Metabolic labelling of cholesteryl glucosides in Helicobacter pylori reveals how the uptake of human lipids enhances bacterial virulence.

Helicobacter pylori infects approximately half of the human population and is the main cause of various gastric diseases. This pathogen is auxotrophic for cholesterol, which it converts upon uptake to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl and 6'-phosphatidyl α-glucosides (CAGs and CPGs). Owing to a lack of sensitive analytical methods, it is not known if CAGs and CPGs play distinct physiological roles or how the acyl chain component affects function. Herein we established a metabolite-labelling method for characterising these derivatives qualitatively and quantitatively with a femtomolar detection limit. The development generated an MS/MS database of CGds, allowing for profiling of all the cholesterol-derived metabolites. The subsequent analysis led to the unprecedented information that these bacteria acquire phospholipids from the membrane of epithelial cells for CAG biosynthesis. The resulting increase in longer or/and unsaturated CAG acyl chains helps to promote lipid raft formation and thus delivery of the virulence factor CagA into the host cell, supporting the idea that the host/pathogen interplay enhances bacterial virulence. These findings demonstrate an important connection between the chain length of CAGs and the bacterial pathogenicity

Cholesteryl α-D-glucoside 6-acyltransferase enhances the adhesion of Helicobacter pylori to gastric epithelium.

Helicobacter pylori, the most common etiologic agent of gastric diseases including gastric cancer, is auxotrophic for cholesterol and has to hijack it from gastric epithelia. Upon uptake, the bacteria convert cholesterol to cholesteryl 6'-O-acyl-α-D-glucopyranoside (CAG) to promote lipid raft clustering in the host cell membranes. However, how CAG appears in the host to exert the pathogenesis still remains ambiguous. Herein we identified hp0499 to be the gene of cholesteryl α-D-glucopyranoside acyltransferase (CGAT). Together with cholesteryl glucosyltransferase (catalyzing the prior step), CGAT is secreted via outer membrane vesicles to the host cells for direct synthesis of CAG. This significantly enhances lipid rafts clustering, gathers adhesion molecules (including Lewis antigens and integrins α5, β1), and promotes more bacterial adhesion. Furthermore, the clinically used drug amiodarone was shown as a potent inhibitor of CGAT to effectively reduce the bacterial adhesion, indicating that CGAT is a potential target of therapeutic intervention

A Systematic Study of the Stability, Safety, and Efficacy of the de novo Designed Antimicrobial Peptide PepD2 and Its Modified Derivatives Against Acinetobacter baumannii

Searching for new antimicrobials is a pressing issue to conquer the emergence of multidrug-resistant (MDR) bacteria and fungi. Antimicrobial peptides (AMPs) usually have antimicrobial mechanisms different from those of traditional antibiotics and bring new hope in the discovery of new antimicrobials. In addition to antimicrobial activity, stability and target selectivity are important concerns to decide whether an antimicrobial peptide can be applied in vivo. Here, we used a simple de novo designed peptide, pepD2, which contains only three kinds of amino acid residues (W, K, L), as an example to evaluate how the residues and modifications affect the antimicrobial activity against Acinetobacter baumannii, stability in plasma, and toxicity to human HEK293 cells. We found that pepI2 with a Leu→Ile substitution can decrease the minimum bactericidal concentrations (MBC) against A. baumannii by one half (4 μg/mL). A D-form peptide, pepdD2, in which the D-enantiomers replaced the L-enantiomers of the Lys(K) and Leu(L) residues, extended the peptide half-life in plasma by more than 12-fold. PepD3 is 3-residue shorter than pepD2. Decreasing peptide length did not affect antimicrobial activity but increased the IC50 to HEK293 cells, thus increased the selectivity index (SI) between A. baumannii and HEK293 cells from 4.7 to 8.5. The chain length increase of the N-terminal acyl group and the Lys→Arg substitution greatly enhanced the hemolytic activity, hence those modifications are not good for clinical application. Unlike colistin, the action mechanism of our peptides relies on negatively charged lipids rather than lipopolysaccharides. Therefore, not only gram-negative bacteria but also gram-positive bacteria can be killed by our peptides

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Application of controllable gene expression system in hair growth

基因轉移到毛囊是一種有潛力的技術來治療皮膚疾病。因為皮膚有非常低的滲透性，所以在活體的皮膚毛囊細胞上，非病毒基因轉殖才被認為是一種困難的方法。在此，我們利用聚乙烯亞胺 (PEI)來包覆人類的端粒酶逆轉錄酶基因 (hTERT)，然後利用塗抹在皮膚表面的方式，可以成功的將hTERT這個基因送到正常大鼠皮膚組織和受傷過的皮膚組織的毛囊細胞，讓這些細胞表現hTERT這個基因。我們發現，在正常皮膚組織的毛囊中，hTERT的表達會使毛囊幹細胞增生，進而加速毛髮的生長。另一方面，在受傷的皮膚組織的毛囊中，hTERT的表達則是會造成毛囊的再生。本研究利用創新的基因轉移的方法，並提供了一種實際的解決方法治療皮膚疾病。Gene transfer to hair follicles is a potential technology to treat skin disease. Non-viral transfection is considered a difficult method to deliver genes into hair follicles in vivo because of the extremely low permeability of skin. We utilized polyethylenimine (PEI) to transfect the human telomerase reverse transcriptase (hTERT) in hair follicle cells in normal and wounding rat skin tissues. We found that expression of hTERT in normal skin tissue could increase the number of proliferating stem cells in hair follicles. On the other hand, expression of hTERT in wounding skin tissue can promote hair follicle de novo formation. These studies might not only reveal an innovative method for gene transfer, but also provide a practical solution for skin disease therapy.目 錄試委員會審定書………………………………………………………………… i謝………………………………………………………………………………….. ii文摘要……………………………………………………………………….. iii文摘要…………………………………………………………………………….. iv. Introduction：…………………………………………………………………. 1~2. Material and method：…………………………………………..…………. 3~13 .1 Antibodies and Chemicals. ………………………………………………. 3~4.2 Magnetic Labeling and Separation of Cell………………………………. 4?5.3 Flow cytometry…………………………………………………………... 5~6.4 Cell culture of hair follicle stem cell…………………………………… 6~8.5 Immunofluorescent staining ………………………...………………… 8~9.6 Colony formation…………………………………………………………... 9.7 Transfection in vitro……………………………………………………… 9~10.8 Cytotoxicity - MTT assay………………………………………………. 10~11.9 Histochemical assay of ß-galactosidase activity…………………………… 11.10 In vivo mouse hair follicle transfection………………………………………. 11.11 Immunohistochemistry staining and H&E staining……………………… 12.12 Counting of hair length……………………………………………………12~13.13 Whole-mount hair follicle neogenesis assay. ………………………… 13. Result……………………………………………………………………… 13~22 .1 Purification of hair follicle stem cells…………………………………… 13~15.2 In vitro transfection efficiency and cytotoxicity of PEI…………………..15~16.3 The function of hTERT in vitro in rat keratinocyte……………………… 17~18.4 In vivo transfection efficiency of PEI………………………… 18~19 .5 The function of hTERT in vivo in rat skin tissue…………….…………...… 19~21.6 hTERT promote de novo follicle formation. ………...…………21~22. Discussions……………………………………………………………… 22~25.1 PEI serves as a promising vector for gene therapy in skin disease……… 22~23.2 TERT serve as a hair growth enhancer…………………………………..23~24.3 TERT and Wnt-signaling pathway………………………………24~25. Conclusion …………………………………………………………………25. Reference ……………………………………………………………… 26~27錄……………………………………………………………………….….…. 28~42 目錄igure 1. Hair follicle stem cells were sorted by MACS………….………28igure 2. The expression of integrin α6…………………………...…………………29igure 3. The expression of integrin ß1……………………………………………30igure 4. The expression of CD34…………………………………………………38igure 5. Clonal growth assays of keratinocytes…………………………………….39igure 6. EGFP reported gene expression in hair follicle stem cells…………….…40igure 7. hTERT reported gene expression in hair follicle stem cells………....41~42igure 8. Cytotoxicity test and transfection efficiency in hair follicle stem cells.…43.igure 9. In vivo transfection of rat hair follicles…………………………………….44igure 10. H&E stain and length of hair after in vivo transfection………………….45igure 11 Immunofluorescent staining for PCNA………………………………….46igure 12. Expression of hTERT potentiates de novo follicle formation at day10......47igure 13. Expression of hTERT potentiates de novo follicle formation at day 40…………………………………………………………………………………48~4

Changes in Near-Infrared Spectroscopy After Congenital Cyanotic Heart Surgery

BackgroundSince oxygen saturation from pulse oximetry (SpO2) and partial pressure of arterial oxygen (PaO2) are observed to improve immediately after surgical correction of cyanotic congenital heart disease (CHD), we postulate that cerebral (CrO2) and somatic (SrO2) oximetry also improves immediately post-correction. We aim to prospectively examine CrO2 and SrO2, before, during, and after surgical correction as well as on hospital discharge in children with cyanotic CHD to determine if and when these variables increase.MethodsThis is a prospective observational trial. Eligibility criteria included children below 18 years of age with cyanotic CHD who required any cardiac surgical procedure. CrO2 and SrO2 measurements were summarized at six time-points for comparison: (1) pre-cardiopulmonary bypass (CPB); (2) during CPB; (3) post-CPB; (4) Day 1 in the pediatric intensive care unit (PICU); (5) Day 2 PICU; and (6) discharge. Categorical and continuous variables are presented as counts (percentages) and median (interquartile range), respectively.ResultsTwenty-one patients were analyzed. 15 (71.4%) and 6 (28.6%) patients underwent corrective and palliative surgeries, respectively. In the corrective surgery group, SpO2 increased immediately post-CPB compared to pre-CPB [99 (98, 100) vs. 86% (79, 90); p < 0.001] and remained in the normal range through to hospital discharge. Post-CPB CrO2 did not change from pre-CPB [72.8 (58.8, 79.0) vs. 72.1% (63.0, 78.3); p = 0.761] and even decreased on hospital discharge [60.5 (53.6, 62.9) vs. 72.1% (63.0, 78.3); p = 0.005]. Post-CPB SrO2 increased compared to pre-CPB [87.3 (77.2, 89.5) vs. 72.7% (65.6, 77.3); p = 0.001] but progressively decreased during PICU stay to a value lower than baseline at hospital discharge [66.9 (57.3, 76.9) vs. 72.7% (65.6, 77.3); p = 0.048].ConclusionCrO2 and SrO2 did not increase after corrective surgery of cyanotic CHD even up to hospital discharge. Future larger studies are required to validate these findings. (This study is registered with ClinicalTrials.gov ID: NCT02417259.

Impact of a nurse-led feeding protocol in a pediatric intensive care unit

Background: To determine effectiveness of a nurse-led, volume-based feeding protocol in our pediatric intensive care unit (PICU), we evaluated patients’ nutrition adequacy pre- and post-protocol implementation. Methods: We conducted an observational study of patients admitted for more than three days in the PICU during pre- and post-feeding protocol periods. We recorded energy and protein intake and feed interruptions in patients started on enteral nutrition over the first seven days of admission. We excluded patients with septic shock requiring more than two inotropes, post-cardiac and post-gastrointestinal surgeries. To determine nutrition adequacy, actual energy and protein intakes were compared with calculated requirements, expressed as percentages. Results: We had a total of 40 patients (20 in the pre- and post-protocol groups, respectively) with median age of 9.4 (interquartile range (IQR) 2.8, 57) months. Median time to feed initiation was similar between groups (20.0 (IQR 17.0, 37.5) vs. 21.5 (IQR 10.5, 27.0) hours, p = 0.516). There was no difference in median energy (55 (IQR 12, 102) vs. 59 (IQR 25, 85) %, p = 0.645) and protein intake (53 (IQR 16, 124) vs. 73 (IQR 22, 137) %, p = 0.069) over the seven-day period between groups; the proportion of patients meeting their energy (10 vs. 35%, p = 0.127) and protein goal (15 vs. 30%, p = 0.451) by day three also did not differ significantly pre- and post-protocol implementation. The most common reasons for feed interruption were intubation/extubation and radiological procedures. Conclusion: Our current feeding protocol did not improve nutrient adequacy. The effectiveness of a more aggressive protocol in units where enteral nutrition is initiated within 24 hours should be investigated

Incidence of Group B Streptococcus early onset sepsis in term neonates with second-line prophylaxis maternal intrapartum antibiotics: A multicenter retrospective study

10.1016/j.ajog.2023.10.035American Journal of Obstetrics and Gynecolog