Improving the antibacterial activity and selectivity of an ultra short peptide by hydrophobic and hydrophilic amino acid stretches

The principle of amino acid stretches tagged at the C terminal of Luecrocin I, which is an ultra-short antibacterial peptide, by tryptophan and arginine or lysine has been reported. The choice of amino acid type at each stretch position depends on the hydrophobic and hydrophilic regions visualized in the helical wheel pattern of Luecrocin I. Oligopeptide tagging should also consider the properties such as positive charge, hydrophobicity, the content of hydrophobic amino acids, polar angle, the properly hydrophilic and hydrophobic facets. Amidation at C terminal and lysine substitute for arginine can increase selectivity between mammalian cells (hemolytic and MTT assay) and bacterial cells tested. KT2 and RT2 which have 53% hydrophobic residues, 7 positive charges, 160° polar angle, ?0.02 (KT2) and ?0.04 (RT2) hydrophobicity were effective against S. typhi DMST 22842, S. epidermidis ATCC 12228, E. coli ATCC 25922 and V. cholerae non-O1, non-O139. The SEM images implied that the antibacterial mechanism of RT2 and KT2 may depend on concentration rather than time. Finally, RT2 and KT2 can be new antibacterial agents or may be further developed for alternative antibiotics.     

Single and Serial Fetal Biometry to Detect Preterm and Term Small- and Large-for-Gestational-Age Neonates: A Longitudinal Cohort Study

ObjectivesTo assess the value of single and serial fetal biometry for the prediction of small- (SGA) and large-for-gestational-age (LGA) neonates delivered preterm or at term.MethodsA cohort study of 3,971 women with singleton pregnancies was conducted from the first trimester until delivery with 3,440 pregnancies (17,334 scans) meeting the following inclusion criteria: 1) delivery of a live neonate after 33 gestational weeks and 2) two or more ultrasound examinations with fetal biometry parameters obtained at ≤36 weeks. Primary outcomes were SGA (<5^th centile) and LGA (>95^th centile) at birth based on INTERGROWTH-21^st gender-specific standards. Fetus-specific estimated fetal weight (EFW) trajectories were calculated by linear mixed-effects models using data up to a fixed gestational age (GA) cutoff (28, 32, or 36 weeks) for fetuses having two or more measurements before the GA cutoff and not already delivered. A screen test positive for single biometry was based on Z-scores of EFW at the last scan before each GA cut-off so that the false positive rate (FPR) was 10%. Similarly, a screen test positive for the longitudinal analysis was based on the projected (extrapolated) EFW at 40 weeks from all available measurements before each cutoff for each fetus.ResultsFetal abdominal and head circumference measurements, as well as birth weights in the Detroit population, matched well to the INTERGROWTH-21^st standards, yet this was not the case for biparietal diameter (BPD) and femur length (FL) (up to 9% and 10% discrepancy for mean and confidence intervals, respectively), mainly due to differences in the measurement technique. Single biometry based on EFW at the last scan at ≤32 weeks (GA IQR: 27.4–30.9 weeks) had a sensitivity of 50% and 53% (FPR = 10%) to detect preterm and term SGA and LGA neonates, respectively (AUC of 82% both). For the detection of LGA using data up to 32- and 36-week cutoffs, single biometry analysis had higher sensitivity than longitudinal analysis (52% vs 46% and 62% vs 52%, respectively; both p<0.05). Restricting the analysis to subjects with the last observation taken within two weeks from the cutoff, the sensitivity for detection of LGA, but not SGA, increased to 65% and 72% for single biometry at the 32- and 36-week cutoffs, respectively. SGA screening performance was higher for preterm (<37 weeks) than for term cases (73% vs 46% sensitivity; p<0.05) for single biometry at ≤32 weeks.ConclusionsWhen growth abnormalities are defined based on birth weight, growth velocity (captured in the longitudinal analysis) does not provide additional information when compared to the last measurement for predicting SGA and LGA neonates, with both approaches detecting one-half of the neonates (FPR = 10%) from data collected at ≤32 weeks. Unlike for SGA, LGA detection can be improved if ultrasound scans are scheduled as close as possible to the gestational-age cutoff when a decision regarding the clinical management of the patient needs to be made. Screening performance for SGA is higher for neonates that will be delivered preterm.     

Epithelial cell-derived neutrophil-activating peptide-78 is present in fetal membranes and amniotic fluid at increased concentrations with intra-amniotic infection and preterm delivery

Intra-amniotic secretion and abundance of epithelial cell-derived neutrophil-activating peptide (ENA)-78, a potent chemoattractant and activator of neutrophils, was studied in the context of term and preterm parturition. Staining of ENA-78 immunoperoxidase was localized predominantly to chorionic trophoblasts and amniotic epithelium in term and preterm gestational membranes, with weaker and less consistent staining in decidual cells. The abundance of ENA-78 in membrane tissue homogenates was significantly increased ( approximately 4-fold) with term labor in amnion (n = 15), and with preterm labor ( approximately 30-fold) in amnion and choriodecidua (n = 31). In amnion tissue homogenate extracts, ENA-78 levels were positively correlated with the degree of leukocyte infiltration (r2 = 0.481). In amniotic fluids, median ENA-78 levels from pregnancies with preterm labor without intra-amniotic infection were significantly lower (P < 0.01 by ANOVA) than those from pregnancies with preterm deliveries with infection; levels in samples derived from term pregnancies were similar before and after labor. Production of ENA-78 by amnion monolayers was stimulated in a concentration-dependent fashion by both interleukin-1beta and tumor necrosis factor alpha. Production of ENA-78 by choriodecidual explants was increased modestly after 2-4 h of exposure to lipopolysaccharide (5 microg/ml). An immunoreactive doublet ( approximately 8 kDa) was detected in choriodecidual explant-conditioned media by immunoblotting. We conclude that ENA-78, derived from the gestational membranes, is present in increased abundance in the amniotic cavity in response to intrauterine infection and, hence, may play a role in the mechanism of infection-driven preterm birth and rupture of membranes secondary to leukocyte recruitment and activation.     

6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking during pregnancy

Neutrophils from pregnant women display reduced neutrophil-mediated effector functions, such as reactive oxygen metabolite (ROM) release. Because the NADPH oxidase and NO synthase produce ROMs and NO, the availability of their substrate NADPH is a potential regulatory factor. NADPH is produced by glucose-6-phosphate dehydrogenase (G-6-PDase) and 6-phosphogluconate dehydrogenase (6-PGDase), which are the first two steps of the hexose monophosphate shunt (HMS). Using immunofluorescence microscopy, we show that 6-PGDase, like G-6-PDase, undergoes retrograde transport to the microtubule-organizing centers in neutrophils from pregnant women. In contrast, 6-PGDase is found in an anterograde distribution in cells from nonpregnant women. However, lactate dehydrogenase distribution is unaffected by pregnancy. Cytochemical studies demonstrated that the distribution of 6-PGDase enzymatic activity is coincident with 6-PGDase Ag. The accumulation of 6-PGDase at the microtubule-organizing centers could be blocked by colchicine, suggesting that microtubules are important in this enzyme's intracellular distribution. In situ kinetic studies reveal that the rates of 6-gluconate turnover are indistinguishable in samples from nonpregnant and pregnant women, suggesting that the enzyme is functionally intact. Resonance energy transfer experiments showed that 6-PGDase and G-6-PDase are in close physical proximity within cells, suggesting the presence of supramolecular enzyme complexes. We suggest that the retrograde trafficking of HMS enzyme complexes during pregnancy influences the dynamics of NADPH production by separating HMS enzymes from glucose-6-phosphate generation at the plasma membrane and, in parallel, reducing ROM and NO production in comparison with fully activated neutrophils from nonpregnant women.     

Purification and Characterization of Antioxidant Peptides from Leukocyte Extract of Crocodylus siamensis

Antioxidant peptides were isolated from the leukocyte extract of the Siamese crocodile, Crocodylus siamensis. Crocodile leukocyte was extracted by a combination of methods including freeze-thawing, acetic acid extraction and homogenization. The peptides in the leukocyte extract were purified by anion exchange chromatography and reversed phase-high performance liquid chromatography. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay was used to evaluate the antioxidant activity of the elution peaks at each purification step. As a result, there were two purified peptides exhibiting strong antioxidant activity in reducing free radicals on DPPH molecules. The amino acid sequences of these peptides were determined by LC-MS/MS as TDVLGLPAK (912.5 Da) and DPNAALPAGPR (1,148.6 Da), and their IC₅₀ values were 153.4 and 95.7 μM, respectively. The results of this study therefore indicate that leukocyte extract of C. siamensis contains peptides with antioxidant activity which could be used as a novel antioxidant.     

A signature of maternal anti-fetal rejection in spontaneous preterm birth: chronic chorioamnionitis, anti-human leukocyte antigen antibodies, and C4d

Background

Chronic chorioamnionitis is found in more than one-third of spontaneous preterm births. Chronic chorioamnionitis and villitis of unknown etiology represent maternal anti-fetal cellular rejection. Antibody-mediated rejection is another type of transplantation rejection. We investigated whether there was evidence for antibody-mediated rejection against the fetus in spontaneous preterm birth.

Methods and Findings

This cross-sectional study included women with (1) normal pregnancy and term delivery (n = 140) and (2) spontaneous preterm delivery (n = 140). We analyzed maternal and fetal sera for panel-reactive anti-HLA class I and class II antibodies, and determined C4d deposition on umbilical vein endothelium by immunohistochemistry. Maternal anti-HLA class I seropositivity in spontaneous preterm births was higher than in normal term births (48.6% vs. 32.1%, p = 0.005). Chronic chorioamnionitis was associated with a higher maternal anti-HLA class I seropositivity (p<0.01), significant in preterm and term birth. Villitis of unknown etiology was associated with increased maternal and fetal anti-HLA class I and II seropositivity (p<0.05, for each). Fetal anti-HLA seropositivity was closely related to maternal anti-HLA seropositivity in both groups (p<0.01, for each). C4d deposition on umbilical vein endothelium was more frequent in preterm labor than term labor (77.1% vs. 11.4%, p<0.001). Logistic regression analysis revealed that chronic chorioamnionitis (OR = 6.10, 95% CI 1.29–28.83), maternal anti-HLA class I seropositivity (OR = 5.90, 95% CI 1.60–21.83), and C4d deposition on umbilical vein endothelium (OR = 36.19, 95% CI 11.42–114.66) were associated with preterm labor and delivery.

Conclusions

A major subset of spontaneous preterm births has a signature of maternal anti-fetal cellular and antibody-mediated rejections with links to fetal graft-versus-host disease and alloimmune reactions.     

Full-Length Human Placental sFlt-1-e15a Isoform Induces Distinct Maternal Phenotypes of Preeclampsia in Mice

Objective

Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring.

Methods

Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia.

Results

Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3±51.7μg/mg vs. 19.3±5.6μg/mg, p = 4.4x10^-2; GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2x10^-2). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR).

Conclusions

A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.     

In Vivo Experiments Reveal the Good,the Bad and the Ugly Faces of sFlt-1 in Pregnancy

Objective

Soluble fms-like tyrosine kinase (sFlt)-1-e15a, a primate-specific sFlt-1-isoform most abundant in the human placenta in preeclampsia, can induce preeclampsia in mice. This study compared the effects of full-length human (h)sFlt-1-e15a with those of truncated mouse (m)sFlt-1(1-3) used in previous preeclampsia studies on pregnancy outcome and clinical symptoms in preeclampsia.

Methods

Mice were injected with adenoviruses or fiber-mutant adenoviruses overexpressing hsFlt-1-e15a, msFlt-1(1-3) or control GFP under the CMV or CYP19A1 promoters on gestational day 8 (GD8) and GD11. Placentas and pups were delivered by cesarean section, and dams were monitored postpartum. Blood pressure was telemetrically recorded. Urine samples were collected with cystocentesis and examined for albumin/creatinine ratios. Tissue specimens were evaluated for transgene as well as endogenous mFlt-1 and msFlt-1-i13 expression. H&E-, Jones- and PAS-stained kidney sections were histopathologically examined. Placental GFP expression and aortic ring assays were investigated with confocal microscopy.

Results

Mean arterial blood pressure (MAP) was elevated before delivery in hsFlt-1-e15a-treated mice compared to controls (GD18: ΔMAP = 7.8 mmHg, p = 0.009), while ΔMAP was 12.8 mmHg (GD18, p = 0.005) in msFlt-1(1-3)-treated mice. Urine albumin/creatinine ratio was higher in hsFlt-1-e15a-treated mice than in controls (GD18, p = 0.04; PPD8, p = 0.03), and msFlt-1(1-3)-treated mice had marked proteinuria postpartum (PPD8, p = 4×10⁻⁵). Focal glomerular changes were detected in hsFlt-1-e15a and msFlt-1(1-3)-treated mice. Aortic ring microvessel outgrowth was decreased in hsFlt-1-e15a (p = 0.007) and msFlt-1(1-3)-treated (p = 0.02) mice. Full-length msFlt-1-i13 expression was unique for the placenta. In hsFlt-1-e15a-treated mice, the number of pups (p = 0.046), total weight of living pups (p = 0.04) and maternal weights (p = 0.04) were higher than in controls. These differences were not observed in truncated msFlt-1(1-3)-treated mice.

Conclusions

Truncated msFlt-1(1-3) simulated the preeclampsia-promoting effects of full-length hsFlt-1. MsFlt-1(1-3) had strong effect on maternal endothelium but not on placentas and embryos. In contrast, hsFlt-1-e15a induced preeclampsia-like symptoms; however, it also increased litter size. In accord with the predominant placental expression of hsFlt-1-e15a and msFlt-1-i13, full-length sFlt-1 may have a role in the regulation of embryonic development. These observations point to the difference in the biological effects of full-length and truncated sFlt-1 and the changes in the effect of full-length sFlt-1 during pregnancy, and may have important implications in the management of preeclampsia.