Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro.

BACKGROUND: Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. METHODS: The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry. RESULTS: MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.

Scorpion toxins interact with nicotinic acetylcholine receptors.

Neurotoxins are among the main components of scorpion and snake venoms. Scorpion neurotoxins affect voltage-gated ion channels, while most snake neurotoxins target ligand-gated ion channels, mainly nicotinic acetylcholine receptors (nAChRs). We report that scorpion venoms inhibit α-bungarotoxin binding to both muscle-type nAChR from Torpedo californica and neuronal human α7 nAChR. Toxins inhibiting nAChRs were identified as OSK-1 (α-KTx family) from Orthochirus scrobiculosus and HelaTx1 (κ-KTx family) from Heterometrus laoticus, both being blockers of voltage-gated potassium channels. With an IC50 of 1.6 µm, OSK1 inhibits acetylcholine-induced current through mouse muscle-type nAChR heterologously expressed in Xenopus oocytes. Other well-characterized scorpion toxins from these families also bind to Torpedo nAChR with micromolar affinities. Our results indicate that scorpion neurotoxins present target promiscuity.

Microfluidic isolation of platelet-covered circulating tumor cells.

The interplay between platelets and tumor cells is known to play important roles in metastasis by enhancing tumor cell survival, tumor-vascular interactions, and escape from immune surveillance. However, platelet-covered circulating tumor cells (CTC) are extremely difficult to isolate due to masking or downregulation of surface epitopes. Here we describe a microfluidic platform that takes advantage of the satellite platelets on the surface of these "stealth" CTCs as a ubiquitous surface marker for isolation. Compared to conventional CTC enrichment techniques which rely on known surface markers expressed by tumor cells, platelet-targeted isolation is generally applicable to CTCs of both epithelial and mesenchymal phenotypes. Our approach first depletes unbound, free platelets by means of hydrodynamic size-based sorting, followed by immunoaffinity-based capture of platelet-covered CTCs using a herringbone micromixing device. This method enabled the reliable isolation of CTCs from 66% of lung and 60% of breast cancer (both epithelial) patient samples, as well as in 83% of melanoma (mesenchymal) samples. Interestingly, we observed special populations of CTCs that were extensively covered by platelets, as well as CTC-leukocyte clusters. Because these cloaked CTCs often escape conventional positive and negative isolation mechanisms, further characterization of these cells may uncover important yet overlooked biological information in blood-borne metastasis and cancer immunology.

Temporal and intrinsic factors of rifampicin tolerance in mycobacteria.

Mycobacteria grow and divide asymmetrically, creating variability in growth pole age, growth properties, and antibiotic susceptibilities. Here, we investigate the importance of growth pole age and other growth properties in determining the spectrum of responses of Mycobacterium smegmatis to challenge with rifampicin. We used a combination of live-cell microscopy and modeling to prospectively identify subpopulations with altered rifampicin susceptibility. We found two subpopulations that had increased susceptibility. At the initiation of treatment, susceptible cells were either small and at early stages of the cell cycle, or large and in later stages of their cell cycle. In contrast to this temporal window of susceptibility, tolerance was associated with factors inherited at division: long birth length and mature growth poles. Thus, rifampicin response is complex and due to a combination of differences established from both asymmetric division and the timing of treatment relative to cell birth.

Microfluidic assay for precise measurements of mouse, rat, and human neutrophil chemotaxis in whole-blood droplets.

Animal models of human disease differ in innate immune responses to stress, pathogens, or injury. Precise neutrophil phenotype measurements could facilitate interspecies comparisons. However, such phenotype comparisons could not be performed accurately with the use of current assays, as they require the separation of neutrophils from blood using species-specific protocols, and they introduce distinct artifacts. Here, we report a microfluidic technology that enables robust characterization of neutrophil migratory phenotypes in a manner independent of the donor species and performed directly in a droplet of whole blood. The assay relies on the particular ability of neutrophils to deform actively during chemotaxis through microscale channels that block the advance of other blood cells. Neutrophil migration is measured directly in blood, in the presence of other blood cells and serum factors. Our measurements reveal important differences among migration counts, velocity, and directionality among neutrophils from 2 common mouse strains, rats, and humans.

Microfluidic platform for measuring neutrophil chemotaxis from unprocessed whole blood.

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

Vietnamese Heterometrus laoticus scorpion venom: evidence for analgesic and anti-inflammatory activity and isolation of new polypeptide toxin acting on Kv1.3 potassium channel.

The scorpion Heterometrus laoticus (Scorpionidae) inhabits Indochinese peninsula and is widely distributed in South-West Vietnam. Since no human fatalities caused by H. laoticus stings were reported, no systematic characterization of the venom was earlier done. In this study we report on biological activity of the venom from H. laoticus caught in Vietnamese province An Giang. The venom manifested a very low acute toxicity with LD50 of about 190 mg/kg body weight in mice at subcutaneous (s.c.) injection and 12 mg/kg at intravenous injection. The venom analgesic effects using tail immersion and writhing tests as well as anti-inflammatory effect using carrageenan test were analyzed at doses of 9.5 and 19 mg/kg at s.c. injections. It was found that at two doses tested H. laoticus venom showed both anti-nociceptive and anti-inflammatory activity. The venom was fractionated by means of gel-filtration and reversed-phase HPLC. As a result several polypeptide toxins were isolated and new toxin hetlaxin was identified. Its amino acid sequence was determined and binding to the extracellular vestibule of the K⁺-conducting pore of Kv1.1 and Kv1.3 potassium channels was studied. Hetlaxin belongs to the scorpion alpha-toxin family and is the first toxin isolated from H. laoticus venom which possesses high affinity (K(i) 59 nM) to Kv1.3 potassium channel.

Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system.

Humans and mice infected with different Plasmodium strains are known to produce microvesicles derived from the infected red blood cells (RBCs), denoted RMVs. Studies in mice have shown that RMVs are elevated during infection and have proinflammatory activity. Here we present a detailed characterization of RMV composition and function in the human malaria parasite Plasmodium falciparum. Proteomics profiling revealed the enrichment of multiple host and parasite proteins, in particular of parasite antigens associated with host cell membranes and proteins involved in parasite invasion into RBCs. RMVs are quantitatively released during the asexual parasite cycle prior to parasite egress. RMVs demonstrate potent immunomodulatory properties on human primary macrophages and neutrophils. Additionally, RMVs are internalized by infected red blood cells and stimulate production of transmission stage parasites in a dose-dependent manner. Thus, RMVs mediate cellular communication within the parasite population and with the host innate immune system.

Magnetic barcode assay for genetic detection of pathogens.

The task of rapidly identifying patients infected with Mycobacterium tuberculosis in resource-constrained environments remains a challenge. A sensitive and robust platform that does not require bacterial isolation or culture is critical in making informed diagnostic and therapeutic decisions. Here we introduce a platform for the detection of nucleic acids based on a magnetic barcoding strategy. PCR-amplified mycobacterial genes are sequence-specifically captured on microspheres, labelled by magnetic nanoprobes and detected by nuclear magnetic resonance. All components are integrated into a single, small fluidic cartridge for streamlined on-chip operation. We use this platform to detect M. tuberculosis and identify drug-resistance strains from mechanically processed sputum samples within 2.5 h. The specificity of the assay is confirmed by detecting a panel of clinically relevant non-M. tuberculosis bacteria, and the clinical utility is demonstrated by the measurements in M. tuberculosis-positive patient specimens. Combined with portable systems, the magnetic barcode assay holds promise to become a sensitive, high-throughput and low-cost platform for point-of-care diagnostics.

Measuring neutrophil speed and directionality during chemotaxis, directly from a droplet of whole blood.

Neutrophil chemotaxis is critical for defense against infections and its alterations could lead to chronic inflammation and tissue injury. The central role that transient alterations of neutrophil chemotaxis could have on patient outcomes calls for its quantification in the clinic. However, current methods for measuring neutrophil chemotaxis require large volumes of blood and are time consuming. To address the need for rapid and robust assays, we designed a microfluidic device that measures neutrophil chemotaxis directly from a single droplet of blood. We validated the assay by comparing neutrophil chemotaxis from finger prick, venous blood and purified neutrophil samples. We found consistent average velocity of (19 ± 6 µm/min) and directionality (91.1%) between the three sources. We quantified the variability in neutrophil chemotaxis between healthy donors and found no significant changes over time. We also validated the device in the clinic and documented temporary chemotaxis deficiencies after burn injuries.

The neutral lipid composition present in the digestive vacuole of Plasmodium falciparum concentrates heme and mediates ß-hematin formation with an unusually low activation energy.

In eukaryotic cells, neutral lipids serve as major energy storage molecules; however, in Plasmodium falciparum, a parasite responsible for causing malaria in humans, neutral lipids may have other functions during the intraerythrocytic stage of the parasite life cycle. Specifically, experimental data suggest that neutral lipid structures behave as a catalyst for the crystallization of hemozoin, a detoxification byproduct of several blood-feeding organisms, including malaria parasites. Synthetic neutral lipid droplets (SNLDs) were produced by depositing a lipid blend solution comprised of mono- and diglycerides onto an aqueous surface. These lipid droplets are able to mediate the production of brown pigments that are morphologically and chemically identical to hemozoin. The partitioning of heme into these SNLDs was examined by employing Nile Red, a lipid specific dye. Soluble ferriprotoporphyrin IX was observed to spontaneously localize to the lipid droplets, partitioning in a pH-dependent manner with an estimated log P of 2.6. Interestingly, the pH profile of heme partitioning closely resembles that of ß-hematin formation. Differential scanning calorimetry and kinetic studies demonstrated that the SNLDs provide a unique environment that promotes hemozoin formation. SNLD-mediated formation of the malaria pigment displayed an activation energy barrier lower than those of individual lipid components. In particular, lipid droplets composed of diglycerides displayed activation barriers lower than those composed of monoglycerides. This difference was attributed to the greater fluidity of these lipids. In conjunction with the known pattern of lipid body proliferation, it is suggested that neutral lipid structures within the digestive vacuole not only are the location of in vivo hemozoin formation but are also essential for the survival of the parasite by functioning as a kinetically competent and site specific mediator for heme detoxification.

Increase on the initial soluble heme levels in acidic conditions is an important mechanism for spontaneous heme crystallization in vitro.

BACKGROUND: Hemozoin (Hz) is a heme crystal that represents a vital pathway for heme disposal in several blood-feeding organisms. Recent evidence demonstrated that ß-hematin (ßH) (the synthetic counterpart of Hz) formation occurs under physiological conditions near synthetic or biological hydrophilic-hydrophobic interfaces. This seems to require a heme dimer acting as a precursor of Hz crystals that would be formed spontaneously in the absence of the competing water molecules bound to the heme iron. Here, we aimed to investigate the role of medium polarity on spontaneous ßH formation in vitro. METHODOLOGY/PRINCIPAL FINDINGS: We assessed the effect of water content on spontaneous ßH formation by using the aprotic solvent dimethylsulfoxide (DMSO) and a series of polyethyleneglycols (PEGs). We observed that both DMSO and PEGs (3.350, 6.000, 8.000, and 22.000) increased the levels of soluble heme under acidic conditions. These compounds were able to stimulate the production of ßH crystals in the absence of any biological sample. Interestingly, the effects of DMSO and PEGs on ßH formation were positively correlated with their capacity to promote previous heme solubilization in acidic conditions. Curiously, a short chain polyethyleneglycol (PEG 300) caused a significant reduction in both soluble heme levels and ßH formation. Finally, both heme solubilization and ßH formation strongly correlated with reduced medium water activity provided by increased DMSO concentrations. CONCLUSIONS: The data presented here support the notion that reduction of the water activity is an important mechanism to support spontaneous heme crystallization, which depends on the previous increase of soluble heme levels.

Crystallization of synthetic haemozoin (beta-haematin) nucleated at the surface of lipid particles.

The mechanism of formation of haemozoin, a detoxification by-product of several blood-feeding organisms including malaria parasites, has been a subject of debate; however, recent studies suggest that neutral lipids may serve as a catalyst. In this study, a model system consisting of an emulsion of neutral lipid particles was employed to investigate the formation of beta-haematin, the synthetic counterpart of haemozoin, at the lipid-water interface. A solution of monoglyceride, either monostearoylglycerol (MSG) or monopalmitoylglycerol (MPG), dissolved in acetone and methanol was introduced to an aqueous surface. Fluorescence, confocal and transmission electron microscopic (TEM) imaging and dynamic light scattering analysis of samples obtained from beneath the surface confirmed the presence of homogeneous lipid particles existing in two major populations: one in the low micrometre size range and the other in the hundred nanometre range. The introduction of haem (Fe(iii)PPIX) to this lipid particle system under biomimetic conditions (37 degrees C, pH 4.8) produced beta-haematin with apparent first-order kinetics and an average half life of 0.5 min. TEM of monoglycerides (MSG or MPG) extruded through a 200 nm filter with haem produced beta-haematin crystals aligned and parallel to the lipid-water interface. These TEM data, together with a model system replacing the lipid with an aqueous organic solvent interface using either methyl laurate or docosane demonstrated that the OH and C[double bond, length as m-dash]O groups are apparently necessary for efficient nucleation. This suggests that beta-haematin crystallizes via epitaxial nucleation at the lipid-water interface through interaction of Fe(iii)PPIX with the polar head group. Once nucleated, the crystal grows parallel to the interface until growth is terminated by the curvature of the lipid particle. The hydrophobic nature of the mature crystal favours an interior transport resulting in crystals aligned parallel to the lipid-water interface and each other, strikingly similar to that seen in malaria parasites.