Selective inhibition of Helicobacter pylori methionine aminopeptidase by azaindole hydroxamic acid derivatives: Design, synthesis, in vitro biochemical and structural studies.

Methionine aminopeptidases (MetAPs) are an important class of enzymes that work co-translationally for the removal of initiator methionine. Chemical inhibition or gene knockdown is lethal to the microbes suggesting that they can be used as antibiotic targets. However, sequence and structural similarity between the microbial and host MetAPs has been a challenge in the identification of selective inhibitors. In this study, we have analyzed several thousands of MetAP sequences and established a pattern of variation in the S1 pocket of the enzyme. Based on this knowledge, we have designed a library of 17 azaindole based hydroxamic acid derivatives which selectively inhibited the MetAP from H. pylori compared to the human counterpart. Structural studies provided the molecular basis for the selectivity.

Spirocyclic sulfonamides with carbonic anhydrase inhibitory and anti-neuropathic pain activity.

A novel series of 4-oxo-spirochromane bearing primary sulfonamide group were synthetized as Carbonic Anhydrase inhibitors (CAIs) and tested for their management of neuropathic pain. Indeed, CAs have been recently validated as novel therapeutic targets in neuropathic pain. All compounds, here reported, showed strong activity against hCA II and hCA VII with KI values in the low or sub-nanomolar range. Two compounds (6d and 6l) showed good neuropathic pain attenuating effects and longer duration than drug reference acetazolamide in an animal model of oxaliplatin induced neuropathy.

Microbial occurrence and antibiotic resistance in ready-to-go food items.

Foodborne pathogens, such as Escherichia coli, and Salmonella, are commonly prevalent in contaminated food products seen through annual food recalls. Excessive use of antibiotics through the past few decades has led to a multitude of antibiotic resistant bacteria, including foodborne pathogens. We investigated microbial occurrence and their antibiotics resistances in ready-to-go food items, i.e. canned food, bagged food, and baby food. A total of 112 isolates were isolated from varying food items, and 21 of these isolates were identified through 16S rRNA sequencing revealing Bacillus sp., Staphylococcus sp. and Micrococcus sp. Bagged food items showed the most microbial diversity as well as the largest colony forming unit (log 20-25 CFU/g). Isolates showed antibiotic resistance to ampicillin, streptomycin, chloramphenicol, and kanamycin at concentrations of 100, 500, and 1000 µg/mL. 57% isolates were ampicillin resistance followed by kanamycin (26%). A variety of microorganisms present in ready-to-go food items may not be pathogenic, however their occurrence and multiple antibiotic resistance (MAR) poses risk of transferring their genes to foodborne pathogens.

Antibiotrophs: The complexity of antibiotic-subsisting and antibiotic-resistant microorganisms.

Widespread overuse of antibiotics has led to the emergence of numerous antibiotic-resistant bacteria; among these are antibiotic-subsisting strains capable of surviving in environments with antibiotics as the sole carbon source. This unparalleled expansion of antibiotic resistance reveals the potent and diversified resistance abilities of certain bacterial strains. Moreover, these strains often possess hypermutator phenotypes and virulence transmissibility competent for genomic and proteomic propagation and pathogenicity. Pragmatic and prospicient approaches will be necessary to develop efficient therapeutic methods against such bacteria and to understand the extent of their genomic adaptability. This review aims to reveal the niches of these antibiotic-catabolizing microbes and assesses the underlying factors linking natural microbial antibiotic production, multidrug resistance, and antibiotic-subsistence.

Human microbiome versus food-borne pathogens: friend or foe.

As food safety advances, there is a great need to maintain, distribute, and provide high-quality food to a much broader consumer base. There is also an ever-growing "arms race" between pathogens and humans as food manufacturers. The human microbiome is a collective organ of microbes that have found community niches while associating with their host and other microorganisms. Humans play an important role in modifying the environment of these organisms through their life choices, especially through individual diet. The composition of an individual's diet influences the digestive system-an ecosystem with the greatest number and largest diversity of organisms currently known. Organisms living on and within food have the potential to be either friends or foes to the consumer. Maintenance of this system can have multiple benefits, but lack of maintenance can lead to a host of chronic and preventable diseases. Overall, this dynamic system is influenced by intense competition from food-borne pathogens, lifestyle, overall diet, and presiding host-associated microbiota.

Extremophiles as sources of inorganic bio-nanoparticles.

Industrial use of nanotechnology in daily life has produced an emphasis on the safe and efficient production of nanoparticles (NPs). Traditional chemical oxidation and reduction methods are seen as inefficient, environmentally unsound, and often dangerous to those exposed and involved in NP manufacturing. However, utilizing microorganisms for biosynthesis of NPs allows efficient green production of a range of inorganic NPs, while maintaining specific size, shape, stability, and dispersity. Microorganisms living under harsh environmental conditions, called "Extremophiles," are one group of microorganisms being utilized for this biosynthesis. Extremophiles' unique living conditions have endowed them with various processes that enable NP biosynthesis. This includes a range of extremophiles: thermophiles, acidophilus, halophiles, psychrophiles, anaerobes, and some others. Fungi, bacteria, yeasts, and archaea, i.e. Ureibacillus thermosphaericus, and Geobacillus stearothermophilus, among others, have been established for NP biosynthesis. This article highlights the extremophiles and methods found to be viable candidates for the production of varying types of NPs, as well as interpreting selective methods used by the organisms to synthesize NPs.

Radiation-resistant extremophiles and their potential in biotechnology and therapeutics.

Extremophiles are organisms able to thrive in extreme environmental conditions. Microorganisms with the ability to survive high doses of radiation are known as radioresistant or radiation-resistant extremophiles. Excessive or intense exposure to radiation (i.e., gamma rays, X-rays, and particularly UV radiation) can induce a variety of mutagenic and cytotoxic DNA lesions, which can lead to different forms of cancer. However, some populations of microorganisms thrive under different types of radiation due to defensive mechanisms provided by primary and secondary metabolic products, i.e., extremolytes and extremozymes. Extremolytes (including scytonemin, mycosporine-like amino acids, shinorine, porphyra-334, palythine, biopterin, and phlorotannin, among others) are able to absorb a wide spectrum of radiation while protecting the organism's DNA from being damaged. The possible commercial applications of extremolytes include anticancer drugs, antioxidants, cell-cycle-blocking agents, and sunscreens, among others. This article aims to review the strategies by which microorganisms thrive in extreme radiation environments and discuss their potential uses in biotechnology and the therapeutic industry. The major challenges that lie ahead are also discussed.

Emergence of antibiotic-resistant extremophiles (AREs).

Excessive use of antibiotics in recent years has produced bacteria that are resistant to a wide array of antibiotics. Several genetic and non-genetic elements allow microorganisms to adapt and thrive under harsh environmental conditions such as lethal doses of antibiotics. We attempt to classify these microorganisms as antibiotic-resistant extremophiles (AREs). AREs develop strategies to gain greater resistance to antibiotics via accumulation of multiple genes or plasmids that harbor genes for multiple drug resistance (MDR). In addition to their altered expression of multiple genes, AREs also survive by producing enzymes such as penicillinase that inactivate antibiotics. It is of interest to identify the underlying molecular mechanisms by which the AREs are able to survive in the presence of wide arrays of high-dosage antibiotics. Technologically, "omics"-based approaches such as genomics have revealed a wide array of genes differentially expressed in AREs. Proteomics studies with 2DE, MALDI-TOF, and MS/MS have identified specific proteins, enzymes, and pumps that function in the adaptation mechanisms of AREs. This article discusses the molecular mechanisms by which microorganisms develop into AREs and how "omics" approaches can identify the genetic elements of these adaptation mechanisms. These objectives will assist the development of strategies and potential therapeutics to treat outbreaks of pathogenic microorganisms in the future.

Ultraviolet-radiation-resistant isolates revealed cellulose-degrading species of Cellulosimicrobium cellulans (UVP1) and Bacillus pumilus (UVP4).

Among extremophiles, microorganisms resistant to ultraviolet radiation (UVR) have been known to produce a variety of metabolites (i.e., extremolytes). We hypothesized that natural microbial flora on elevated land (hills) would reveal a variety of UVR-resistant extremophiles and polyextremophiles with modulated proteins and enzymes that had biotechnological implications. Microorganisms Cellulosimicrobium cellulans UVP1 and Bacillus pumilus UVP4 were isolated and identified using 16S rRNA sequencing, and showed extreme UV resistance (1.03 × 106 and 1.71 × 105 J/m², respectively) from elevated land soil samples along with unique patterns of protein expression under UVR and non-UVR. A broad range of cellulolytic activity on carboxymethyl cellulose agar plates in C. cellulans UVP1 and B. pumilus UVP4 was revealed at varying pH, temperature, and inorganic salt concentration. Further, the microbial strain B. pumilus UVP4 showed the basic characteristics of a novel group: polyextremophiles with significance in bioenergy.

Weedy lignocellulosic feedstock and microbial metabolic engineering: advancing the generation of 'Biofuel'.

Lignocellulosic materials are the most abundant renewable organic resources (~200 billion tons annually) on earth that are readily available for conversion to ethanol and other value-added products, but they have not yet been tapped for the commercial production of fuel ethanol. The lignocellulosic substrates include woody substrates such as hardwood (birch and aspen, etc.) and softwood (spruce and pine, etc.), agro residues (wheat straw, sugarcane bagasse, corn stover, etc.), dedicated energy crops (switch grass, and Miscanthus etc.), weedy materials (Eicchornia crassipes, Lantana camara etc.), and municipal solid waste (food and kitchen waste, etc.). Despite the success achieved in the laboratory, there are limitations to success with lignocellulosic substrates on a commercial scale. The future of lignocellulosics is expected to lie in improvements of plant biomass, metabolic engineering of ethanol, and cellulolytic enzyme-producing microorganisms, fullest exploitation of weed materials, and process integration of the individual steps involved in bioethanol production. Issues related to the chemical composition of various weedy raw substrates for bioethanol formation, including chemical composition-based structural hydrolysis of the substrate, need special attention. This area could be opened up further by exploring genetically modified metabolic engineering routes in weedy materials and in biocatalysts that would make the production of bioethanol more efficient.

Applications of proteomic technologies for understanding the premature proteolysis of CFTR.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, which encodes an ATP-dependent anion channel. Disease-causing mutations can affect channel biogenesis, trafficking or function, and result in reduced ion transport at the apical surface of many tissues. The most common CFTR mutation is a deletion of phenylalanine at position 508 (DeltaF508), which results in a misfolded protein that is prematurely targeted for degradation. This article focuses on how proteomic approaches have been utilized to explore the mechanisms of premature proteolysis in CF. Additionally, we emphasize the potential for proteomic-based technologies in expanding our understanding of CF pathophysiology and therapeutic approaches.

Proteomics: a strategy to understand the novel targets in protein misfolding and cancer therapy.

Proteins carry out important functions as they fold themselves. Protein misfolding occurs during different biochemical processes and may lead to the development of diseases such as cancer, which is characterized by genetic instability. The cancer microenvironment exposes malignant cells to a variety of stressful conditions that may further promote protein misfolding. Tumor development and progression often arises from mutations that interfere with the appropriate function of tumor-suppressor proteins and oncogenes. These may be due to alteration of catalytic activity of the protein, loss of binding sites for effector proteins or alterations of the native folded protein conformation. Src family kinases, p53, mTOR and C-terminus of HSC70 interacting protein (CHIPs) are some examples associated with protein misfolding and tumorigenesis. Molecular chaperones, such as heat-shock protein (HSP)70 and HSP90, assist protein folding and recognize target misfolded proteins for degradation. It is likely that this misfolding in cancer is linked by common principles, and may, therefore, present an exciting possibility to identify common targets for therapeutic intervention. Here we aim to review a number of examples that show how alterations in the folding of tumor-suppressor proteins or oncogenes lead to tumorigenesis. The possibility of targeting the targets to repair or degrade protein misfolding in cancer therapy is discussed.

Using genomics to develop novel antibacterial therapeutics.

As the genomics era matures, the availability of complete microbial genome sequences is facilitating computational approaches to understand bacterial genomes and DNA structure/function relationships. From the genome of pathogens, we can derive invaluable information on potential targets for new antimicrobial agents. Advancements in high-throughput 'omics' technologies and the availability of multiple isolates of the same species have significantly changed the time frame and scope for identifying novel therapeutic targets. This article aims to discuss selected aspects of the bacterial genome, and advocates 'omics'-based techniques to advance the discovery of new therapeutic targets against extracellular bacterial pathogens.

Chemical rescue of deltaF508-CFTR mimics genetic repair in cystic fibrosis bronchial epithelial cells.

In a previous study of sodium 4-phenylbutyrate (4-PBA)-responsive proteins in cystic fibrosis (CF) IB3-1 bronchial epithelial cells, we identified 85 differentially expressed high abundance proteins from whole cellular lysate (Singh, O. V., Vij, N., Mogayzel, P. J., Jr., Jozwik, C., Pollard, H. B., and Zeitlin, P. L. (2006) Pharmacoproteomics of 4-phenylbutyrate-treated IB3-1 cystic fibrosis bronchial epithelial cells. J. Proteome Res. 5, 562-571). In the present work we hypothesize that a subset of heat shock proteins that interact with cystic fibrosis transmembrane conductance regulator (CFTR) in common during chemical rescue and genetic repair will identify therapeutic networks for targeted intervention. Immunocomplexes were generated from total cellular lysates, and three subcellular fractions (endoplasmic reticulum (ER), cytosol, and plasma membrane) with anti-CFTR polyclonal antibody from CF (IB3-1), chemically rescued CF (4-PBA-treated IB3-1), and genetically repaired CF (IB3-1/S9 daughter cells repaired by gene transfer with adeno-associated virus-(wild type) CFTR). CFTR-interacting proteins were analyzed on two-dimensional gels and identified by mass spectrometry. A set of 16 proteins known to act in ER-associated degradation were regulated in common and functionally connected to the protein processing, protein folding, and inflammatory response. Some of these proteins were modulated exclusively in ER, cytosol, or plasma membrane. A subset of 4-PBA-modulated ER-associated degradation chaperones (GRP94, HSP84, GRP78, GRP75, and GRP58) was observed to associate with the immature B form of CFTR in ER. HSP70 and HSC70 interacted with the C band (mature form) of CFTR at the cell surface. We conclude that chemically rescued CFTR associates with a specific set of HSP70 family proteins that mark therapeutic interactions and can be useful to correct both ion transport and inflammatory phenotypes in CF subjects.

Medical Device Sterilization and Reprocessing in the Era of Multidrug-Resistant (MDR) Bacteria: Issues and Regulatory Concepts.

The emergence of multidrug-resistant (MDR) bacteria threatens humans in various health sectors, including medical devices. Since formal classifications for medical device sterilization and disinfection were established in the 1970's, microbial adaptation under adverse environmental conditions has evolved rapidly. MDR microbial biofilms that adhere to medical devices and recurrently infect patients pose a significant threat in hospitals. Therefore, it is essential to mitigate the risk associated with MDR outbreaks by establishing novel recommendations for medical device sterilization, in a world of MDR. MDR pathogens typically thrive on devices with flexible accessories, which are easily contaminated with biofilms due to previous patient use and faulty sterilization or reprocessing procedures. To prevent danger to immunocompromised individuals, there is a need to regulate the classification of reprocessed medical device sterilization. This article aims to assess the risks of improper sterilization of medical devices in the era of MDR when sterilization procedures for critical medical devices are not followed to standard. Further, we discuss key regulatory recommendations for consistent sterilization of critical medical devices in contrast to the risks of disinfection reusable medical devices.

Extracellular Synthesis and Characterization of Silver Nanoparticles from Alkaliphilic Pseudomonas sp.

Bio-nanotechnology offers eco-friendly processes for the synthesis of stable nanoparticles (NPs). We hypothesized that microorganisms isolated from the root nodules of leguminous plants would biosynthesize silver (Ag) bio-nanoparticles. Clover root nodules enriched with nutrient broth (NB) produced four distinct colonies on NA plates. Microbial colonies were purified by repeated streaking and designated as SS6, SS7, SS8, and SS9 for identification using 16S rRNA sequencing. Four species of Pseudomonas were identified with a similarity score of over 99% using the EZ Taxon search engine, and tested for extracellular biosynthesis of AgNPs. Microorganism Pseudomonas taiwanensis-SS8 with alkaliphilic growth characteristics reduced the AgNO3 solution into AgNPs in the shortest time period. AgNPs were characterized using UV-Vis spectrophotometry and electron and transmission electron microscopy. A number of physical (i.e., temperature and time) and chemical (i.e., pH and growth media) parameters were optimized. An efficient polydispersal biosynthesis of AgNPs at pH 8-9 after 48 hrs in NB growth medium was observed. In addition, the AgNPs showed antimicrobial properties against 16 commonly occurring pathogenic microorganisms.

Proteome of synaptosome-associated proteins in spinal cord dorsal horn after peripheral nerve injury.

Peripheral nerve injury may lead to neuroadaptive changes of cellular signals in spinal cord that are thought to contribute to central mechanisms underlying neuropathic pain. Here we used a 2-DE-based proteomic technique to determine the global expression changes of synaptosome-associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury (SNI). The fifth lumbar dorsal horns ipsilateral to SNI or sham surgery were harvested on day 14 post-surgery, and the total soluble and synaptosomal fractions were isolated. The proteins derived from the synaptosomal fraction were resolved by 2-DE. We identified 27 proteins that displayed different expression levels after SNI, including proteins involved in transmission and modulation of noxious information, cellular metabolism, membrane receptor trafficking, oxidative stress, apoptosis, and degeneration. Six of the 27 proteins were chosen randomly and further validated in the synaptosomal fraction by Western blot analysis. Unexpectedly, Western blot analysis showed that only one protein in the total soluble fraction exhibited a significant expression change after SNI. The data indicate that peripheral nerve injury changes not only protein expression but also protein subcellular distribution in dorsal horn cells. These changes might participate in the central mechanism that underlies the maintenance of neuropathic pain.

High-dimensional biology to comprehend hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and is the third leading cause of death from cancer. The diverse etiology, high morbidity/mortality, lack of diagnostic markers for early diagnosis and the highly variable clinical course of HCC have hindered advances in diagnosis and treatment. Microsatellite instability, chromosomal aberrations, mutations in key cell cycle genes and epigenetic changes have been reported in HCC. Availability of modern technologies advance 'high-dimensional biology' (HDB), a term that refers to the simultaneous study of the genetic variants (genome), transcription (mRNA; transcriptome), peptides and proteins (proteomics), and metabolites (metabolomics) for the intermediate products of metabolism of an organ, tissue or organism. The growing interest in omics-based research has enabled the simultaneous examination of thousands of genes, transcripts and proteins of interest, with high-throughput techniques and advanced analytical tools for data analysis. The use of each approach towards functional omics has lead to the classification of HCC into molecular subgroups. Here we review the use of HDB as a tool for the identification of markers for screening, diagnosis, molecular classification and the discovery of new therapeutic drug targets of HCC. With the extensive use of HDB, it may be possible in the near future, to have custom-made therapeutic regimens for HCC based on the molecular subtype, ultimately leading to an improved survival of HCC patients.

Bioremediation of radionuclides: emerging technologies.

A large quantity of radioactive waste is being generated as the byproduct of atomic energy and related programs worldwide. There are multiple radioactive waste dumping sites, that, if exposed to the general population, may cause serious life-threatening disorders. Currently, no efficient technology is available that can store the radioactive wastes with adequate safety. Therefore, bioremediation of radionuclides/radioactive waste is an unavoidable necessity that has been tried using biotransformation, bioaccumulation, biosorption, biostimulation, and bioaugmentaion, with limited success. Genetic engineering has been implemented to develop an organism that can effectively detoxify radionuclides along with other organic pollutants present as co-contaminants in the radioactive waste sites. However, the lack of system-wide information regarding factors regulating growth and metabolism of microbial communities can be conquered by newly seeded "-omics"-based technologies, viz. transcriptomics and proteomics. Studies combining functional transcriptomics and proteomics would create a system-wide approach studying the microbial metabolism in radionuclides detoxification.

Iridium(I)-catalyzed stereospecific decarboxylative allylic amidation of chiral branched benzyl allyl imidodicarboxylates.

Ir(I)-catalyzed decarboxylative allylic amidation of chiral branched benzyl allyl imidodicarboxylates has been shown to proceed with complete retention of enantiomeric purity and configuration. The transformation is stereospecific and appears to be quite general, accommodating a wide range of R groups.