Significance of Amino Acid(S) During Clostridium Difficile Spore Germination

Clostridium difficile spore germination is critical for the transmission of disease. C. difficile spores germinate in response to cholic acid derivatives, such as taurocholate (TA), and amino acids, such as glycine or alanine. Many endospore-forming bacteria embed alanine racemases into their spore coats and these enzymes are thought to convert the L-alanine germinant into D-alanine, a spore germination inhibitor. C. difficile packages the Alr2 alanine racemase into the spore. Here, I describe my findings that alr2 mutant spores more readily germinate in response to L-alanine as a co-germinant. Surprisingly, D-alanine also functioned as a co-germinant. Finally, I demonstrated that L- and D-serine are also co-germinants for C. difficile spores and C. difficile Alr2 can accommodate both alanine and serine as substrates. During the analysis on Alr2, I found that D-alanine can function as a co-germinant for C. difficile spores at 37 ºC but not at 25 ºC. Because most germination assays are conducted at room temperature, I tested the ability of other amino acids to act as co-germinants with TA at 37 ºC and found that many amino acids previously categorized as non-co-germinants are actually co-germinants at 37 ºC. Even though C. difficile spore germination is known to require an amino acid co-germinant, the amino acid spore germinant receptor was unknown. In search of the amino acid germinant receptor, I used EMS mutagenesis as a strategy to generate mutants with altered requirements for the amino acid co-germinant, similar to the strategy used previously to identify the bile acid receptor, CspC. Surprisingly, I identified strains that do not require amino acids as co-germinants, and the mutant spores germinated in response to TA alone. Upon sequencing these mutants, I identified non-isogenic mutations in yabG. For C. difficile, the YabG protease is critical for the processing of CspBA to CspB and CspA and preproSleC to proSleC during spore formation. A defined yabG mutant exacerbated the EMS mutant phenotype. Moreover, I found that various mutations in cspA caused spores to germinate in the presence of TA alone without the requirement of an amino acid. Thus, my study provides evidence that apart from regulating the CspC levels in the spore, CspA is important for recognition of amino acids as co-germinants during C. difficile spore germination and that two pseudoproteases (CspC and CspA) function as the C. difficile germinant receptors

Multi-functional Bio-synthetic Hybrid Nanostructures for Enhanced Cellular Uptake, Endosomal Escape and Targeted Delivery Toward Diagnostics and Therapeutics

Applications of nanotechnology in medicine, also known as nanomedicine, is a rapidly growing field as it holds great potential in the development of novel therapeutics toward treatment of various diseases. Shell crosslinked knedel-like nanoparticles (SCKs) that are self assembled from amphiphilic block copolymers into polymeric micelles followed by crosslinking selectively throughout the shell domain have been investigated as theranostic agents for the delivery of nucleic acids and incorporation of imaging probes. The main focus of this dissertation is to design and develop unique multifunctional bio-synthetic hybrid nanoparticles that can carry agents for radiolabeling, moieties for inducing stealth properties to minimize protein adsorption in vivo, ligands for site-specific targeting, therapeutic payloads, and are optimized for efficient delivery of cargoes intracellularly and to the target sites toward constructing novel nanoscopic objects for therapy and diagnosis. Alteration of polymeric building blocks of the nanoparticles provides opportunities for precise control over the sizes, shapes, compositions, structures and properties of the nanoparticles. To ensure ideal performance of nanoparticles as theranostic agents, it is critical to ensure high intracellular bioavailability of the therapeutic payload conjugated to nanoparticles. Special efforts were made by employing well-defined multi-step polymerization and polymer modification reactions that involved conjugation of peptide nucleic acids (PNAs) to chain terminus of poly(ethylene glycol) (PEG) chain grafts such that they were presented at the outermost surface of SCKs. Additionally, chemical modification reactions were performed on the polymer backbone to integrate positive charges onto the shell of the nanoparticles to afford cationic SCKs (cSCKs) for facilitating cellular entry and electrostatic interactions with negatively charged nucleic acids. Covalent conjugation of F3, a tumor homing peptide, post-assembly of the nanoparticles enhanced cellular uptake and knockdown of nucleolin (a shuttling protein overexpressed at the sites of angiogenesis) and thus inhibiting tumor cell growth. Furthermore, these polymer precursors of the cSCKs were modified with partial to full incorporation of histamines to facilitate their endosomal escape for efficient delivery into the cytosol. The cSCKs were further templated onto high aspect ratio anionic cylinders to form hierarchically-assembled nanostructures that bring together individual components with unique functions, such as one carrying a therapeutic payload and the other with sites for radiolabeling. These higher order nanoobjects enhance circulation in vivo, have capabilities to package nucleic acids electrostatically and contain sites for radiolabeling, providing an overall advantage over the individual components, which could each facilitate only one or the other of the combined functions. Hierarchically-assembled nanostructures were investigated for their cellular uptake, transfection behavior and radiolabeling efficiency, as the next generation of theranostic agents

Prognostic role of immune checkpoint regulators in cholangiocarcinoma : a pilot study

Cholangiocarcinoma (CCA) is a hepatobiliary malignancy associated with steadily increasing incidence and poor prognosis. Ongoing clinical trials are assessing the effectiveness and safety of a few immune checkpoint inhibitors (ICIs) in CCA patients. However, these ICI treatments as monotherapies may be effective for a proportion of patients with CCA. The prevalence and distribution of other immune checkpoints (ICs) in CCA remain unclear. In this pilot study, we screened databases of CCA patients for the expression of 19 ICs and assessed the prognostic significance of these ICs in CCA patients. Notably, expression of immune modulator IDO1 and PD-L1 were linked with poor overall survival, while FASLG and NT5E were related to both worse overall survival and progression-free survival. We also identified immune modulators IDO1, FASLG, CD80, HAVCR2, NT5E, CTLA-4, LGALS9, VTCN1 and TNFRSF14 that synergized with PD-L1 and correlated with worse patient outcomes. In vitro studies revealed that the expression of ICs was closely linked with aggressive CCA subpopulations, such as cancer stem cells and cells undergoing TGF-β and TNF-α-mediated epithelial-to-mesenchymal transition. These findings suggest that the aforementioned IC molecules may serve as potential prognostic biomarkers and drug targets in CCA patients, leading to lasting and durable treatment outcomes. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. **Please note that there are multiple authors for this article therefore only the name of the first 5 including Federation University Australia affiliate “Prashanth Prithviraj, Revati Sharma, George Kannourakis” is provided in this record*

Combined inhibition of TGF-beta 1-induced EMT and PD-L1 silencing re-sensitizes hepatocellular carcinoma to sorafenib treatment

Hepatocellular carcinoma (HCC) is the most common type of primary hepatic malignancy. HCC is one of the leading causes of cancer deaths worldwide. The oral multi-tyrosine kinase inhibitor Sorafenib is the standard first-line therapy in patients with advanced unresectable HCC. Despite the significant survival benefit in HCC patients post treatment with Sorafenib, many patients had progressive disease as a result of acquiring drug resistance. Circumventing resistance to Sorafenib by exploring and targeting possible molecular mechanisms and pathways is an area of active investigation worldwide. Epithelial-to-mesenchymal transition (EMT) is a cellular process allowing epithelial cells to assume mesenchymal traits. HCC tumour cells undergo EMT to become immune evasive and develop resistance to Sorafenib treatment. Immune checkpoint molecules control immune escape in many tumours, including HCC. The aim of this study is to investigate whether combined inhibition of EMT and immune checkpoints can re-sensitise HCC to Sorafenib treatment. Post treatment with Sorafenib, HCC cells PLC/PRF/5 and Hep3B were monitored for induction of EMT and immune checkpoint molecules using quantitative reverse transcriptase (qRT)- PCR, western blot, immunofluorescence, and motility assays. The effect of combination treatment with SB431542, a specific inhibitor of the transforming growth factor (TGF)-β receptor kinase, and siRNA mediated knockdown of programmed cell death protein ligand-1 (PD-L1) on Sorafenib resistance was examined using a cell viability assay. We found that three days of Sorafenib treatment activated EMT with overexpression of TGF-β1 in both HCC cell lines. Following Sorafenib exposure, increase in the expression of PD-L1 and other immune checkpoints was observed. SB431542 blocked the TGF-β1-mediated EMT in HCC cells and also repressed PD-L1 expression. Likewise, knockdown of PD-L1 inhibited EMT. Moreover, the sensitivity of HCC cells to Sorafenib was enhanced by combining a blockade of EMT with SB431542 and knockdown of PD-L1 expression. Sorafenib-induced motility was attenuated with the combined treatment of SB431542 and PD-L1 knockdown. Our findings indicate that treatment with Sorafenib induces EMT and expression of immune checkpoint molecules, which contributes to Sorafenib resistance in HCC cells. Thus, the combination treatment strategy of inhibiting EMT and immune checkpoint molecules can re-sensitise HCC cells to Sorafenib

The Use of Financial Literacy for Growing Personal Finance

Financial literacy played an important role for everyone in managing personal finances.This research aimed to determine how the level of financial literacy in students S1 Facultyof Economics and Business, Universitas Pasundan and investigate what factors are influencingit. The observed respondents were students from the Faculty of Economics andBusiness, Universitas Pasundan. The research data was collected through questionnaires,descriptive analysis, and test multinomial logit. Based on the results of the researchshowed that the level of financial literacy from undergraduate students UniversitasPasundan was in the low category. Financial literacy was determined by gender, GreaterAcademic Achievement (GPA), parental education level, and parental income level;,whereas for age, year of study and residence do not contribute to the research model. Theresults of this study were expected to support personal financial planning of students inimproving the skills of reading, analyzing, and managing their own finances, thus avoidingthe daily financial problems

Economics of production and marketing of banana in Chitwan district, Nepal

The study was conducted in several place of Chitwan, Nepal from where 90 representative farmers and 20 traders were selected randomly as sample. The farmers were categorized into small, medium and large farm based on land holding size. The finding showed average productivity was maximum in medium size farm. Total cost of banana cultivation per bigha (1.5 bigha = 1 hector) was NRs. 239031 (106 NRs. = $1) excluding land rent. The share of planting material was 12.66%, manure 13.55%, fertilizer 16.49%, micro-nutrient 2.59%, pesticides 10.54%, labor 19.48%, animal/ machine 19.23%, marketing & transport cost 2%, and miscellaneous 3.46%. The average return rate found to be highest in large farm and lowest in small farm. The average profit of banana cultivation found to be NRs. 131902.3 per bigha. The average benefit cost ratio was 1.55 in the study area. Producer–Collector–Wholesaler–Retailer–Consumer was the most prevalent marketing channel, through which 94.74% volume was traded. Disease and pest infestation was the major production problem and fluctuation in price for marketing. The average share of banana on annual income for farmers found to be 41.34 %, indicating commercial cultivation in study area

Molecular Docking and ADMET Prediction of Compounds from Piper longum L. Detected by GC-MS Analysis in Diabetes Management

Medicinal plants have been utilized traditionally for therapeutics against various diseases. This study focuses on identifying bioactive compounds present in the fruit of hexane, ethyl acetate, and methanol extract of Piper longum L. through GC-MS analysis. The molecular level computational exploration of its phytocompounds against diabetes through molecular docking and ADMET prediction was carried out. The results of the GC-MS analysis unveiled the presence of 33 different phytochemicals and the Molecular docking revealed that 5,6-dihydroergosterol, β-sitosterol, and piperine demonstrated better binding affinities of -9.7 kcal/mol, -9.5 kcal/mol, and -7.9 kcal/mol, respectively with α-amylase (PDB ID: 2QV4) and -9.1 kcal/mol, -9.4 kcal/mol and -8.1 kcal/mol respectively with α-glucosidase (PDB ID: 5ZCC). Most of the docked compounds exhibited significant binding with the receptor protein greater than that of the reference drugs (miglitol, voglibose, and metformin). Moreover, the ADMET predictions suggested that the compounds were comparative with the reference drugs. These phytochemicals may be considered promising candidates for addressing diabetes by interfering with the normal functioning of α-amylase and α-glucosidase. The study recommends additional in vitro and in vivo experiments to validate the preliminary in silico results

Reexamining the Germination Phenotypes of Several Clostridium difficile Strains Suggests Another Role for the CspC Germinant Receptor

Clostridium difficile spore germination is essential for colonization and disease. The signals that initiate C. difficile spore germination are a combination of taurocholic acid (a bile acid) and glycine. Interestingly, the chenodeoxycholic acid class (CDCA) bile acids competitively inhibit taurocholic acid-mediated germination, suggesting that compounds that inhibit spore germination could be developed into drugs that prophylactically prevent C. difficile infection or reduce recurring disease. However, a recent report called into question the utility of such a strategy to prevent infection by describing C. difficile strains that germinated in the apparent absence of bile acids or germinated in the presence of the CDCA inhibitor. Because the mechanisms of C. difficile spore germination are beginning to be elucidated, the mechanism of germination in these particular strains could yield important information on how C. difficile spores initiate germination. Therefore, we quantified the interaction of these strains with taurocholic acid and CDCA, the rates of spore germination, the release of DPA from the spore core, and the abundance of the germinant receptor complex (CspC, CspB, and SleC). We found that strains previously observed to germinate in the absence of taurocholic acid correspond to more potent 50% effective concentrations (EC(50) values; the concentrations that achieve a half-maximum germination rate) of the germinant and are still inhibited by CDCA, possibly explaining the previous observations. By comparing the germination kinetics and the abundance of proteins in the germinant receptor complex, we revised our original model for CspC-mediated activation of spore germination and propose that CspC may activate spore germination and then inhibit downstream processes. IMPORTANCE Clostridium difficile forms metabolically dormant spores that persist in the health care environment. In susceptible hosts, C. difficile spores germinate in response to certain bile acids and glycine. Blocking germination by C. difficile spores is an attractive strategy to prevent the initiation of disease or to block recurring infection. However, certain C. difficile strains have been identified whose spores germinate in the absence of bile acids or are not blocked by known inhibitors of C. difficile spore germination (calling into question the utility of such strategies). Here, we further investigate these strains and reestablish that bile acid activators and inhibitors of germination affect these strains and use these data to suggest another role for the C. difficile bile acid germinant receptor

pH-Triggered reversible morphological inversion of orthogonally-addressable poly(3-acrylamidophenylboronic acid)-block-poly(acrylamidoethylamine) micelles and their shell crosslinked nanoparticles

Functionally-responsive amphiphilic core-shell nanoscopic objects, capable of either complete or partial inversion processes, were produced by the supramolecular assembly of pH-responsive block copolymers, without or with covalent crosslinking of the shell layer, respectively. A new type of well-defined, dual-functionalized boronic acid- and amino-based diblock copolymer poly(3-acrylamidophenylboronic acid)30-block-poly(acrylamidoethylamine)25 (PAPBA30-b-PAEA25) was synthesized by sequential reversible addition-fragmentation chain transfer (RAFT) polymerization and then assembled into cationic micelles in aqueous solution at pH 5.5. The micelles were further cross-linked throughout the shell domain comprised of poly(acrylamidoethylamine) by reaction with a bis-activated ester of 4,15-dioxo-8,11-dioxa-5,14-diazaoctadecane-1,18-dioic acid, upon increase of the pH to 7, to different cross-linking densities (2%, 5% and 10%), forming well-defined shell cross-linked nanoparticles (SCKs) with hydrodynamic diameters of ca. 50 nm. These smart micelles and SCKs presented switchable cationic, zwitterionic and anionic properties, and existed as stable nanoparticles with high positive surface charge at low pH (pH = 2, zeta potential ~ +40 mV) and strong negative surface charge at high pH (pH = 12, zeta potential ~ −35 mV). 1H NMR spectroscopy, X-ray photoelectron spectroscopy (XPS), dynamic light scattering (DLS), transmission electron microscopy (TEM), atomic force microscopy (AFM), and zeta potential, were used to characterize the chemical compositions, particle sizes, morphologies and surface charges. Precipitation occurred near the isoelectric points (IEP) of the polymer/particle solutions, and the IEP values could be tuned by changing the shell cross-linking density. The block copolymer micelles were capable of full reversible morphological inversion as a function of pH, by orthogonal protonation of the PAEA and hydroxide association with the PAPBA units, whereas the SCKs underwent only reptation of the PAPBA chain segments through the crosslinked shell of PAEA as the pH was elevated. Further, these nanomaterials also showed D-glucose-responsive properties

Monitoring Immune Checkpoint Regulators as Predictive Biomarkers in Hepatocellular Carcinoma

The global burden of hepatocellular carcinoma (HCC), one of the frequent causes of cancer-related deaths worldwide, is rapidly increasing partly due to the limited treatment options available for this disease and recurrence due to therapy resistance. Immune checkpoint inhibitors that are proved to be beneficial in the treatment of advanced melanoma and other cancer types are currently in clinical trials in HCC. These ongoing trials are testing the efficacy and safety of a few select checkpoints in HCC. Similar to observations in other cancers, these immune checkpoint blockade treatments as monotherapy may benefit only a fraction of HCC patients. Studies that assess the prevalence and distribution of other immune checkpoints/modulatory molecules in HCC have been limited. Moreover, robust predictors to identify which HCC patients will respond to immunotherapy are currently lacking. The objective of this study is to perform a comprehensive evaluation on different immune modulators as predictive biomarkers to monitor HCC patients at high risk for poor prognosis. We screened publically available HCC patient databases for the expression of previously well described immune checkpoint regulators and evaluated the usefulness of these immune modulators to predict high risk, patient overall survival and recurrence. We also identified the immune modulators that synergized with known immune evasion molecules programmed death receptor ligand-1 (PD-L1), programmed cell death protein-1 (PD-1), and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and correlated with worse patient outcomes. We evaluated the association between the expression of epithelial-to-mesenchymal transition (EMT) markers and PD-L1 in HCC patient tumors. We also examined the relationship of tumor mutational burden with HCC patient survival. Notably, expression of immune modulators B7-H4, PD-L2, TIM-3, and VISTA were independently associated with worse prognosis, while B7-H4, CD73, and VISTA predicted low recurrence-free survival. Moreover, the prognosis of patients expressing high PD-L1 with high B7-H4, TIM-3, VISTA, CD73, and PD-L2 expression was significantly worse. Interestingly, PD-L1 expression in HCC patients in the high-risk group was closely associated with EMT marker expression and prognosticates poor survival. In HCC patients, high tumor mutational burden (TMB) predicted worse patient outcomes than those with low TMB