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In an age-appropriate remote learning activity developed by a researcher-teacher team, junior high school students learned about the link between human health and food safety. The students were introduced to basic concepts about the innate immune system and food safety through a learning activity that focused on peanut allergens in food products. The students (a) learned how to distinguish between the concepts of immunity and allergy; (b) learned about cross-contamination and the link between allergies and food safety; (c) learned what antibodies are and how they can be used for science; (d) applied basic knowledge about the immune system and food safety to screen for peanut residues in suspect non-peanut food products using a commercial test kit; and (e) applied basic knowledge about the immune system and food safety to create individual poster presentations on other types of allergies, such as soy or dairy allergies.

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive bacterium Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide masarimycin as a bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 8 µM. The diamide inhibited detergent-induced autolysis in a concentration-dependent manner, indicating perturbation of peptidoglycan degradation as the mode-of-action. Cell based screening of masarimycin against a panel of autolysin mutants, identified a higher MIC against a ΔlytB strain lacking an endo-N-acetylglucosaminidase involved in cell division. Subsequent biochemical and phenotypic analyses suggested that the higher MIC was due to an indirect interaction with LytB. Further analysis of changes to the cell surface in masarimycin treated cells identified the overexpression of several moonlighting proteins, including elongation factor Tu which is implicated in regulating cell shape. Checkerboard assays using masarimycin in concert with additional antibiotics identified an antagonistic relationship with the cell wall targeting antibiotic fosfomycin, which further supports a cell wall mode-of-action.

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive bacterium Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide masarimycin as a bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 8 µM. The diamide inhibited detergent-induced autolysis in a concentration-dependent manner, indicating perturbation of peptidoglycan degradation as the mode-of-action. Cell based screening of masarimycin against a panel of autolysin mutants, identified a higher MIC against a ΔlytB strain lacking an endo-N-acetylglucosaminidase involved in cell division. Subsequent biochemical and phenotypic analyses suggested that the higher MIC was due to an indirect interaction with LytB. Further analysis of changes to the cell surface in masarimycin treated cells identified the overexpression of several moonlighting proteins, including elongation factor Tu which is implicated in regulating cell shape. Checkerboard assays using masarimycin in concert with additional antibiotics identified an antagonistic relationship with the cell wall targeting antibiotic fosfomycin, which further supports a cell wall mode-of-action.

© The Author(s) 2020., Streptococcus pneumoniae choline kinase (sChoK) has previously been proposed as a drug target, yet the effectiveness of the first and only known inhibitor of sChoK, HC-3, is in the millimolar range. The aim of this study was thus to further validate sChoK as a potential therapeutic target by discovering more powerful sChoK inhibitors. LDH/PK and colorimetric enzymatic assays revealed two promising sChoK inhibitor leads RSM-932A and MN58b that were discovered with IC50 of 0.5 and 150 μM, respectively, and were shown to be 2–4 magnitudes more potent than the previously discovered inhibitor HC-3. Culture assays showed that the minimum inhibitory concentration (MIC) of RSM-932A and MN58b for S. pneumoniae was 0.4 μM and 10 μM, respectively, and the minimum lethal concentration (MLC) was 1.6 μM and 20 μM, respectively. Western blot monitoring of teichoic acid production revealed differential patterns in response to each inhibitor. In addition, both inhibitors possessed a bacteriostatic mechanism of action, and neither interfered with the autolytic effects of vancomycin. Cells treated with MN58b but not RSM-932A were more sensitive to a phosphate induced autolysis with respect to the untreated cells. SEM studies revealed that MN58b distorted the cell wall, a result consistent with the apparent teichoic acid changes. Two novel and more highly potent putative inhibitors of sChoK, MN58b and RSM-932A, were characterized in this study. However, the effects of sChoK inhibitors can vary at the cellular level. sChoK inhibition is a promising avenue to follow in the development of therapeutics for treatment of S. pneumoniae., This research was funded in part by the NIFA through the Agricultural Research Program at North Carolina Agricultural and Technical State University (Evans-Allen Program, project number NC.X-291-5-15-170-1). We would also like to express our gratitude to the group of Yuxing Chen of the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences at the University of Science and Technology of China for their generous donation of the LicA construct.

In this study, we modified reinforced clostridial medium (RCM) to selectively enumerate and isolate Lactobacillus delbrueckii ssp. bulgaricus, a probiotic and important starter culture in the dairy industry. The disparity in the reported carbohydrate fermentation pattern of L. delbrueckii ssp. bulgaricus was used to develop a growth medium not only selective for L. delbrueckii ssp. bulgaricus but significantly inhibitory to the growth of other lactic acid bacteria. A recently modified RCM (mRCM) was optimized for this study by the addition of 0.5% fructose, 0.5% dextrose, 1% maltose, and 0.25% sodium pyruvate while replacing lactose as a carbohydrate source. The cell recovery and bacterial counts of L. delbrueckii ssp. bulgaricus in tested products (pure L. delbrueckii ssp. bulgaricus strains, starter culture, probiotic supplements, and yogurt) using our mRCM with sodium pyruvate (mRCM-PYR) were significantly higher than in the recently modified RCM and the common de Man, Rogosa, and Sharpe (MRS) culture medium. The growth of other lactic acid bacteria (Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus reuteri) and Bifidobacteria was retarded in this modified medium compared with their growth in MRS and mRCM. This result is a significant improvement in the enumeration and differentiation of L. delbrueckii ssp. bulgaricus in mRCM-PYR compared with the results in MRS and mRCM where the high background growth of similar species interferes with the accuracy of bacterial population counts. Our results thus suggest that mRCM-PYR could be recommended as a reliable alternative growth medium for the selective enumeration and isolation of L. delbrueckii ssp. bulgaricus in a mixed culture.

In an age-appropriate activity developed by a researcher-teacher team working together under the auspices of the American Chemical Science Coaches Program, middle school students at the Greensboro Montessori School (1) learned about the microbiological basis of fermentation, (2) learned about the chemical changes that take place during the fermentation of milk into yogurt, (3) applied this basic knowledge to designing and implementing experiments to test different conditions for culturing yogurt, (4) assessed the outcomes of different culturing conditions, and (5) developed a method for producing yogurt. This exercise includes hypothesis formulation, experimental design, and hypothesis testing and serves as an example of how empirically derived knowledge can be applied to the design of a food product.

The study reported in this research communication evaluates the chemical (solvents) and mechanical (sonication, bead-beater) extraction methods to determine the maximum recovery of β-galactosidase from L. bulgaricus spp. Among all extraction techniques, sonication-assisted extraction yielded the highest amounts of enzyme activity (between 1892–2156 Miller Units) in cell-free extract (supernatant). Interestingly, solvent extracted enzyme activities were found to be very low (between 83–153 Miller Units) in supernatant. SDS-polyacrylamide gel electrophoresis and the total protein determination showed that mechanical methods can completely lyse the cells. Our results thus demonstrated that the mechanical extraction method of sonication is the best one for recovering the maximum amount of lactase from L. bulgaricus strains.

The SARS-COVID-19 pandemic has resulted in over 6 million deaths worldwide (>967,000 deaths in the U.S. alone). Importantly, this pandemic has had a disproportionate impact on the food industry, including the meat processing industry. As a result, the health of food processing workers has been negatively impacted, leading to high numbers of SARS-COVID-19 cases at food processing facilities and surrounding communities. Resulting shutdowns have also led to product shortages for consumers and economic losses to the industry. In response, the food processing industry and public health agencies have prioritized continued food access over identifying evidence-based best practices for controlling airborne viral threats (AVTs). Consequently, the lack of high -quality evidence has made it more difficult to identify appropriate control measures that could prevent current and future airborne viral threats (AVT) in food processing facilities. Without evidence-based best practices, the food processing industry will remain vulnerable to future AVT events. Therefore, there is a pressing need for timely research on the spread of AVT in food processor settings. The aim of this article is to summarize the epidemiological knowledge regarding the impact COVID-19 has had on the food industry and the meat industry in particular and to emphasize the need for empirical research into the factors that contribute to the spread of airborne viruses in these settings in order to secure these facilities from future AVTs.

The current SARS-COVID-19 crisis has demonstrated the dangers that airborne virus (AV) pandemics pose to the health of all workers (particularly in the meat processing industry), the economic health of the food industry, and food security. The impact that the current pandemic has had on the food industry points to the need for a proactive rather than reactive approach towards preventing future AV outbreaks. Such a proactive approach should be based on empirical assessments of current AV food safety practices and the development of more robust practices tailored to the culture and needs of the food industry. Moreover, a proactive approach is necessary in order to better prepare the food industry for future AV outbreaks, protect the health of workers, reduce disparities in AV occupational health risks, and enhance the safety of the food supply chain. The aim of this review is to make the case for a new food safety research paradigm that incorporates the intensive study of airborne viruses under conditions that simulate food industry work environments.

This research was funded by CSIC, grant number PIE202020E041; and in part by the NIFA through the Agricultural Research Program at North Carolina Agricultural and Technical State University (Evans-Allen Program, project number NC.X-291-5-15-170-1)., Choline kinase (ChoK) is a cytosolic enzyme that catalyzes the phosphorylation of choline to form phosphorylcholine (PCho) in the presence of ATP and magnesium. ChoK is required for the synthesis of key membrane phospholipids and is involved in malignant transformation in a large variety of human tumours. Active compounds against ChoK have been identified and proposed as antitumor agents. The ChoK inhibitory and antiproliferative activities of symmetrical bispyridinium and bisquinolinium compounds have been defined using quantitative structure–activity relationships (QSARs) and structural parameters. The design strategy followed in the development of the most active molecules is presented. The selective anticancer activity of these structures is also described. One promising anticancer compound has even entered clinical trials. Recently, ChoKα inhibitors have also been proposed as a novel therapeutic approach against parasites, rheumatoid arthritis, inflammatory processes, and pathogenic bacteria. The evidence for ChoKα as a novel drug target for approaches in precision medicine is discussed., CSIC, grant number PIE202020E041, NIFA through the Agricultural Research Program at North Carolina Agricultural and Technical State University (Evans-Allen Program, project number NC.X-291-5-15-170-1)

To prevent the growth of food pathogens and food spoilage organisms, antimicrobials are used to disinfect surfaces or are added to food. In light of the important role that antibiotics and antimicrobials play in food safety, food science students need a deep understanding of how these chemicals function. We describe here a combined wet lab and in silico laboratory experience designed to help students visualize two biochemical concepts explaining antibiotic activity: (i) bacteriolytic versus bacteriostatic activity and (ii) competitive versus noncompetitive inhibition. This laboratory experience was implemented for students enrolled in the Introduction to Food Science course in Family and Consumer Sciences at North Carolina A&T State University.

Despite renewed interest, development of chemical biology methods to study peptidoglycan metabolism has lagged in comparison to the glycobiology field in general. To address this, a panel of diamides were screened against the Gram-positive pathogen Streptococcus pneumoniae to identify inhibitors of bacterial growth. The screen identified the diamide fgkc as a narrow spectrum bacteriostatic inhibitor of S. pneumoniae growth with an MIC of 7.8 μM. The diamide inhibited detergent-induced autolysis in a concentration dependent manner indicating peptidoglycan degradation as the mode-of-action. Genetic screening of autolysin mutants suggested LytB, an endo-N-acetylglucosaminidase, involved in cell division as the potential target. Surprisingly, biochemical, and phenotypic analysis contradicted the genetic screen results. Phenotypic studies with the Δlytb strain illustrate the difference between genetic and chemical inactivation of autolysins. These findings suggest that meta-phenotypes including autolytic activity, cell morphology, and genetic screening can be the result of the complex interaction of one or more possible pathways that are connected to cell wall metabolism.

The study reported in this research communication evaluates the chemical (solvents) and mechanical (sonication, bead-beater) extraction methods to determine the maximum recovery of β-galactosidase from L. bulgaricus spp. Among all extraction techniques, sonication-assisted extraction yielded the highest amounts of enzyme activity (between 1892-2156 Miller Units) in cell-free extract (supernatant). Interestingly, solvent extracted enzyme activities were found to be very low (between 83-153 Miller Units) in supernatant. SDS-polyacrylamide gel electrophoresis and the total protein determination showed that mechanical methods can completely lyse the cells. Our results thus demonstrated that the mechanical extraction method of sonication is the best one for recovering the maximum amount of lactase from L. bulgaricus strains.

To examine whether choline and its derivatives can be used to preserve viable cells of Lactobacillus reuteri in autolytic models. A phosphate-induced autolytic model in de Man, Rogosa and Sharpe medium (MRS) was used. Viable cell counts were determined by plated on MRS-agar. Choline and hemicholinium-3 (HC-3) significantly blocked autolysis of L. reuteri at 360 mM and 4 mM, respectively. Viable cell counts corroborated these observations. Importantly, autolytically induced cells treated with choline and hemicholinium-3 were significantly more viable then even non-induced cells. Over-production of a known autolytic protein, spirosin, was not attenuated in the presence of choline and hemicholinium-3. Inducing autolysis and then blocking it with choline and its analogs is a promising approach for retaining the viability of L. reuteri cells.

Prebiotics are nondigestible food ingredients that have beneficial effects on human health. Recently, these ingredients have garnered increasing attention, particularly for their role in feeding beneficial bacteria within the gastrointestinal tract. In addition, synbiotic (combination of pre- and probiotics) approaches have been studied as therapeutic strategies in several clinical conditions and have shown the potential to improve host health. This chapter provides an overview of how prebiotics and synbiotics can modulate the gut microbiota within the context of disease prevention and the promotion of good health. The chapter also highlights the concerns regarding potential stimulating effect of prebiotics on other harmful bacteria.

The purpose of this study was to investigate the bactericidal activity of copper (Cu, 10 ppm) and ascorbic acid (AA, 0.3% v/v) mixture against Staphylococcus aureus (S. aureus) ATCC 25923 (methicillin-susceptible) and ATCC 700698 (methicillin-resistant) strains. Approximately 5.5 log10 CFU/ml of each strain was individually inoculated into brain heart infusion (BHI) broth containing Cu, AA, and a Cu-AA mixture, and a time-kill assay was conducted to assess the bactericidal activity. In order to determine the efficacy of Cu-AA mixture as a surface decontaminating solution, the cells were first attached to glass slides (~6.5–7.5 log10 CFU per slide) and treated with a Cu-AA mixture for 1 min. The inner membrane permeability of the S. aureus strains was determined by measuring β-Galactosidase activity. Similarly, changes in the protein profile of treated bacterial cells were analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The time-kill study revealed >3.0 log10 CFU/ml reduction in both strains within 24 h of incubation at 37 °C. Attached cells on glass slides were also reduced by > 3.0 log10 CFU per slide after 1 min of exposure to the Cu-AA mixture. The results indicate that the Cu-AA mixture was bactericidal against S. aureus strains and the damage to the inner cell membrane could be attributed to this effect.

Aldose reductase (AKR1B1) is a critical drug target because of its involvement in diabetic complications, inflammation, and tumorigenesis. However, to date, development of clinically useful inhibitors has been largely unsuccessful. Cyclopentenone prostaglandins (cyPGs) are reactive lipid mediators that bind covalently to proteins and exert anti-inflammatory and antiproliferative effects in numerous settings. By pursuing targets for modification by cyPGs we have found that the cyPG PGA1 binds to and inactivates AKR1B1. A PGA1-AKR1B1 adduct was observed, both by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by SDS-PAGE using biotinylated PGA1 (PGA1-B). Insight into the molecular interactions between AKR1B1 and PGA1 was advanced by molecular modeling. This anticipated the addition of PGA1 to active site Cys298 and the potential reversibility of the adduct, which was supported experimentally. Indeed, loss of biotin label from the AKR1B1-PGA1-B adduct was favored by glutathione, indicating a retro-Michael reaction, which unveils new implications of cyPG-protein interaction. PGA1 elicited only marginal inhibition of aldehyde reductase (AKR1A1), considered responsible for the severe adverse effects of many AKR1B1 inhibitors. Interestingly, other prostaglandins (PGs) inhibited the enzyme, including non-electrophilic PGE1 and PGE2, currently used in clinical practice. Moreover, both PGA1 and PGE1 reduced the formation of sorbitol in an ex-vivo model of diabetic cataract to an extent comparable to that attained by the known AKR inhibitor epalrestat. Taken together, these results highlight the role of PGs as AKR1B1 inhibitors and the interest in PG-related molecules as leads for the development of novel pharmacological tools.

Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5′-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

The long-term objective of this project is to utilize the I-domain protein for the α-subunit of LFA-1 to target drugs to lymphocytes by binding to ICAM receptors on the cell surface. The short-term goal is to provide proof-of-concept that I-domain conjugated to small molecules can still bind to and uptake by ICAM-1 on the surface of lymphocytes (i.e., Raji cells). To accomplish this goal, the I-domain protein was labeled with FITC at several lysine residues to produce the FITC-I-domain and CD spectroscopy showed that the FITC-I-domain has a secondary structure similar to that of the parent I-domain. The FITC-I-domain was taken up by Raji cells via receptor-mediated endocytosis and its uptake can be blocked by anti-I-domain mAb but not by its isotype control. Antibodies to ICAM-1 enhance the binding of I-domain to ICAM-1, suggesting it binds to ICAM-1 at different sites than the antibodies. The results indicate that fluorophore modification does not alter the binding and uptake properties of the I-domain protein. Thus, I-domain could be useful as a carrier of drug to target ICAM-1-expressing lymphocytes.

Using chemical and artificial preservation methods to keep food safe is becoming an issue with consumers. Therefore, it is important to discover antimicrobials from natural sources for use in food safety applications. The objective of the present study was to establish screening system for natural inhibitors of choline kinase (ChoK), a known antimicrobial target. A previously developed dual choline/phosphocholine colorimetric method was used to determine the relative strength of 3 choline kinase inhibitors: Hemocholinium-3, RSM-928A, and MN58 . Whole cell extracts containing the choline kinase of Streptococcus pneumoniae (sChoK) was used as a model. The measured IC50 values of these drugs were >2700 µM, 0.54 µM, and 170-225 µM, respectively. Importantly, not every step of the colorimetric method could be used in the case of every inhibitor, since each had its own particular reactive profile with the colorimetric dyes, which could have lead to confounded measurements. However, in every case, the system was flexible enough to measure choline or phosphocholine, if not both metabolites. We establish here that this dual choline/phosphocholine system is flexible enough to measure the IC50 any possible inhibitor. This colorimetric method is an ideal benchtop method for screening for natural inhibitors of bacterial ChoKs.

Leukocyte-function associated antigen-1 (LFA-1) is an alpha(L)beta(2) chain integrin expressed on the surface of endothelial cells that modulates the behavior of leukocytes by mediating their adhesion to other cells through its interaction to cell-surface ligands. The most important ligand of LFA-1 is ICAM-1 which is expressed on the surface of endothelial cells. The interaction between LFA-1 and ICAM-1 is involved in inflammatory responses and is therefore implicated in inflammatory pathologies and autoimmune diseases; and, in addition, it is involved in many cancer processes. In light of this, there is great interest in developing small molecule, orally available, inhibitors of the LFA-1/ICAM-1 interaction. A structurally diverse collection of small molecule inhibitors has been characterized and developed either to bind the IDAS site of the alpha(L) I-domain or to the MIDAS of the beta2 I-like domain. In this review, a summary of the structure and regulation of LFA-1 will be given, followed by a description of the different classes of inhibitors that have been described to date.

We have investigated the mechanism of action of inhibition of the choline kinase of P. falciparum ( p.f. -ChoK) by two inhibitors of the human ChoKα, MN58b and RSM-932A, which have previously been shown to be potent antitumoral agents. The efficacy of these inhibitors against p.f. -ChoK is investigated using enzymatic and in vitro assays. While MN58b may enter the choline/phosphocholine binding site, RSM-932A appears to have an altogether novel mechanism of inhibition and is synergistic with respect to both choline and ATP. A model of inhibition for RSM-932A in which this inhibitor traps p.f. -ChoK in a phosphorylated intermediate state blocking phosphate transfer to choline is presented. Importantly, MN58b and RSM-932A have in vitro inhibitory activity in the low nanomolar range and are equally effective against chloroquine-sensitive and chloroquine-resistant strains. RSM-932A and MN58b significantly reduced parasitemia and induced the accumulation of trophozoites and schizonts, blocking intraerythrocytic development and interfering with parasite egress or invasion, suggesting a delay of the parasite maturation stage. The present data provide two new potent structures for the development of antimalarial compounds and validate p.f. -ChoK as an accessible drug target against the parasite.

The integrin leukocyte function associated antigen 1 (LFA-1) binds the intercellular adhesion molecule 1 (ICAM-1) by its αL-chain inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report the structure-based design, small-scale synthesis, and biological activity evaluation of a novel family of LFA-1 antagonists. The design led to the synthesis of a family of highly substituted homochiral pyrrolidines with antiproliferative and antimetastatic activity in a murine model of colon carcinoma, as well as potent antiadhesive properties in several cancer cell lines in the low micromolar range. NMR analysis of their binding to the isolated I-domain shows that they bind to the I-domain allosteric site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled. © 2013 American Chemical Society.

Lactic acid bacteria (LAB) are used by industry to produce fermented food products. The standard media used to cultivate LABs is DeMan Rogosa Sharp (MRS). However, it is expensive. Alternative low-cost media must be developed for industrial use. A good source for growth media components are by-products generated during the production of agricultural goods, such as dates. Our objective was to investigate the use of date by-products for cultivating Lactobacillus reuteri. Date palm extract (DPE) was prepared by pressing fresh date fruits for one week and diluting 1:2 with diH2O, centrifuging at 4696 x g and 4°C 25 min, and autoclaving the supernatant at 110°C for 15 min. An MRS-based buffer solution was added to DPE make a date palm medium (DPM). DPM was then enriched with various amounts phytone peptone (0, 0.2, 0.4, 0.6, and 0.8 %, w/v). The enriched DPMs were used to cultivate three strains of Lactobacillus reuteri: DSM 20016, CF2-7F, and SD 2112. Our results showed that in the DPM minus phytone peptone, bacterial counts reached 3.18 ± 0.5 log CFU/mL. Addition of lower amounts of phytone peptone did not improve bacterial growth. However, DPM medium supplemented with 0.8% phytone peptone improved the bacterial counts, which reached 6.94 ± 0.1 log CFU/mL, similar to what was observed with MRS (7.90± 0.24 log CFU/mL). There was no significant difference in the growth of LAB in MRS and phytone peptone enriched media DPM (p > 0.05). Date by-products are potentially alternative low cost components of LAB growth media.

In the present study, we determined the effects of long-term exposure to aspirin on the growth and functionality of Lactobacillus rhamnosus. One isolated colony of L. rhamnosus was propagated three times in deMan, Rogosa and Sharpe (MRS) broth and incubated at 37°C. The active strain was then harvested and washed with 0.1% peptone water. Cells were transferred into 9 mL MRS broth containing approximately 6 mg/mL of aspirin, vigorously mixed and incubated for 4 h. The cells were further harvested, transferred into MRS broth and incubated at 37°C. The exposure protocol was repeated for five sequential transfers a week, and the exposed strain cell was surface plated onto MRS agar containing equal amount of aspirin. One isolated colony of aspirin exposed L. rhamnosus from an aspirin containing agar plate was further activated in MRS broth. This procedure was repeated sequentially for 12 consecutive weeks. Bacterial populations, beta-galactosidase activity (beta-gal), and protein expression were determined. Bacterial cells unexposed to aspirin were used as the control. Our results showed that L. rhamnosus could survive after long term exposure to a sub-inhibitory concentration of aspirin. The average beta-gal activity of L. rhamnosus in unexposed cells was 153 ± 2.5 Gal U.; however, beta-gal activity was completely inhibited in exposed cells throughout the exposure period. There was approximately 54% more protein expressed in the long term aspirin-exposed strain compared to the short term exposed (4 h) strain as determined by BCA assay. In addition, the SDS-page gel indicated the synthesis of more protein in the exposed L. rhamnosus. The onset of common or chronic illnesses, especially among the elderly population, is often treated with common medications. However, regular the intake of medical drugs such as aspirin could affect beneficial gut microflora. Therefore, foods containing probiotics such as yogurt and other functional foods are important for the maintenance of optimal gastrointestinal health.

There are 15 known laminins, which differ in the isoforms of the three chains that assemble into the cross-shape molecules that are observed by electron microscopy. The amino acid sequences of the rod-like portion of the long arm have long been recognized as having a potential for coiled-coil structure formation; however, an experimental determination of its structure is hampered by the complexity of laminin, a multidomain, heterotrimeric, and glycosilated 800 kDa molecule. Here, we have investigated the coiled-coil structure potential of laminin to evaluate its distribution along the long arm, the presence of conserved patterns, and differences between natural and non-natural isoforms. With these aims, we have analysed the sequences of each laminin chain in the context of the three-chain assemblies to yield an overall score of coiled-coil potential for the 15 natural laminins and for the other 30 possible but non-detected ones. The potential has been calculated with two different existing methods to exclude algorithm specific biases and with different chain alignments to evaluate the dependency of the results on uncertainties in the specific alignment along the domain. The analysis shows that the distribution of the potential is discontinuous, highly fragmented along the arm, without a common pattern except for a higher potential at the C-terminus, and that natural and non-natural laminins cannot be distinguished based on their coiled-coil potential, indicating that other factors are responsible for the selection of chain assembly.

The integrin Leukocyte Function-associated Antigen-1 (LFA-1) recognizes and binds the Intercellular Adhesion Molecule-1 (ICAM-1) by its L chain Inserted domain (I-domain). This interaction plays a key role in cancer and other diseases. We report here the rational in silico design, small scale synthesis and biological activity evaluation of a novel family of potent LFA-1 antagonists. The structure-based design led to the synthesis of a family of highly substituted homoquiral pyrrolidines that mimic key residues of the ICAM-1 moiety. Some of these compounds showed both antiproliferative and anti-metastatic activity in a murine model of melanoma, as well as potent anti-adhesive properties in several cancer cell lines in the low micromolar range. The molecular mechanism of action of selected antagonists have been investigated by NMR analysis of their binding to the isolated I-domain of LFA-1. We show that the compounds bind to the I-domain Allosteric Site (IDAS), the binding site of other allosteric LFA-1 inhibitors. These results provide evidence of the potential therapeutic value of a new set of LFA-1 inhibitors, whose further development is facilitated by a synthetic strategy that is versatile and fully stereocontrolled Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C46.

We have used nuclear magnetic resonance to characterize the binding site of two intercellular adhesion molecule-1 derived cyclic peptides, cIBC and cIBR, to the I-domain of leukocyte function-associated antigen-1. These peptides inhibit the leukocyte function-associated antigen-1/intercellular adhesion molecule-1 interaction known to play a key role in autoimmune diseases and cancer metastasis. Perturbation of the chemical shifts and intensities of the nuclear magnetic resonance signals corresponding to a number of residues of the I-domain of leukocyte function-associated antigen-1 show that both peptides bind to the I-domain allosteric site, the binding site of I-domain allosteric inhibitors such as lovastatin, and therefore the peptides probably also act as allosteric inhibitors of leukocyte function-associated antigen-1. Molecular models of the interaction of these two cyclic peptides with leukocyte function-associated antigen-1 I-domain show that the binding mode of the three molecules are analogous: the hydrophobic residues of the peptides remain buried and occupy the same positions as the apolar groups of lovastatin, while the peptides regions containing the most polar residues are flexible and primarily exposed to the solvent. These results suggest an allosteric mechanism for the inhibitory effect on T-cell adhesion displayed by both peptides, which exhibit potential as therapeutic agents.