Small Molecular Weight Molecules Research Articles

Predicting ion mobility collision cross sections and assessing prediction variation by combining conventional and data driven modeling

The use of collision cross section (CCS) values derived from ion mobility studies is proving to be an increasingly important tool in the characterization and identification of molecules detected in complex mixtures. Here, a novel machine learning (ML) based method for predicting CCS integrating both molecular modeling (MM) and ML methodologies has been devised and shown to be able to accurately predict CCS values for singly charged small molecular weight molecules from a broad range of chemical classes. The model performed favorably compared to existing models, improving compound identifications for isobaric analytes in terms of ranking and assigning identification probability values to the annotation. Furthermore, charge localization was seen to be correlated with CCS prediction accuracy and with gas-phase proton affinity demonstrating the potential to provide a proxy for prediction error based on chemical structural properties. The presented approach and findings represent a further step towards accurate prediction and application of computationally generated CCS values.

Removal of urea in ultrapure water system by urease-coated reverse osmosis membrane

Among the various substances found in the feed source for the production of ultrapure water (UPW), urea is challenging to remove because it is a small molecular weight molecule that is not easily oxidized and does not carry a charge under neutral pH conditions. Urease enzyme, found in various organisms such as plants and bacteria, catalyze the hydrolysis of urea into carbon dioxide and ammonia. In this study, urease was immobilized on the polyamide layer of a reverse osmosis (RO) membrane to remove urea in UPW systems. The removal efficiency of urea by urease-coated RO membrane showed up to 27.9 % higher urea removal efficiency compared to the pristine membrane. This increase in urea removal can be attributed to both physical and biological effects from the urease coating on the membrane. Firstly, urease on the membrane surface can act as an additional physical barrier for urea to pass through. Secondly, urea can be hydrolyzed by the enzyme when it passes through the urease-coated RO membrane. In a two-pass RO system typical for UPW production, the removal of urea by a urease-coated membrane would be enhanced by twofold. This overall method can significantly increase the removal efficiency of urea in UPW systems, especially when considering the compounded removal by the urease coating, rejection by RO, and additional reactions by other treatment processes. Moreover, urea in UPW systems can be removed without the installment of additional processes by simply coating urease on the existing RO membranes.

Cannabidiol Inhibits Tumorigenesis in Cisplatin-Resistant Non-Small Cell Lung Cancer via TRPV2.

Simple SummaryDrug resistance is the key factor contributing to the therapeutic failure of lung cancer and the deaths related to lung cancer. Our study demonstrated that small molecular weight non-psychotropic phytochemical, cannabidiol (CBD), inhibits growth and metastasis of drug-resistant non-small cell lung cancer cells (NSCLC) cells in-vitro and in-vivo. We further discovered that CBD mediates its anti-cancer effects in part via an ion channel receptor, TRPV2, present on lung adenocarcinoma. Moreover, we showed that CBD induces apoptosis of cisplatin-resistant cells by modulating oxidative stress pathways. Overall, these studies indicate that CBD could be used as a promising therapeutic strategy in TRPV2 expressing cisplatin-resistant NSCLC.Chemotherapy forms the backbone of current treatments for many patients with advanced non-small-cell lung cancer (NSCLC). However, the survival rate is low in these patients due to the development of drug resistance, including cisplatin resistance. In this study, we developed a novel strategy to combat the growth of cisplatin-resistant (CR) NSCLC cells. We have shown that treatment with the plant-derived, non-psychotropic small molecular weight molecule, cannabidiol (CBD), significantly induced apoptosis of CR NSCLC cells. In addition, CBD treatment significantly reduced tumor progression and metastasis in a mouse xenograft model and suppressed cancer stem cell properties. Further mechanistic studies demonstrated the ability of CBD to inhibit the growth of CR cell lines by reducing NRF-2 and enhancing the generation of reactive oxygen species (ROS). Moreover, we show that CBD acts through Transient Receptor Potential Vanilloid-2 (TRPV2) to induce apoptosis, where TRPV2 is expressed on human lung adenocarcinoma tumors. High expression of TRPV2 correlates with better overall survival of lung cancer patients. Our findings identify CBD as a novel therapeutic agent targeting TRPV2 to inhibit the growth and metastasis of this aggressive cisplatin-resistant phenotype in NSCLC.

Kinetic Study of Encapsulated β-Carotene Degradation in Dried Systems: A Review.

β-Carotene serves as a precursor of vitamin A and provides relevant health benefits. To overcome the low bioavailability of β-carotene from natural sources, technologies have been designed for its encapsulation in micro- and nano-structures followed by freeze-drying, spray-drying, supercritical fluid-enhanced dispersion and electrospraying. A technological challenge is also to increase β-carotene stability, since due to its multiple conjugated double bonds, it is particularly prone to oxidation. This review analyzes the stability of β-carotene encapsulated in different dried micro- and nano-structures by comparing rate constants and activation energies of degradation. The complex effect of water activity and glass transition temperature on degradation kinetics is also addressed, since the oxidation process is remarkably dependent on the glassy or collapsed state of the matrix. The approaches to improve β-carotene stability, such as the development of inclusion complexes, the improvement of the performance of the interface between air and oil phase in which β-carotene was dissolved by application of biopolymer combinations or functionalization of natural biopolymers, the addition of hydrophilic small molecular weight molecules that reduce air entrapped in the powder and the co-encapsulation of antioxidants of various polarities are discussed and compared, in order to provide a rational basis for further development of the encapsulation technologies.

Cardiovascular Benefits of Extended-Time Nocturnal Hemodialysis.

Hemodialysis (HD) remains the most utilized treatment for End-Stage Kidney Disease (ESKD) globally, mainly as conventional HD administered in 4 h sessions thrice weekly. Despite advances in HD delivery, patients with ESKD carry a heavy cardiovascular morbidity and mortality burden. This is associated with cardiac remodeling, left ventricular hypertrophy (LVH), myocardial stunning, hypertension, decreased heart rate variability, sleep apnea, coronary calcification and endothelial dysfunction. Therefore, intensive HD regimens closer to renal physiology were developed. They include longer, more frequent dialysis or both. Among them, Nocturnal Hemodialysis (NHD), carried out at night while asleep, provides efficient dialysis without excessive interference with daily activities. This regimen is closer to the physiology of the native kidneys. By providing increased clearance of small and middle molecular weight molecules, NHD can ameliorate uremic symptoms, control hyperphosphatemia and improve quality of life by allowing a liberal diet and free time during the day. Lastly, it improves reproductive biology leading to successful pregnancies. Conversion from conventional to NHD is followed by improved blood pressure control with fewer medications, regression of LVH, improved LV function, improved sleep apnea, and stabilization of coronary calcifications. These beneficial effects have been associated, among others, with better extracellular fluid volume control, improved endothelial- dependent vasodilation, decreased total peripheral resistance, decreased plasma norepinephrine levels and restoration of heart rate variability. Some of these effects represent improvements in outcomes used as surrogates of hard outcomes related to cardiovascular morbidity and mortality. In this review, we consider the cardiovascular effects of NHD.

Hemocompatible hemoadsorbent for effective removal of protein-bound toxin in serum

Resin hemoperfusion is a life-saving treatment for drug intoxication or hepatic failure of patients. However, current resin adsorbents exhibit a limited hemocompatibility or low adsorption efficiency, representing a major roadblock to successful clinical applications. In this work, we developed a hemocompatible and effective hemoadsorbent based on polystyrene resin (H103) microparticles encapsulated in anti-biofouling zwitterionic poly(carboxybetaine) (PCB) hydrogels. Apart from a strong mechanical stability, this PCB-based adsorbent (PCB-H103) exhibited excellent hemocompatibility (hemolysis ratio was ∼0.64%), which was attributed to the anti-biofouling property of PCB hydrogel. In addition, it can efficiently adsorb both small and middle molecular weight molecules in phosphate-buffered saline, and the efficiencies were significantly higher than poly(ethylene glycol) methacrylate-based and poly(2-hydroxyethyl methacrylate)-based adsorbent counterparts, indicating the favorable permeability of PCB hydrogel coating. More importantly, PCB-H103 could effectively remove protein-bound toxins including phenol red and bilirubin in bovine serum albumin solution or even in 100% fetal bovine serum (FBS). In 100% FBS, the adsorption capacity of PCB-H103 towards bilirubin was 8.3 times higher than that of pristine clinical-scale resin beads. Findings in this work may provide a new strategy for the development of modern resin hemoperfusion technology.

Early Stage Discovery and Validation of Pharmacological Chaperones for the Correction of Protein Misfolding Diseases.

Pharmacological chaperones are small molecular weight molecules that bind specifically to protein targets and stabilize unstable and misfolded conformations. In particular, there is an increasing interest in the application of this type of compounds for the correction of genetic conformational disorders, which are caused by mutations leading to protein instability. The discovery of compounds with pharmacological chaperone ability is customarily initiated by a high-throughput screening of chemical libraries searching for stabilizing binders. However, there is no established consensus for the subsequent steps. Therefore, here, we introduce an example of a successful protocol directed to the discovery of pharmacological chaperones with potential for the therapeutic correction of phenylketonuria, a defect caused by mutations in the enzyme phenylalanine hydroxylase.

Role of Osmolytes and their Transporter Systems in Pathogen Survival and Pathogenicity.

Pathogenic microbes routinely keep encountering different types of stress in their environment such as high osmolarity, high temperature, pH fluctuations in host gut etc. They tend to acquire certain small molecular weight molecules, termed osmolytes, so as to handle these challenges and survive in harsh conditions. These osmolytes include some which are self-synthesized by the microorganisms, while majority of them are imbibed from the external environment via osmolyte transporters. In this review, we have discussed work done on osmolytes and their transport systems, which influence upon sustenance and virulence of the pathogens under the given stress conditions. Osmolytes and their transport systems play vital role in efficient maintenance of cell turgidity and electrolyte levels for proper cell functioning. These molecules don't disturb normal metabolic processes within the microbial cell at all; it rather stabilizes the macromolecules, such as nucleic acids and proteins. Besides, these osmolytes also empower the microbes with the pathogenicity potential under harsh conditions such as salt, pH, temperature stress, and the efficient host immunity. Exploring avenues with respect to osmolyte transport systems is the need of the hour especially in this time where we are facing the evolution of antibiotic resistance in pathogens. Most interestingly, a detailed study of bacterial stress responses would prove to be useful in comprehending what these pathogens face in the host microenvironment, whereby we can manipulate the pathogen survival in human gut, and hence address the menace of pathogenic resistance in humans as well as animals.

MALDI coupled to modified traveling wave ion mobility mass spectrometry for fast enantiomeric determination.

In this work, the use of MALDI traveling wave ion mobility spectrometry-mass spectrometry (MALDI-TWIMS-MS) for stereoselective structural analysis of direct cleavage and identification of 2-substituted piperidines obtained through solid-phase asymmetric synthesis by using heterogeneous 8-phenylmenthyl-based chiral auxiliary resins. A strategy for gas-phase chiral and structural characterization of small molecular weight molecules by using MALDI-IMS-MS technique is discussed. Because both MALDI and IMS do not directly offer chiral resolution, an easy methodology by adding a chiral phase is described to carry out in situ online ion/molecule complexation with different chiral analytes inside the mass spectrometer. Piperidine enantiomers were resolved, and separation obtained shows dependence of surface areas. To corroborate this assumption and elucidate the separation mechanism to accomplish an analytical technique by which fast determination of the chirality of molecules may be determined for a wide range organic compound applications, it was performed DFT calculations to determine the cross-sectional areas of proton-bound dimer complexes. Drift times are affected by cross-sectional areas, correlating bigger times with bigger molecular volumes during the ion mobility experiments of proton-bound dimer complexes.

Chemoreception of botanical nematicides by Meloidogyne incognita and Caenorhabditis elegans

ABSTRACTPlant-parasitic nematodes, such as Meloidogyne incognita, cause serious damage to various agricultural crops worldwide, and their control necessitates environmentally safe measures. We have studied the effects of plant secondary metabolites on M. incognita locomotion, as it is an important factor affecting host inoculation inside the soil. We compared the effects to the respective behavioral responses of the model saprophytic nematode Caenorhabditis elegans. The tested botanical nematicides, all reported to be active against Meloidogyne sp. in our previous works, are small molecular weight molecules (acids, alcohols, aldehydes, and ketones). Here, we specifically report on the attractant or repellent properties of trans-anethole, (E,E)-2,4-decadienal, (E)-2-decenal, fosthiazate, and 2-undecanone. The treatments for both nematode species were made at sublethal concentration levels, namely, 1 mM (<EC50), and the chemical controls used for the experiments were the commercial nematicides fosthiazate and oxamyl. According to our results, trans-anethole, decenal, and oxamyl attract C. elegans, while 2-undecanone strongly attracts M. incognita. These findings can be of use in the development of nematicidal formulations, contributing to the disruption of nematode chemotaxis to root systems.

Efficient zinc uptake is critical for the ability of Pseudomonas aeruginosa to express virulence traits and colonize the human lung

We have recently shown that Pseudomonas aeruginosa, an opportunistic pathogen that chronically infects the lungs of patients with cystic fibrosis (CF) and other forms of lung disease, is extremely efficient in recruiting zinc from the environment and that this capability is required for its ability to cause acute lung infections in mice. To verify that P. aeruginosa faces zinc shortage when colonizing the lungs of human patients, we analyzed the expression of three genes that are highly induced under conditions of zinc deficiency (zrmA, dksA2 and rpmE2), in bacteria in the sputum of patients with inflammatory lung disease. All three genes were expressed in all the analyzed sputum samples to a level much higher than that of bacteria grown in zinc-containing laboratory medium, supporting the hypothesis that P. aeruginosa is under zinc starvation during lung infections. We also found that the expression of several virulence traits that play a central role in the ability of P. aeruginosa to colonize the lung is affected by disruption of the most important zinc importing systems. Virulence features dependent on zinc intake include swarming and swimming motility and the ability to form biofilms. Furthermore, alterations in zinc assimilation interfere with the synthesis of the siderophore pyoverdine, suggesting that zinc recruitment could modulate iron uptake and affect siderophore-mediated cell signaling. Our results reveal that zinc uptake is likely to play a key role in the ability of P. aeruginosa to cause chronic lung infections and strongly modulates critical virulence traits of the pathogen. Taking into account the recent discovery that zinc uptake in P. aeruginosa is promoted by the release of a small molecular weight molecule showing high affinity for zinc, our data suggest novel and effective possibilities to control lung infections by these bacteria.

Removal of uremic toxins by renal replacement therapies: a review of current progress and future perspectives

Accumulation of uremic toxins induces various uremia-related complications in patients with chronic kidney disease, particularly those undergoing dialysis treatment. Direct interactions of uremic toxins with organ tissues are thought to be a major pathophysiological mechanism for disease; for example, indoxyl sulfate reacts directly with macrophages and accelerates atherosclerosis. The removal of sufficient volume of uremic toxins will prevent uremia-related complications in dialysis patients. Hemodialysis with the use of a high-flux dialysis membrane, long or frequent treatment, and increased blood/dialysate flow has improved removal of water-soluble small molecular weight uremic toxins. Middle molecular weight molecules are removed more effectively with hemodialysis using a high-flux membrane, hemodiafiltration and hemofiltration, and a direct hemoperfusion method using β2-microglobulin adsorption column, which is useful in reducing serum β2-microglobulin levels as well as improving dialysis-related amyloidosis-induced clinical symptoms. With improvements in dialysis therapies, removal of low and middle molecular weight water-soluble molecules has improved; however, conventional dialysis treatment is limited in its ability to remove protein-bound uremic toxins (PBUTs). Recent findings suggest that adsorption treatments using oral charcoal adsorbent, mixed matrix membrane hollow fiber, and additive charcoal in the dialysate, in addition to conventional dialysis treatment, may effectively remove a substantial amount of PBUTs. Further improvement of renal replacement therapy, including dialysis and additional therapeutic strategies, is needed for better clearance of small and middle molecular weight molecules and PBUTs, which will lead to improved survival and quality of life for dialysis-dependent chronic kidney disease patients.

Water-based preparation of spider silk films as drug delivery matrices

The main focus of this work was to obtain a drug delivery matrix characterized by biocompatibility, water insolubility and good mechanical properties. Moreover the preparation process has to be compatible with protein encapsulation and the obtained matrix should be able to sustain release a model protein. Spider silk proteins represent exceptional natural polymers due to their mechanical properties in combination with biocompatibility. As both hydrophobic and slowly biodegrading biopolymers, recombinant spider silk proteins fulfill the required properties for a drug delivery system. In this work, we present the preparation of eADF4(C16) films as drug delivery matrices without the use of any organic solvent. Water-based spider silk films were characterized in terms of protein secondary structure, thermal stability, zeta-potential, solubility, mechanical properties, and water absorption and desorption. Additionally, this study includes an evaluation of their application as a drug delivery system for both small molecular weight drugs and high molecular weight molecules such as proteins. Our investigation focused on possible improvements in the film's mechanical properties including plasticizers in the film matrix. Furthermore, different film designs were prepared, such as: monolayer, coated monolayer, multilayer (sandwich), and coated multilayer. The release of the model protein BSA from these new systems was studied. Results indicated that spider silk films are a promising protein drug delivery matrix, capable of releasing the model protein over 90days with a release profile close to zero order kinetic. Such films could be used for several pharmaceutical and medical purposes, especially when mechanical strength of a drug eluting matrix is of high importance.

Astrocytic β1-integrin affects cellular composition of murine blood brain barrier in the cerebral cortex

The blood brain barrier (BBB) is composed of endothelial cells, astrocytes, and pericytes and maintains functional homeostasis by regulating transport of ions, fluid and cells between blood and neural tissue. The cellular and molecular pathways that contribute to the formation of the BBB in the developing brain have not been fully deciphered. β1-integrin (β1-itg) within endothelial cells is known to play a critical role in vasculogenesis. However, the role of astrocytic β1-itg in BBB development is not known. Our study used a mouse glial fibrillary acidic protein (GFAP)-cre transgenic line to selectively ablate β1-itg within astrocytes. We found that deletion of astrocytic β1-itg had a striking effect on the different cell types that form the BBB. Mutant mice had a decreased density of aquaporin-4 immunoreactivity within the perivascular astrocytic end-feet. We also found decreases in immunoreactivity for vimentin and CD-31 within endothelial cells. These changes were not accompanied by functional changes in BBB under physiological conditions as assessed by extravasation of large and small molecular weight molecules. However, mutant mice had an increased incidence of severe cystic injury in response to neonatal hypoxia. Our findings show that astrocytic β1-itg has an important role in defining cellular properties of the blood brain barrier in the cerebral cortex.

A pretargeting system for tumor PET imaging and radioimmunotherapy.

Labeled antibodies, as well as their fragments and antibody-derived recombinant constructs, have long been proposed as general vectors to target radionuclides to tumor lesions for imaging and therapy. They have indeed shown promise in both imaging and therapeutic applications, but they have not fulfilled the original expectations of achieving sufficient image contrast for tumor detection or sufficient radiation dose delivered to tumors for therapy. Pretargeting was originally developed for tumor immunoscintigraphy. It was assumed that directly-radiolabled antibodies could be replaced by an unlabeled immunoconjugate capable of binding both a tumor-specific antigen and a small molecular weight molecule. The small molecular weight molecule would carry the radioactive payload and would be injected after the bispecific immunoconjugate. It has been demonstrated that this approach does allow for both antibody-specific recognition and fast clearance of the radioactive molecule, thus resulting in improved tumor-to-normal tissue contrast ratios. It was subsequently shown that pretargeting also held promise for tumor therapy, translating improved tumor-to-normal tissue contrast ratios into more specific delivery of absorbed radiation doses. Many technical approaches have been proposed to implement pretargeting, and two have been extensively documented. One is based on the avidin-biotin system, and the other on bispecific antibodies binding a tumor-specific antigen and a hapten. Both have been studied in preclinical models, as well as in several clinical studies, and have shown improved targeting efficiency. This article reviews the historical and recent preclinical and clinical advances in the use of bispecific-antibody-based pretargeting for radioimmunodetection and radioimmunotherapy of cancer. The results of recent evaluation of pretargeting in PET imaging also are discussed.

Aptamer-functionalized porous phospholipid nanoshells for direct measurement of Hg(2+) in urine.

A porous phospholipid nanoshell (PPN) sensor functionalized with a specific aptamer sensor agent was prepared for rapid detection of Hg(2+) in human urine with minimal sample preparation. Aptamer sensors provide an important class of optical transducers that can be readily and reproducibly synthesized. A key limitation of aptamer sensors, and many other optical sensors, is the potential of biofouling or biodegradation when used in complex biological matrices such as serum or urine, particularly when high levels of nucleases are present. We prepared Hg(2+)-responsive, PPN-encapsulated aptamer sensors that overcome these limitations. PPNs provide a protective barrier to encapsulate the aptamer sensor in an aqueous environment free of diffusional restrictions encountered with many polymer nanomaterials. The unique porous properties of the PPN membrane enable ready and rapid transfer of small molecular weight ions and molecules into the sensor interior while minimizing the macromolecular interactions between the transducer and degradants or interferents in the exterior milieu. Using Hg(2+)-responsive, PPN-encapsulated aptamer sensors, we were able to detect sub-100ppb (chronic threshold limit from urine test) Hg(2+) in human urine with no sample preparation, whereas free aptamer sensors yielded inaccurate results due to interferences from the matrix. The PPN architecture provides a new platform for construction of aptamer-functionalized sensors that target low molecular weight species in complex matrices, beyond the Hg(2+) demonstrated here.

The interaction between nanoparticles and biological barriers

Biological barriers are truly one of the most perfect biological machines to have been studied. They perform a primary function of preventing undesired access to sensitive organs, while allowing for limited and selected passage across. They thereby manage access of a variety of molecules and assemblies with an efficiency and fidelity that no man-made engineered filter or barrier can even approach. Without such a barrier, for example the lungs, our bloodstream and subsequently all our organs would be exposed to a multitude of dangerous inhalation scenarios. Most delicate of all, the brain would be unable to function in the face of a variety of infections and other challenges. The basis of the success of the biological barrier is that the primary component of the barrier is composed of an array of tightly joined cells backed up by an array of other cells involved in clearance, immunity and other processes. Thus, while some very small molecular weight molecules can diffuse or passively partition across the barriers, most molecules and other undesirable larger assemblies are excluded, or removed, by active biological processes. Those larger molecules that pass the barrier must traffic across using active energy dependent biological processes, during which they are exposed to many intracellular checks and analyses. Naturally, the very success of biological barriers in excluding passage can present very significant (arguably, currently often insuperable) challenges for the delivery of therapies across the barriers. For example, many of the more intractable human brain-related diseases ranging from neurodegeneration, persistent drug cocktail resistant viral infection (HIV for example), and glioblastomas, are exacerbated by the difficulty of gaining therapeutic access to the brain. Great inconvenience and expense is added to numerous other therapeutic scenarios in which biological barriers have to be crossed before drugs can act. Nanomedicine, were it to allow us to address these challenges, would by this achievement alone change the face of medicine, and justify the endeavour. However, a survey of recent efforts suggests that nanoparticles, while they offer important developmental directions, do not automatically, and, naively applied, will not lead to immediate simplistic solutions, based on size alone. Certainly nanoparticles are exceptional in that, due to their

Nanomaterials enhanced surface plasmon resonance for biological and chemical sensing applications

The main challenge for all electrical, mechanical and optical sensors is to detect low molecular weight (less than 400 Da) chemical and biological analytes under extremely dilute conditions. Surface plasmon resonance sensors are the most commonly used optical sensors due to their unique ability for real-time monitoring the molecular binding events. However, their sensitivities are insufficient to detect trace amounts of small molecular weight molecules such as cancer biomarkers, hormones, antibiotics, insecticides, and explosive materials which are respectively important for early-stage disease diagnosis, food quality control, environmental monitoring, and homeland security protection. With the rapid development of nanotechnology in the past few years, nanomaterials-enhanced surface plasmon resonance sensors have been developed and used as effective tools to sense hard-to-detect molecules within the concentration range between pmol and amol. In this review article, we reviewed and discussed the latest trend and challenges in engineering and applications of nanomaterials-enhanced surface plasmon resonance sensors (e.g., metallic nanoparticles, magnetic nanoparticles, carbon-based nanomaterials, latex nanoparticles and liposome nanoparticles) for detecting "hard-to-identify" biological and chemical analytes. Such information will be viable in terms of providing a useful platform for designing future ultrasensitive plasmonic nanosensors.

Chimerization of lactoferricin and lactoferrampin peptides strongly potentiates the killing activity against Candida albicans1This article is part of a Special Issue entitled Lactoferrin and has undergone the Journal's usual peer review process.

Bovine lactoferrin harbors 2 antimicrobial sequences (LFcin and LFampin), situated in close proximity in the N1-domain. To mimic their semi parallel configuration we have synthesized a chimeric peptide (LFchimera) in which these sequences are linked in a head-to-head fashion to the α- and ε-amino group, respectively, of a single lysine. In line with previously described bactericidal effects, this peptide was also a stronger candidacidal agent than the antimicrobial peptides LFcin17–30 and LFampin265–284, or a combination of these 2. Conditions that strongly reduced the candidacidal activities of LFcin17–30 and LFampin265–284, such as high ionic strength and energy depletion, had little influence on the activity of LFchimera. Freeze-fracture electron microscopy showed that LFchimera severely affected the membrane morphology, resulting in disintegration of the membrane bilayer and in an efflux of small and high molecular weight molecules such as ATP and proteins. The differential effects displayed by the chimeric peptide and a mixture of its constituent peptides clearly demonstrate the synergistic effect of linking these peptides in a fashion that allows a similar spatial arrangement as in the parent protein, suggesting that in bovine lactoferrrin the corresponding fragments act in concert in its candidacidal activity.

Metabolomics and histology on exactly the same ex vivo prostate biopsy.

166 Background: Histopathology is the standard method for diagnosis and histological grading and staging of cancer. Molecular biomarkers are associated with cancer aggressiveness. One drawback to some molecular studies is that the portion of the tissue to be analyzed is processed in a manner that is destructive to the tissue. We present here a new method for performing analysis of small molecule biomarkers and histology in exactly the same biopsy tissue. The process, molecular Preservation by Extraction and Fixation (mPREF), allows traditional histological examination to be combined with qualitative and quantitative analysis of small molecule biomarkers. Methods: 18 gauge core biopsies were obtained ex vivo from prostatectomy specimens. One core biopsy/specimen container was immersed in aqueous alcohol. After 12-24 hrs, biopsies were transferred to UMFIX, and the original alcohol retained. Histologic sections of biopsies were stained by routine methods and the immunohistochemical (IHC) stain, PIN4. Small molecular weight molecules in the retained alcohol were examined by a mass spectrometry-based method. Results: Light Microscopy: Tumor and grade were evaluable and IHC for PIN4 performed well. Metabolomics of Retained Alcohol: From a single core biopsy, 260 named biochemical compounds and their relative concentrations WERE detected. 83 were different between the cancer and non-cancer biopsy extracts (p&lt; .05). 18 of the 20 common amino acids, and a number of long chain fatty acids and phospholipids were increased. Highest fold changes, 4- to 6-fold, were observed in cysteine, dihomo-linoleate, docosapentaenoate, N-acetylaspartate, N-acetylglucosamine, uracil, xanthine, and 1-stearoylglycerophosphoinositol. A sub-set of candidate biomarkers were differentially expressed in pT3 vs pT2 disease. Conclusions: MPREF allows for both histology and detection of small molecules in exactly the same tissue. The results suggest a higher metabolic state in cancer as compared with non-cancer containing biopsy tissues. Differential expression of small molecules from pT2 vs pT3 disease suggests that quantitative prognostic molecular and histologic data can be obtained from a single core biopsy.