Binding of Hepatitis B Virus Pre-S1 Domain-Derived Synthetic Myristoylated Peptide to Scavenger Receptor Class B Type 1 with Differential Properties from Sodium Taurocholate Cotransporting Polypeptide

(1) Background: The myristoylated pre-S1 peptide (Myr47) synthesized to mimic pre-S1 domain (2-48) in large (L) surface protein of hepatitis B virus (HBV) prevents HBV infection to hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP). We previously demonstrated that yeast-derived nanoparticles containing L protein (bio-nanocapsules: BNCs) bind scavenger receptor class B type 1 (SR-B1). In this study, we examined the binding of Mry47 to SR-B1. (2) Methods: The binding and endocytosis of fluorescence-labeled Myr47 to SR-B1 (and its mutants)-green fluorescence protein (GFP) fusion proteins expressed in HEK293T cells were analyzed using flow cytometry and laser scanning microscopy (LSM). Various ligand-binding properties were compared between SR-B1-GFP and NTCP-GFP. Furthermore, the binding of biotinylated Myr47 to SR-B1-GFP expressed on HEK293T cells was analyzed via pull-down assays using a crosslinker and streptavidin-conjugated beads. (3) Conclusions: SR-B1 bound not only Myr47 but also its myristoylated analog and BNCs, but failed to bind a peptide without myristoylation. However, NTCP only bound Myr47 among the ligands tested. Studies using SR-B1 mutants suggested that both BNCs and Myr47 bind to similar sites of SR-B1. Crosslinking studies indicated that Myr47 binds preferentially SR-B1 multimer than monomer in both HEK293T and HepG2 cells.

Pretreatment of Garlic Oil Extracts Hampers Epithelial Damage in Cell Culture Model of Peptic Ulcer Disease

Background and Objectives: Peptic ulcer disease is a chronic disease affecting up to 10% of the world’s population. Proton pump inhibitors, such as lansoprazole are the gold standard in the treatment of ulcer disease. However, various studies have shown the effectiveness of garlic oil extracts in the treatment of ulcer disease. A cellular model can be established in the human gastric cell line by sodium taurocholate. The aim of this study was to explore the effects of garlic oil extracts pretreatment and LPZ addition in the cell culture model of peptic ulcer disease by examining oxidative stress and F-actin distribution. Materials and Methods: Evaluation was performed by determination of glutathione and prostaglandin E2 concentrations by ELISA; human gastric cell line proliferation by cell counting; expression of ATP-binding cassette, sub-family G, member 2; nuclear factor kappa B subunit 2 by RT PCR; and F-actin cytoskeleton visualization by semi-quantification of Rhodamine Phalloidin stain. Results: Our results showed significant reduction of cell damage after sodium taurocholate incubation when the gastric cells were pretreated with lansoprazole (p < 0.001) and increasing concentrations of garlic oil extracts (p < 0.001). Pretreatment with lansoprazole and different concentrations of garlic oil extracts increased prostaglandin E2 and glutathione concentrations in the cell culture model of peptic ulcer disease (p < 0.001). Positive correlation of nuclear factor kappa B subunit 2 (p < 0.01) with lansoprazole and garlic oil extracts pretreatment was seen, while ATP-binding cassette, sub-family G, member 2 expression was not changed. Treatment with sodium taurocholate as oxidative stress on F actin structure was less pronounced, although the highest concentration of garlic oil extracts led to a statistically significant increase of total amount of F-actin (p < 0.001). Conclusions: Hence, pretreatment with garlic oil extracts had gastroprotective effect in the cell model of peptic ulcer disease. However, further experiments are needed to fully elucidate the mechanism of this protective role.

Presence of sodium taurocholate co-transporting polypeptide and Hepatitis B replication marker on placenta: Another home for the virus

Background: The role of sodium taurocholate co-transporting polypeptide (NTCP), in facilitating the binding of Hepatitis B virus (HBV) on surface of hepatocytes is well documented. Expression of NTCP in extra hepatic cells may make these cells susceptible to HBV infection and support cellular proliferation akin to hepatocytes. Placental replication of HBV is not well explored. In this study we have assessed the expression of NTCP and HBV replication markers (HBeAg, HBcAg, and HBV DNA) in placental cells, to investigate if these cells act as host for HBV. Methods: Fourty one HBsAg+ve pregnant women along with 10 healthy controls were enrolled after obtaining informed consent. The HBV DNA in placenta was detected by qPCR using primers for X and core ORF. Expression of NTCP in placenta was analyzed by qRT-PCR and further investigated by immunohistochemistry (IHC) along with HBV replication biomarkers, HBeAg, and HBcAg. Results: HBsAg positive subjects were divided in two groups on the basis of viral load [High Viral Load (HVL) Group; viral load ≥2000IU/ml, Low Viral Load (LVL) Group; viral load <2000IU/ml] according to INASL guidelines 2018. HBV infected females showed increased expression of NTCP in trophoblasts of placenta compared to control group (HVL 3.69,SE 0.13 Vs Control 1.74,SE 0.15, p=0.0117). Furthermore, significant difference in NTCP expression was also observed between HVL and LVL group (HVL 3.69,SE 0.13 Vs LVL 1.98,SE 0.17, p=0.022) and positively correlated with the maternal HBV DNA load. Membranous and/or cytoplasmic immunostaining of NTCP, and cytoplasmic staining of HBeAg and HBcAg in trophoblasts along with presence of HBV DNA indicated that trophoblasts are not only susceptible to HBV infection but may also be a site for viral replication. Conclusions: This is the pioneer study, which demonstrates expression of NTCP on placenta which may facilitate the entry of HBV. Furthermore, the study establishes the presence of HBeAg in placenta of patients without circulating HBeAg, indicating these cells may act as replication host/reservoir. This pioneering finding hints at the possibility of exploring the potential of NTCP blocking strategies in preventing vertical transmission of HBV.

Changes in noradrenaline and dopamine levels under oxygen debt conditions in the brain of rats during experimental acute pancreatitis

This study’s aim was to assess the level of catecholamines, i.e., noradrenaline and dopamine, under oxygen debt conditions in the brain of experimental animals in which acute pancreatitis was experimentally induced. Catecholamines play the role of neurotransmitters and neuromediators. They are responsible for the regulation of motor and emotional processes, take part in the regulation of hormonal activities, sleep, wakefulness, concentration, attention, and learning processes. The experiment also determined the oxygen tension as an indicator of respiratory failure and the activity of amylase and lipase in the development of the inflammatory process. The animals on which the experiment was conducted were Wistar rats (140 animals) divided into 3 research groups: control (C) animals (n = 30), healthy (H) animals (n = 30), and operated (O) animals (n = 80). The determination of amylase, lipase, oxygen pressure, NA, and DO levels were performed at hours 2, 6, 12, 24, and 48 of the experiment. The animals in group C had an injection needle inserted to investigate only the effects of mechanical damage to the organs. On the other hand, the animals in group O had a 5% solution of sodium taurocholate introduced into the common bile-pancreatic duct. The research conducted shows that the most significant changes in NA and DO levels were observed on the first day of the experiment. The concentrations of the above catecholamines were statistically significantly correlated with the level of amylase in the blood. The peak of dopamine was observed between the 6th and 12th hours of the experiment, while the lowest concentration of noradrenaline was observed at the 6th hour of the experiment.

Importance of Bile Composition for Diagnosis of Biliary Obstructions

Determination of the cause of a biliary obstruction is often inconclusive from serum analysis alone without further clinical tests. To this end, serum markers as well as the composition of bile of 74 patients with biliary obstructions were determined to improve the diagnoses. The samples were collected from the patients during an endoscopic retrograde cholangiopancreatography (ERCP). The concentration of eight bile salts, specifically sodium cholate, sodium glycocholate, sodium taurocholate, sodium glycodeoxycholate, sodium chenodeoxycholate, sodium glycochenodeoxycholate, sodium taurodeoxycholate, and sodium taurochenodeoxycholate as well as bile cholesterol were determined by HPLC-MS. Serum alanine aminotransferase (ALT), aspartate transaminase (AST), and bilirubin were measured before the ERCP. The aim was to determine a diagnostic factor and gain insights into the influence of serum bilirubin as well as bile salts on diseases. Ratios of conjugated/unconjugated, primary/secondary, and taurine/glycine conjugated bile salts were determined to facilitate the comparison to literature data. Receiver operating characteristic (ROC) curves were determined, and the cut-off values were calculated by determining the point closest to (0,1). It was found that serum bilirubin was a good indicator of the type of biliary obstruction; it was able to differentiate between benign obstructions such as choledocholithiasis (at the concentration of >11 µmol/L) and malignant changes such as pancreatic neoplasms or cholangiocarcinoma (at the concentration of >59 µmol/L). In addition, it was shown that conjugated/unconjugated bile salts confirm the presence of an obstruction. With lower levels of conjugated/unconjugated bile salts the possibility for inflammation and, thus, neoplasms increase.

Na+-Taurocholate Co-Transporting Polypeptide (NTCP) in Livers, Function, Expression Regulation, and Potential in Hepatitis B Treatment

Chronic hepatitis B virus (HBV) infection has become one of the leading causes of liver cirrhosis and hepatocellular carcinoma globally. The discovery of sodium taurocholate co-transporting polypeptide (NTCP), a solute carrier, as a key receptor for HBV and hepatitis D virus (HDV) has opened new avenues for HBV treatment. Additionally, it has led researchers to generate hepatoma cell lines (including HepG2-NTCP and Huh-7-NTCP) susceptible to HBV infection in vitro, hence, paving the way to develop and efficiently screen new and novel anti-HBV drugs. This review summarizes the history, function and critical findings regarding NTCP as a viral receptor for HBV/HDV, and it also discusses recently developed drugs targeting NTCP.

1037. Efficacy of Germinants and Omadacycline for Preventing Clostridioides difficile Relapse in a Murine Model

Background Clostridioides difficile is labeled one of five urgent pathogens by the CDC. The urgency is related to the high burden of disease, limited effective antimicrobials, and recurrent C. difficile infections (rCDI) from residual spores (Fig. 1). Impervious to antibiotics, C. difficile spores could be induced into vegetative cells by germinants, namely taurocholate, for antibiotic targeting. This study aims to evaluate spore reservoir eradication through applying germinants with antibiotics. Figure 1. Schematic of the infectious life cycle of C. difficile and treatment opportunities Noah Budi, Nasia Safdar, Warren E Rose, Treatment issues in recurrent Clostridioides difficile infections and the possible role of germinants, FEMS Microbes, Volume 1, Issue 1, September 2020, xtaa001, https://doi.org/10.1093/femsmc/xtaa001 Methods A published murine model of rCDI using C57BL/6 mice and 1 x 105C. difficile spores (VPI 10463) with modification was used (Fig. 2). Six hours after inoculation, mice received 1.5 mg vancomycin (VAN, n=10) or 0.25 mg omadacycline (OMC, n=10) daily by oral gavage until day 4 or either with germinant (G) solution (8 mg of sodium taurocholate, 10 mg of taurine, 0.2 mg of sodium docusate, and 1.72 mg of calcium gluconate) given concomitantly on days 1 to 3 (OMC+G, n=9 and VAN+G, n=8). As a positive control, five mice did not receive antibiotics after spores. To induce rCDI, clindamycin was given on days 10 to 12. Survival, clinical scoring (CS), and weight loss (WL) were recorded until day 15. Fecal samples were taken to measure toxin production and spore shedding. Mice that died prior to day 15, were too sick to provide samples, or had positive stool culture were considered positive for day 15 spore shedding. Fisher’s exact test was used. Figure 2: Experimental Design Antibiotic water consisted of kanamycin 0.4 mg/ml, gentamicin 0.035 mg/mL, colistin 850 U/mL, metronidazole 0.215 mg/mL, and vancomycin 0.045 mg/mL given noon to noon on specified days. Clindamycin IP injections given as weight based dose of 10 mg/kg. Results Survival is summarized in Figure 3. Both OMC and VAN had 60% survival by day 15 while OMC+G and VAN+G had 100% (p=0.004). Germinant CS and WL were similar to respective antibiotic alone groups until day 8; OMC overall had less severe disease than VAN (Figure 4). Toxin production on day 10 was lower in OMC than VAN, but absent from OMC+G and VAN+G. On day 15, 100% of VAN mice were spore positive compared to 60% with OMC (p=0.087). No mice receiving germinants (OMC+G or VAN+G) were spore positive (p&lt; 0.0001). Figure 3. Survival Percentage Figure 4. Clinical Scoring and Weight Loss Conclusion Germinant/antibiotic combinations improved survival in a rCDI mouse model compared to antibiotics alone. Germinants did not induce toxin production when combined with OMC or VAN and eliminated the spore reservoir at the end of treatment. This provides basis for further study of germinants combined with antibiotics to reduce rCDI. Disclosures Warren Rose, PharmD, MPH, Merck (Grant/Research Support)Paratek (Grant/Research Support, Advisor or Review Panel member)

Selective binding of small molecules to Vibrio cholerae DsbA offers a starting point for the design of novel antibacterials

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446 ± 10 µM. The same benzimidazole compound has ~8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD > 3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveals the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.