Heterogenous Expression and Purification of Lipid II Flippase from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target. OBJECTIVE: In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment. METHODS: SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied. RESULTS: SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ. CONCLUSION: The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer.

Development of a bioengineered Erwinia chrysanthemi asparaginase to enhance its anti-solid tumor potential for treating gastric cancer.

Asparaginase has been traditionally applied for only treating acute lymphoblastic leukemia due to its ability to deplete asparagine. However, its ultimate anticancer potential for treating solid tumors has not yet been unleashed. In this study, we bioengineered Erwinia chrysanthemi asparaginase (ErWT), one of the US Food and Drug Administration-approved types of amino acid depleting enzymes, to achieve double amino acid depletions for treating a solid tumor. We constructed a fusion protein by joining an albumin binding domain (ABD) to ErWT via a linker (GGGGS)5 to achieve ABD-ErS5. The ABD could bind to serum albumin to form an albumin-ABD-ErS5 complex, which could avoid renal clearance and escape from anti-drug antibodies, resulting in a remarkably prolonged elimination half-life of ABD-ErS5. Meanwhile, ABD-ErS5 did not only deplete asparagine but also glutamine for ∼2 weeks. A biweekly administration of ABD-ErS5 (1.5 mg/kg) significantly suppressed tumor growth in an MKN-45 gastric cancer xenograft model, demonstrating a novel approach for treating solid tumor depleting asparagine and glutamine. Multiple administrations of ABD-ErS5 did not cause any noticeable histopathological abnormalities of key organs, suggesting the absence of acute toxicity to mice. Our results suggest ABD-ErS5 is a potential therapeutic candidate for treating gastric cancer.

Increased Prevalence of Alcohol-Related Gastrointestinal and Liver Diseases During the COVID-19 Pandemic.

BACKGROUND: Higher prevalence of alcohol-related gastrointestinal (GI) and liver diseases (ARGLDs) were anecdotally reported during the COVID-19 pandemic, but little published evidence exists. METHODS: A healthcare system audit of inpatient GI consults was performed during the pandemic's lockdown phase (3/23/2020-5/10/2020, n=558) and reopening phase (6/1/2020-7/19/2020, n=711) with comparison to those timeframes in 2019. RESULTS: Consult volume decreased by 27.7% during the lockdown, but the proportion of ARGLDs increased by 59.6% (p=0.03). This trend continued during reopening, with potentially more severe disease as more patients required endoscopic intervention. Patients with alcoholic hepatitis during reopening were younger compared to the lockdown. CONCLUSIONS: Our study demonstrates increased prevalence and severity of ARGLDs amongst younger individuals during the COVID-19 pandemic. This increase started during the lockdown but worsened despite relaxation of restrictions. Systems to increase screening for and treatment of alcohol use disorder as society recovers from the pandemic remain imperative.

CON: Should patients with nonalcoholic steatohepatitis fibrosis undergo bariatric surgery as a primary treatment?

Content available: Author Interview and Audio Recording.

A Predictive Model for the Diagnostic and Therapeutic Yield of Colonoscopy Performed for Lower Gastrointestinal Bleeding.

BACKGROUND: Although colonoscopy is routinely performed for the management of lower gastrointestinal bleeding (LGIB), the quality of evidence supporting its use is poor and its yield for active bleeding or malignancy is low in practice. METHODS: We conducted a retrospective analysis of all adult patients who underwent colonoscopies for LGIB at our hospital system between January 1, 2015, and December 31, 2019. A statistical model was built on a cohort of 5195 cases using multiple logistic regressions to predict the detection of various colonoscopy findings and the use of different colonoscopy maneuvers. The model was converted into a risk scoring system, named the TYPICAL Index, and was validated against a separate cohort of 914 cases. RESULTS: Active bleeding was only seen in 3.8% of colonoscopies performed for LGIB and endoscopic hemostasis was applied in 43.7% of actively bleeding lesions. Malignant-appearing lesions were detected in 2.5% of LGIB procedures and all cases of proximal colonic mass were identified in subjects age above 60. The TYPICAL Index, derived from age, gender, hemoglobin, creatinine, international normalized ratio, and prior colonoscopy for LGIB has a concordance statistic of 0.71 and a negative predictive value of 93.8% for potential bleeding sources requiring hemostasis, malignant-appearing lesions, or active bleeding on colonoscopy when applied using a threshold of 6. CONCLUSIONS: Active bleeding and malignancies are rarely encountered and endoscopic hemostasis is seldom required during colonoscopy performed for LGIB. We developed and validated a risk scoring system to identify cases of low predicted diagnostic and therapeutic yield and to guide clinical decision-making.

NPO at Midnight: Reassessing Unnecessary Pre-Endoscopy Fasting.

INTRODUCTION: Endoscopy with sedation is a common inpatient procedure. "NPO after midnight" remains the prevailing fasting practice despite ASA guidelines indicating specific fasting times. This quality improvement project aims to assess patient discomfort with the "NPO after midnight" order versus implementation of specific NPO times. METHODS: Patients in the inpatient wards scheduled for endoscopy after 1 pm the following day were recruited. The gastroenterology services designated specific NPO times per ASA guidelines for the post-intervention group. Each participant completed a survey qualifying hunger, thirst, and discomfort levels. Pearson's chi-squared analysis was performed. RESULTS: NPO duration was reduced in the post-intervention group with significant improvement in thirst, hunger, and discomfort levels. A shortened preoperative fasting period did not lead to increase in procedural complications. CONCLUSION: Despite ASA guidelines, the practice of keeping patients NPO after midnight remains pervasive, resulting in unnecessarily prolonged fasting and patient discomfort. Implementing specific diet recommendations reduces duration of NPO and improves patient comfort and overall satisfaction.

Clinical implications, diagnosis, and management of diabetes in patients with chronic liver diseases.

Diabetes mellitus (DM) negatively affects the development and progression of chronic liver diseases (CLD) of various etiologies. Concurrent DM and CLD are also associated with worse clinical outcomes with respect to mortality, the occurrence of hepatic decompensation, and the development of hepatocellular carcinoma (HCC). Unfortunately, early diagnosis and optimal treatment of DM can be challenging, due to the lack of established clinical guidelines as well as the medical complexity of this patient population. We conducted an exploratory review of relevant literature to provide an up-to-date review for internists and hepatologists caring for this patient population. We reviewed the epidemiological and pathophysiological associations between DM and CLD, the impact of insulin resistance on the progression and manifestations of CLD, the pathogenesis of hepatogenic diabetes, as well as the practical challenges in diagnosis and monitoring of DM in this patient population. We also reviewed the latest clinical evidence on various pharmacological antihyperglycemic therapies with an emphasis on liver disease-related clinical outcomes. Finally, we proposed an algorithm for managing DM in patients with CLD and discussed the clinical and research questions that remain to be addressed.

Correction: Aspartate ß-hydroxylase expression promotes a malignant pancreatic cellular phenotype.

[This corrects the article DOI: 10.18632/oncotarget.2840.].

Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase.

We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1), heat shock protein 90 (HSP90), and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.

Anti-Tumor Effects of Second Generation ß-Hydroxylase Inhibitors on Cholangiocarcinoma Development and Progression.

Cholangiocarcinoma (CCA) has a poor prognosis due to widespread intrahepatic spread. Aspartate ß-hydroxylase (ASPH) is a transmembrane protein and catalyzes the hydroxylation of aspartyl and asparaginyl residues in calcium binding epidermal growth factor (cbEGF)-like domains of various proteins, including Notch receptors and ligands. ASPH is highly overexpressed (>95%) in human CCA tumors. We explored the molecular mechanisms by which ASPH mediated the CCA malignant phenotype and evaluated the potential of ASPH as a therapeutic target for CCA. The importance of expression and enzymatic activity of ASPH for CCA growth and progression was examined using shRNA "knockdown" and a mutant construct that reduced its catalytic activity. Second generation small molecule inhibitors (SMIs) of ß-hydroxylase activity were developed and used to target ASPH in vitro and in vivo. Subcutaneous and intrahepatic xenograft rodent models were employed to determine anti-tumor effects on CCA growth and development. It was found that the enzymatic activity of ASPH was critical for mediating CCA progression, as well as inhibiting apoptosis. Mechanistically, ASPH overexpression promoted Notch activation and modulated CCA progression through a Notch1-dependent cyclin D1 pathway. Targeting ASPH with shRNAs or a SMI significantly suppressed CCA growth in vivo.

Activation of signal transduction pathways during hepatic oncogenesis.

BACKGROUND AND AIMS: Understanding the molecular pathogenesis of hepatocellular carcinoma (HCC) is essential to identify therapeutic targets. A hepatitis B virus (HBV) related double transgenic murine model was developed. METHODS: Liver specific expression of HBV X protein (HBx) and insulin receptor substrate 1 (IRS1) was achieved and transgenic mice were followed from birth to age 21 months. Liver and tumor tissue were assessed for histologic changes as well as activation of signal transduction pathways by qRT-PCR and multiplex ELISA protein assays. RESULTS: Overexpression of HBx and IRS1 stimulates liver cell proliferation in the double transgenic mice. Only the male mice developed HCC starting at age 15-18 months. The IN/IGF1/IRS1/MAPK/ERK and IN/IGF1/IRS1/PI3K/AKT/GSK3ß cascades were activated early (6-9 months) in the liver followed by WNT/ß-catenin and Notch signaling. Aspartate ß-hydroxylase (ASPH) was found to link these upstream growth factor signaling pathways to downstream Notch activation in tumor tissues. CONCLUSIONS: Sustained overexpression of HBx and IRS1 led to constitutive activation of a tripartite growth factor signal transduction cascade in the liver and was necessary and sufficient to promote HCC development and progression.

Aspartate ß-Hydroxylase expression promotes a malignant pancreatic cellular phenotype.

Pancreatic cancer (PC) is one of the leading causes of cancer related deaths due to aggressive progression and metastatic spread. Aspartate ß-hydroxylase (ASPH), a cell surface protein that catalyzes the hydroxylation of epidermal growth factor (EGF)-like repeats in Notch receptors and ligands, is highly overexpressed in PC. ASPH upregulation confers a malignant phenotype characterized by enhanced cell proliferation, migration, invasion and colony formation in vitro as well as PC tumor growth in vivo. The transforming properties of ASPH depend on enzymatic activity. ASPH links PC growth factor signaling cascades to Notch activation. A small molecule inhibitor of ß-hydroxylase activity was developed and found to reduce PC growth by downregulating the Notch signaling pathway. These findings demonstrate the critical involvement of ASPH in PC growth and progression, provide new insight into the molecular mechanisms leading to tumor development and growth and have important therapeutic implications.

Fluorescent TEM-1 ß-lactamase with wild-type activity as a rapid drug sensor for in vitro drug screening.

We report the development of a novel fluorescent drug sensor from the bacterial drug target TEM-1 ß-lactamase through the combined strategy of Val216→Cys216 mutation and fluorophore labelling for in vitro drug screening. The Val216 residue in TEM-1 is replaced with a cysteine residue, and the environment-sensitive fluorophore fluorescein-5-maleimide is specifically attached to the Cys216 residue in the V216C mutant for sensing drug binding at the active site. The labelled V216C mutant has wild-type catalytic activity and gives stronger fluorescence when ß-lactam antibiotics bind to the active site. The labelled V216C mutant can differentiate between potent and impotent ß-lactam antibiotics and can distinguish active-site binders from non-binders (including aggregates formed by small molecules in aqueous solution) by giving characteristic time-course fluorescence profiles. Mass spectrometric, molecular modelling and trypsin digestion results indicate that drug binding at the active site is likely to cause the fluorescein label to stay away from the active site and experience weaker fluorescence quenching by the residues around the active site, thus making the labelled V216C mutant to give stronger fluorescence in the drug-bound state. Given the ancestor's role of TEM-1 in the TEM family, the fluorescent TEM-1 drug sensor represents a good model to demonstrate the general combined strategy of Val216→Cys216 mutation and fluorophore labelling for fabricating tailor-made fluorescent drug sensors from other clinically significant TEM-type ß-lactamase variants for in vitro drug screening.

A cell-surface ß-hydroxylase is a biomarker and therapeutic target for hepatocellular carcinoma.

UNLABELLED: Hepatocellular carcinoma (HCC) has a poor prognosis as a result of widespread intra- and extrahepatic metastases. There is an urgent need to understand signaling cascades that promote disease progression. Aspartyl-(asparaginyl)-ß-hydroxylase (ASPH) is a cell-surface enzyme that generates enhanced cell motility, migration, invasion, and metastatic spread in HCC. We hypothesize that inhibition of its enzymatic activity could have antitumor effects. Small molecule inhibitors (SMIs) were developed based on the crystal structure of the ASPH catalytic site followed by computer-assisted drug design. Candidate compounds were tested for inhibition of ß-hydroxylase activity and selected for their capability to modulate cell proliferation, migration, invasion, and colony formation in vitro and to inhibit HCC tumor growth in vivo using orthotopic and subcutaneous murine models. The biological effects of SMIs on the Notch signaling cascade were evaluated. The SMI inhibitor, MO-I-1100, was selected because it reduced ASPH enzymatic activity by 80% and suppressed HCC cell migration, invasion, and anchorage-independent growth. Furthermore, substantial inhibition of HCC tumor growth and progression was observed in both animal models. The mechanism(s) for this antitumor effect was associated with reduced activation of Notch signaling both in vitro and in vivo. CONCLUSIONS: These studies suggest that the enzymatic activity of ASPH is important for hepatic oncogenesis. Reduced ß-hydroxylase activity generated by the SMI MO-I-1100 leads to antitumor effects through inhibiting Notch signaling cascade in HCC. ASPH promotes the generation of an HCC malignant phenotype and represents an attractive molecular target for therapy of this fatal disease.

LRH1 promotes pancreatic cancer metastasis.

The transcriptional factor liver receptor homolog 1 (LRH1) regulates pancreatic development, and may participate in pancreatic oncogenesis through activation of growth factor signaling transduction cascades. We measured transcriptional activity of ß-catenin in response to LRH1 stimulation by a Topflash reporter assay. The pancreatic cancer (PC) phenotype was then characterized by cell migration, wound healing, invasion, and sphere formation in vitro, as well as tumor formation and distant metastatic spread in vivo. We compared results between vector control and LRH1-overexpressing stable PC cell lines. In addition, tumor burden, angiogenesis, histologic characteristics, and hepatic spread were assessed in orthotopic and experimental liver metastatic murine models. Expression of downstream LRH1 related genes was evaluated by Western blot and immunohistochemistry in PC cell lines and human tumor specimens. Specific inhibition of LRH1 expression and function was accomplished by shRNAs "knockdown" experiments. It was found that LRH1 enhanced transcriptional activity of ß-catenin and the expression of downstream target genes (c-Myc, MMP2/9), as well as promoted migration, wound healing, invasion, and sphere formation of PC cell lines. Specific inhibition of LRH1 by shRNAs reduced cell migration, invasion, sphere formation and expression of c-Myc and MMP2/9 target genes. Mice injected with LRH1 overexpressing stable PC cells developed tumors with increased size and exhibited striking hepatic metastatic spread. More important, LRH1 was overexpressed in PC tumors compared to adjacent normal pancreas. Our findings demonstrate that LRH1 overexpression is associated with increased PC growth and metastatic spread, indicating that LRH1-targeted therapy could inhibit tumor progression.

LRH1 as a driving factor in pancreatic cancer growth.

Liver receptor homolog 1 (LRH1), directs the development and differentiation of embryonic pancreas, and is overexpressed in pancreatic cancer (PC). We hypothesized that LRH1 promotes PC growth. Cell proliferation and tumorigenicity in nude mice were compared between empty vector-transfected (control) and stable LRH1-overexpressed PC cell lines. The subsequent tumor burden, vasculature development, and histologic features were evaluated. LRH1 overexpression enhanced the expression of downstream target genes (cyclin D1/E1) and stimulated cell proliferation in PC cell lines. LRH1 upregulated cyclin E1 truncated T1/T2 isoforms expression which may occur through ERα-calpain1 signaling. Compared with the control, LRH1 overexpressing stable cells generated tumors with increased weight, proliferation index and enhanced angiogenesis. Cyclin D1/E1 and calpain1 were overexpressed in human PC tumors compared to adjacent normal pancreas. These observations demonstrate that LRH1 promotes PC growth and angiogenesis, suggesting that LRH1 is a driving factor in tumorigenesis and may serve as a potential therapeutic target.

Identification of a new class of FtsZ inhibitors by structure-based design and in vitro screening.

The Filamenting temperature-sensitive mutant Z (FtsZ), an essential GTPase in bacterial cell division, is highly conserved among Gram-positive and Gram-negative bacteria and thus considered an attractive target to treat antibiotic-resistant bacterial infections. In this study, a new class of FtsZ inhibitors bearing the pyrimidine-quinuclidine scaffold was identified from structure-based virtual screening of natural product libraries. Iterative rounds of in silico studies and biological evaluation established the preliminary structure-activity relationships of the new compounds. Potent FtsZ inhibitors with low micromolar IC50 and antibacterial activity against S. aureus and E. coli were found. These findings support the use of virtual screening and structure-based design for the rational development of new antibacterial agents with innovative mechanisms of action.

Role of rpoS in Escherichia coli O157:H7 strain H32 biofilm development and survival.

The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS(+) wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH(2)O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log(10) in sterile ddH(2)O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log(10) in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS(+) and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.

Increased structural flexibility at the active site of a fluorophore-conjugated beta-lactamase distinctively impacts its binding toward diverse cephalosporin antibiotics.

The Ω-loop at the active site of ß-lactamases exerts significant impact on the kinetics and substrate profile of these enzymes by forming part of the substrate binding site and posing as steric hindrance toward bulky substrates. Mutating certain residues on the Ω-loop has been a general strategy for molecular evolution of ß-lactamases to expand their hydrolytic activity toward extended-spectrum antibiotics through a mechanism believed to involve enhanced structural flexibility of the Ω-loop. Yet no structural information is available that demonstrates such flexibility or its relation to substrate profile and enzyme kinetics. Here we report an engineered ß-lactamase that contains an environment-sensitive fluorophore conjugated near its active site to probe the structural dynamics of the Ω-loop and to detect the binding of diverse substrates. Our results show that this engineered ß-lactamase has improved binding kinetics and positive fluorescence signal toward oxyimino-cephalosporins, but shows little such effect to non-oxyimino-cephalosporins. Structural studies reveal that the Ω-loop adopts a less stabilized structure, and readily undergoes conformational change to accommodate the binding of bulky oxyimino-cephalosporins while no such change is observed for non-oxyimino-cephalosporins. Mutational studies further confirm that this substrate-induced structural change is directly responsible for the positive fluorescence signal specific to oxyimino-cephalosporins. Our data provide mechanistic evidence to support the long-standing model that the evolutionary strategy of mutating the Ω-loop leads to increased structural flexibility of this region, which in turn facilitates the binding of extended spectrum ß-lactam antibiotics. The oxyimino-cephalosporin-specific fluorescence profile of our engineered ß-lactamase also demonstrates the possibility of designing substrate-selective biosensing systems.

Benzothiazole-substituted benzofuroquinolinium dye: a selective switch-on fluorescent probe for G-quadruplex.

A new switch-on fluorescent probe containing the natural product cryptolepine analogue benzofuroquinolinium moiety (binding scaffold) and a benzothiazole moiety (signalling unit) shows a remarkable fluorescence enhancement selective for the G-quadruplex nucleic acid structure. Binding studies revealed that the highly selective response of the fluorescent probe arises from end-stack binding to G-quadruplex.