen) and PHSL WNV IgM ELISA 52 Specificity 1 Submitted for in-house WNV IgM ELISA testing February through April 2003; negative for WNV IgM 73 Specificity 2 PCR + for enteroviruses 28 PCR + for varicella-zoster virus 11 PCR + for JC virus 7 PCR + for herpes simplex virus type 1 3 PCR + for herpes simplex virus type 2 5 PCR + for Epstein-Barr virus 3 PCR + for cytomegalovirus 2 PCR + for human herpesvirus 6 1 Open in a separate window CSF specimens evaluated WNV IgM capture ELISA kit.

Techniques: Enzyme-linked Immunosorbent Assay, Polymerase Chain Reaction

Journal: Peptides

Article Title: An angiotensin-converting enzyme-2-derived heptapeptide GK-7 for SARS-CoV-2 spike blockade

doi: 10.1016/j.peptides.2021.170638

Figure Lengend Snippet: Diagram depicting the design of short peptides derived from ACE2. The extracellular domain of ACE2 is presented as ribbon structure. EQ-10, KT-11, LD-11, NH-11, and ED-10 are derived from the N-terminal helix of ACE2. GK-7 is extracted from the linker between β3 and β4 of ACE2. The critical residues contributing to the interaction of ACE2 with SARS-CoV-2 spike in the peptides are shown in sticks and colored red.

Article Snippet: SARS-CoV-2 spike pseudovirions (20 μL, 1.98 × 10 7 TU/mL) preincubated with increasing concentrations of GK-7 (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 75, and 100 μg/mL), LD-11 (0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 75, 100, and 200 μg/mL), or ACE2 (10108-H02H, Sino Biological, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, and 1.2 μg/mL) at 37 °C for 1 h were added to the cells, followed by 12 h of coincubation.

Techniques: Derivative Assay

Journal: Peptides

Article Title: An angiotensin-converting enzyme-2-derived heptapeptide GK-7 for SARS-CoV-2 spike blockade

doi: 10.1016/j.peptides.2021.170638

Figure Lengend Snippet: Inhibitory effects of GK-7 on SARS-CoV-2 S1 adherence to cells. ( A ) Western blotting detection of SARS-CoV-2 S1 binding to A549 cells in the absence and presence of 50 μg/mL LD-11, ED-10, and GK-7. β-actin was used as the reference. ( B ) Dose-dependent suppression of GK-7 on SARS-CoV-2 S1 binding to A549 cells. ( C ) Immunofluorescence microscopy revealing the protective effect of GK-7 on A549 cells exposed to SARS-CoV-2 S1 (red). Nuclei were stained with DAPI (blue). The scale bar indicates 50 μm.

Article Snippet: SARS-CoV-2 spike pseudovirions (20 μL, 1.98 × 10 7 TU/mL) preincubated with increasing concentrations of GK-7 (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 75, and 100 μg/mL), LD-11 (0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 75, 100, and 200 μg/mL), or ACE2 (10108-H02H, Sino Biological, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, and 1.2 μg/mL) at 37 °C for 1 h were added to the cells, followed by 12 h of coincubation.

Techniques: Western Blot, Binding Assay, Immunofluorescence, Microscopy, Staining

Journal: Peptides

Article Title: An angiotensin-converting enzyme-2-derived heptapeptide GK-7 for SARS-CoV-2 spike blockade

doi: 10.1016/j.peptides.2021.170638

Figure Lengend Snippet: Antiviral action of GK-7 against SARS-CoV-2 spike pseudovirion infection. ( A ) Dose-dependent antiviral activity of GK-7, ( B ) LD-11, and ( C ) ACE2 against pseudovirions. Results are shown as mean ± standard deviation (SD). The fitted curves obtained by nonlinear regression are in burgundy.

Article Snippet: SARS-CoV-2 spike pseudovirions (20 μL, 1.98 × 10 7 TU/mL) preincubated with increasing concentrations of GK-7 (0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 75, and 100 μg/mL), LD-11 (0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 75, 100, and 200 μg/mL), or ACE2 (10108-H02H, Sino Biological, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, and 1.2 μg/mL) at 37 °C for 1 h were added to the cells, followed by 12 h of coincubation.

Techniques: Infection, Activity Assay, Standard Deviation