High-Pressure Mechanistic Insight into Bioinorganic NO Chemistry.

Pressure is one of the most important parameters controlling the kinetics of chemical reactions. The ability to combine high-pressure techniques with time-resolved spectroscopy has provided a powerful tool in the study of reaction mechanisms. This review is focused on the supporting role of high-pressure kinetic and spectroscopic methods in the exploration of nitric oxide bioinorganic chemistry. Nitric oxide and other reactive nitrogen species (RNS) are important biological mediators involved in both physiological and pathological processes. Understanding molecular mechanisms of their interactions with redox-active metal/non-metal centers in biological targets, such as cofactors, prosthetic groups, and proteins, is crucial for the improved therapy of various diseases. The present review is an attempt to demonstrate how the application of high-pressure kinetic and spectroscopic methods can add additional information, thus enabling the mechanistic interpretation of various NO bioinorganic reactions.

Analysis of the influence of EDTA-treated reference samples on forensic bloodstain age estimation.

The age estimation of blood traces provides important leads for the chronological assessment of criminal events and their reconstruction. To determine bloodstain age, experimental comparative data from a laboratory environment are used. Under these conditions the utilization of anticoagulants such as EDTA helps to suppress the blood clotting mechanism to allow the examination over a longer time period. This unnatural prevention of blood coagulation is highly questionable when estimating bloodstain age, since the blood's physical and chemical properties are altered. For this reason, the authors determined actual influence of EDTA on blood spectra over time in order to formulate a statement as to whether this effect can be measured. Human and porcine blood samples were aged under controlled conditions. The resulting UV/VIS spectra were separated into their individual components using signal separation techniques, allowing the changes in the ratios of the individual hemoglobin derivatives to be observed over time. The results show a significant influence of EDTA on the conversion of oxyhemoglobin to methemoglobin and a minor influence on the conversion of methemoglobin to hemichrome within the relevant time range of 5-100 h. The use of EDTA thus slows down the aging process of blood spots. To illustrate the great influence of EDTA, spectra of untreated pig blood samples were included as comparison data. These show that the difference between EDTA-treated and untreated blood samples is as great as the difference between human blood and pig blood. As a consequence of our findings experimental comparative data for the age estimation of bloodstains should never result from EDTA-treated blood.

Extracellular vesicles from thalassemia patients carry iron-containing ferritin and hemichrome that promote cardiac cell proliferation.

Extracellular vesicles (EVs) are bioactive, submicron-sized membrane vesicles released from all cell types upon activation or apoptosis. EVs including microparticles (MPs) and exosomes have emerged as important mediators of cell-to-cell communication in both normal and pathological states including thalassemia (thal). However, the role of EVs derived from ß-thal patients with iron overload (+ IO) and without iron overload (-IO) on cardiac cells is unclear. We hypothesized plasma EVs in thal patients containing ferritin (iron storage protein) and a denaturated hemoglobin-hemichrome that induce cardiac cell proliferation. The origins and numbers of EVs isolated from plasma of normal, thal (+ IO), and (- IO) patients were compared and determined for their iron and iron-containing proteins along with their effects on cardiac and endothelial cells. Data shows that MPs were originated from many cell sources with marked numbers of platelet origin. Only the number of RBC-derived MPs in thal (+ IO) patients was significantly high when compared to normal controls. Although MPs derived from both normal and thal patients promoted cardiac cell proliferation in a dose-dependent manner, only exosomes from thal patients promoted cardiac cell proliferation compared to the untreated. Moreover, the exosomes from thal (+ IO) potentially induce higher cardiac cell proliferation and angiogenesis in terms of tube number than thal (- IO) and normal controls. Interestingly, ferritin content in the exosomes isolated from thal (+ IO) was higher than that found in the MPs isolated from the same patient. The exosomes of thal patients with higher serum ferritin level also contained greater level of ferritin inside the exosomes. Apart from ferritin, there were trends of increasing hemichrome and iron presented in the plasma EVs and EV-treated H9C2 cells. Findings from this study support the hypothesis that EVs from ß-thal patients carry iron-load proteins that leads to the induction of cardiac cell proliferation.

Rapid and quantitative antimalarial drug efficacy testing via the magneto-optical detection of hemozoin.

Emergence of resistant Plasmodium species makes drug efficacy testing a crucial part of malaria control. Here we describe a novel assay for sensitive, fast and simple drug screening via the magneto-optical detection of hemozoin, a natural biomarker formed during the hemoglobin metabolism of Plasmodium species. By quantifying hemozoin production over the intraerythrocytic cycle, we reveal that hemozoin formation is already initiated by ~ 6-12 h old ring-stage parasites. We demonstrate that the new assay is capable of drug efficacy testing with incubation times as short as 6-10 h, using synchronized P. falciparum 3D7 cultures incubated with chloroquine, piperaquine and dihydroartemisinin. The determined 50% inhibitory concentrations agree well with values established by standard assays requiring significantly longer testing time. Accordingly, we conclude that magneto-optical hemozoin detection provides a practical approach for the quick assessment of drug effect with short incubation times, which may also facilitate stage-specific assessment of drug inhibitory effects.

Rapid phenotyping towards personalized malaria medicine.

Malaria is major public health concerns which continues to claim the lives of more than 435,000 people each year. The challenges with anti-malarial drug resistance and detection of low parasitaemia forms an immediate barrier to achieve the fast-approaching United Nations Sustainable Development Goals of ending malaria epidemics by 2030. In this Opinion article, focusing on the recent published technologies, in particularly the nuclear magnetic resonance (NMR)-based diagnostic technologies, the authors offer their perspectives and highlight ways to bring these point-of-care technologies towards personalized medicine. To this end, they advocate an open sourcing initiative to rapidly close the gap between technological innovations and field implementation.

Surface-enhanced resonance Raman spectroscopy of heme proteins on a gold grid electrode.

The combination of surface-enhanced resonance Raman spectroscopy (SERRS) and electrochemistry is an ideal tool to study the redox process of the heme proteins and is often performed on silver electrodes. In this manuscript, we present an approach using a microstructured gold surface that serves as the electrochemical working electrode, and at the same time, acts as SERS active substrate. The cell requires a micromolar concentration of sample at the electrode surface. Even if the performance of the gold grid as SERS substrate exhibited a smaller enhancement factor than expected for silver, oxidized and reduced spectra of proteins (Сyt c, Hb and Mb) monolayers could be obtained and the characteristic redox dependent shifts of the marker bands ν19, ν4 and ν10 were seen. The easy modification protocol and the higher stability of the gold electrode towards oxidative currents are the advantages of the present spectroeletrochemical cell. Finally, FDTD simulations confirm that the roughness of the gold grid has an effect on the Raman enhancement of the adsorbed proteins.

Portable Device for Optical Quantification of Hemozoin in Diluted Blood Samples.

OBJECTIVE: This paper focuses on a novel and portable device prototype with optical detectors to quickly and efficiently detect hemozoin (Hz) in blood, aiming at malaria diagnostics. METHODS: Taking advantage of the particular features of malaria parasite in infected blood, particularly the Hz formation, the main innovation described is a portable device for the optical quantification of parasitic Hz in blood, through optical absorbance spectrophotometry. This device comprises detection chambers for fluidic samples, an optical emission and detection system, and a power supply system to provide autonomy. The working principle is based on colorimetric detection, by absorbance, at six specific wavelengths. A detection algorithm relates the absorbance values at all wavelengths to quantify the Hz concentration, thus working as a biomarker of malaria presence and stage. RESULTS: Under the tested conditions, e.g., in fluidic samples containing synthetic Hz, hemoglobin, and diluted whole blood, the device detected Hz above 1 µg/mL concentrations with 100% sensitivity and 96.3% specificity. CONCLUSION: This paper features an autonomous, portable, 1-min analysis time, and low-cost per test device, without the need for samples, centrifugation, allowing the use of whole blood. SIGNIFICANCE: The presented device is a step ahead for meeting the growing clinical demands for reliable, rapid, portable, and quantitative malaria diagnosis.

Quantitative imaging of intraerythrocytic hemozoin by transient absorption microscopy.

Hemozoin, the heme detoxification end product in malaria parasites during their growth in the red blood cells (RBCs), serves as an important marker for diagnosis and treatment target of malaria disease. However, the current method for hemozoin-targeted drug screening mainly relies on in vitro ß-hematin inhibition assays, which may lead to false-positive events due to under-representation of the real hemozoin crystal. Quantitative in situ imaging of hemozoin is highly desired for high-throughput screening of antimalarial drugs and for elucidating the mechanisms of antimalarial drugs. We present transient absorption (TA) imaging as a high-speed single-cell analysis platform with chemical selectivity to hemozoin. We first demonstrated that TA microscopy is able to identify ß-hematin, the artificial form of hemozoin, from the RBCs. We further utilized time-resolved TA imaging to in situ discern hemozoin from malaria-infected RBCs with optimized imaging conditions. Finally, we quantitatively analyzed the hemozoin amount in RBCs at different infection stages by single-shot TA imaging. These results highlight the potential of TA imaging for efficient antimalarial drug screening and drug mechanism investigation.

Insufficient sensitivity of laser desorption-time of flight mass spectrometry-based detection of hemozoin for malaria screening.

Laser desorption-time of flight (LD-TOF) mass spectrometry-based detection of hemozoin was assessed for its performance characteristics as a rapid screening test for malaria. In spite of good specificity of >95%, poor sensitivity of 80.2% for microscopically positive samples makes the easy-to-apply and rapid approach unsuitable for the routine diagnostic setting.

The paramagnetic properties of malaria pigment, hemozoin, yield clues to a low-cost system for its trapping and determination.

The binding of malaria pigment, hemozoin, by a gradient magnetic field has been investigated in a manual trapping column system. Two types of magnetic filling have been tested to produce field gradients: nickel-plated steel wires, wrapped around a steel core, and superparamagnetic microbeads. The latter system allows an efficient trapping (> 80%) of ß-hematin (a synthetic pigment with physical and paramagnetic properties analogous to those of hemozoin). Tests with a Plasmodium falciparum 3D7 culture indicate that hemozoin is similarly trapped. Off-line optical spectroscopy measurements present limited sensitivity as the hemozoin we detected from in vitro cultured parasites would correspond to only a theoretical 0.02% parasitemia (1000 parasites/µL). Further work needs to be undertaken to reduce this threshold to a practical detectability level. Based on these data, a magneto-chromatographic on-line system with reduced dead volumes is proposed as a possible low-cost instrument to be tested as a malaria diagnosis system.

Rapid Diagnostic for Point-of-Care Malaria Screening.

Despite significant success in therapeutic development, malaria remains a widespread and deadly infectious disease in the developing world. Given the nearly 100% efficacy of current malaria therapeutics, the primary barrier to eradication is lack of early diagnosis of the infected population. However, there are multiple strains of malaria. Although significant efforts and resources have been invested in developing antibody-based diagnostic methods for Plasmodium falciparum, a rapid and easy to use screening method capable of detecting all malaria strains has not been realized. Yet, until the entire malaria-infected population receives treatment, the disease will continue to impact society. Here, we report the development of a portable, magneto-optic technology for early stage malaria diagnosis based on the detection of the malaria pigment, hemozoin. Using ß-hematin, a hemozoin mimic, we demonstrate detection limits of <0.0081 µg/mL in 500 µL of whole rabbit blood with no additional reagents required. This level corresponds to <26 parasites/µL, a full order of magnitude below clinical relevance and comparable to or less than existing technologies.

Parasites under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research.

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.

Differential induction of malaria liver pathology in mice infected with Plasmodium chabaudi AS or Plasmodium berghei NK65.

BACKGROUND: Cerebral malaria and severe anaemia are the most common deadly complications of malaria, and are often associated, both in paediatric and adult patients, with hepatopathy, whose pathogenesis is not well characterized, and sometimes also with acute respiratory distress syndrome (ARDS). Here, two species of murine malaria, the lethal Plasmodium berghei strain NK65 and self-healing Plasmodium chabaudi strain AS which differ in their ability to cause hepatopathy and/or ARDS were used to investigate the lipid alterations, oxidative damage and host immune response during the infection in relation to parasite load and accumulation of parasite products, such as haemozoin. METHODS: Plasma and livers of C57BL/6J mice injected with PbNK65 or PcAS infected erythrocytes were collected at different times and tested for parasitaemia, content of haemozoin and expression of tumour necrosis factor (TNF). Hepatic enzymes, antioxidant defenses and lipids content and composition were also evaluated. RESULTS: In the livers of P. berghei NK65 infected mice both parasites and haemozoin accumulated to a greater extent than in livers of P. chabaudi AS infected mice although in the latter hepatomegaly was more prominent. Hepatic enzymes and TNF were increased in both models. Moreover, in P. berghei NK65 infected mice, increased lipid peroxidation, accumulation of triglycerides, impairment of anti-oxidant enzymes and higher collagen deposition were detected. On the contrary, in P. chabaudi AS infected mice the antioxidant enzymes and the lipid content and composition were normal or even lower than uninfected controls. CONCLUSIONS: This study demonstrates that in C57BL/6J mice, depending on the parasite species, malaria-induced liver pathology results in different manifestations, which may contribute to the different outcomes. In P. berghei NK65 infected mice, which concomitantly develop lethal acute respiratory distress syndrome, the liver tissue is characterized by an excess oxidative stress response and reduced antioxidant defenses while in P. chabaudi AS infected mice hepatopathy does not lead to lipid alterations or reduction of antioxidant enzymes, but rather to inflammation and cytokine burst, as shown earlier, that may favour parasite killing and clearance of the infection. These results may help understanding the different clinical profiles described in human malaria hepatopathy.

Multispectral Atomic Force Microscopy-Infrared Nano-Imaging of Malaria Infected Red Blood Cells.

Atomic force microscopy-infrared (AFM-IR) spectroscopy is a powerful new technique that can be applied to study molecular composition of cells and tissues at the nanoscale. AFM-IR maps are acquired using a single wavenumber value: they show either the absorbance plotted against a single wavenumber value or a ratio of two absorbance values. Here, we implement multivariate image analysis to generate multivariate AFM-IR maps and use this approach to resolve subcellular structural information in red blood cells infected with Plasmodium falciparum at different stages of development. This was achieved by converting the discrete spectral points into a multispectral line spectrum prior to multivariate image reconstruction. The approach was used to generate compositional maps of subcellular structures in the parasites, including the food vacuole, lipid inclusions, and the nucleus, on the basis of the intensity of hemozoin, hemoglobin, lipid, and DNA IR marker bands, respectively. Confocal Raman spectroscopy was used to validate the presence of hemozoin in the regions identified by the AFM-IR technique. The high spatial resolution of AFM-IR combined with hyperspectral modeling enables the direct detection of subcellular components, without the need for cell sectioning or immunological/biochemical staining. Multispectral-AFM-IR thus has the capacity to probe the phenotype of the malaria parasite during its intraerythrocytic development. This enables novel approaches to studying the mode of action of antimalarial drugs and the phenotypes of drug-resistant parasites, thus contributing to the development of diagnostic and control measures.

Towards a needle-free diagnosis of malaria: in vivo identification and classification of red and white blood cells containing haemozoin.

BACKGROUND: Optical detection of circulating haemozoin has been suggested as a needle free method to diagnose malaria using in vivo microscopy. Haemozoin is generated within infected red blood cells by the malaria parasite, serving as a highly specific, endogenous biomarker of malaria. However, phagocytosis of haemozoin by white blood cells which persist after the infection is resolved presents the potential for false positive diagnosis; therefore, the focus of this work is to identify a feature of the haemozoin signal to discriminate between infected red blood cells and haemozoin-containing white blood cells. METHODS: Conventional brightfield microscopy of thin film blood smears was used to analyse haemozoin absorbance signal in vitro. Cell type and parasite maturity were morphologically determined using colocalized DAPI staining. The ability of features to discriminate between infected red blood cells and haemozoin-containing white blood cells was evaluated using images of smears from subjects infected with two species of Plasmodium, Plasmodium yoelii and Plasmodium falciparum. Discriminating features identified by blood smear microscopy were characterized in vivo in P. yoelii-infected mice. RESULTS: Two features of the haemozoin signal, haemozoin diameter and normalized intensity difference, were identified as potential parameters to differentiate infected red blood cells and haemozoin-containing white blood cells. Classification performance was evaluated using the area under the receiver operating characteristic curve, with area under the curve values of 0.89 for the diameter parameter and 0.85 for the intensity parameter when assessed in P. yoelii samples. Similar results were obtained from P. falciparum blood smears, showing an AUC of 0.93 or greater for both classification features. For in vivo investigations, the intensity-based metric was the best classifier, with an AUC of 0.91. CONCLUSIONS: This work demonstrates that size and intensity features of haemozoin absorbance signal collected by in vivo microscopy are effective classification metrics to discriminate infected red blood cells from haemozoin-containing white blood cells. This reduces the potential for false positive results associated with optical imaging strategies for in vivo diagnosis of malaria based on the endogenous biomarker haemozoin.

Biochemistry of malaria parasite infected red blood cells by X-ray microscopy.

Red blood cells infected by the malaria parasite Plasmodium falciparum are correlatively imaged by tomography using soft X-rays as well as by scanning hard nano-X-ray beam to obtain fluorescence maps of various elements such as S and Fe. In this way one can deduce the amount of Fe bound either in hemoglobin or in hemozoin crystals in the digestive vacuole of the malaria parasite as well as determine the hemoglobin concentrations in the cytosols of the red blood cell and of the parasite. Fluorescence map of K shows that in the parasite's schizont stage the K concentration in the red blood cell cytosol is diminished by a factor of seven relative to a pristine red blood cell but the total amount of K in the infected red blood cell is the same as in the pristine red blood cell.

Quantitative characterization of hemozoin in Plasmodium berghei and vivax.

The incidence and global distribution of chloroquine resistant (CR) Plasmodium vivax infection has increased since emerging in 1989. The mechanism of resistance in CR P. vivax has not been defined. The resistance likely relates to the formation and disposition of hemozoin as chloroquine's primary mechanism of action involves disruption of hemozoin formation. CR P. berghei strains, like CR P. vivax strains, are confined to reticulocyte host cells and reportedly they do not accumulate appreciable intraerythrocytic hemozoin. Reports comparing hemozoin production between P. vivax strains and CR to chloroquine sensitive (CS) P. berghei are absent. Here we compare in vivo patterns of hemozoin formation and distribution in blood, spleen and liver tissue of male Swiss mice infected with CS or CR P. berghei not treated with chloroquine and CR P. berghei also treated with chloroquine. Light microscopy, laser desorption mass spectrometry and a colorimetric hemozoin assay detect trace hemozoin in the blood of CR P. berghei infected mice but significant hemozoin accumulation in liver and spleen tissue. Field emission in lens scanning electron microscopy reveals CR P. berghei hemozoin crystals are morphologically smaller but similar to those formed by CS parasites. CR P. berghei produces approximately five-fold less total hemozoin than CS strain. Lipid analysis of CS and CR P. berghei sucrose gradient purified bloodstage hemozoin indicates a similar lipid environment around the isolated hemozoin, predominately monopalmitic glycerol and monostearic glycerol. In contrast to CR and CS P. berghei, colorimetric hemozoin analysis of P. vivax strains indicates similar amounts of hemozoin are produced despite differing chloroquine sensitivities. These results suggest CR P. berghei forms significant hemozoin which accumulates in liver and spleen tissues and that the P. vivax chloroquine resistance mechanism differs from P. berghei.

Organic Cyanide Decorated SERS Active Nanopipettes for Quantitative Detection of Hemeproteins and Fe3+ in Single Cells.

It is challenging to develop a robust nanoprobe for real-time operational and accurate detection of heavy metals in single cells. Fe-CN coordination chemistry has been well studied to determine the structural characteristics of hemeproteins by different techniques. However, the frequently used cyanide ligands are inorganic molecules that release cyanide anion under particular conditions and cause cyanide poisoning. In the present study, organic cyanide (4-mercaptobenzonitrile, MBN) was utilized for the first time in developing a facile nanoprobe based on surface-enhanced Raman scattering (SERS) for quantitative detection of hemeproteins (oxy-Hb) and trivalent iron (Fe3+) ions. The nanoprobe prepared by coating the glass capillary tip (100 nm) with a thin gold film, which enables highly localized study in living cell system. The cyanide stretching vibration in MBN was highly sensitive and selective to Fe3+ and oxy-Hb with excellent binding affinity (Kd 0.4 pM and 0.1 nM, respectively). The high sensitivity of the nanoprobe to analyte (Fe3+) was attributed to the two adsorption conformations (-SH and -CN) of MBN to the gold surface. Therefore, MBN showed an exceptional dual-peak (2126 and 2225 cm-1) behavior. Furthermore, the special Raman peaks of cyanide in 2100-2300 cm-1 (silent region of SERS spectra) are distinguishable from other biomolecules characteristic peaks. The selective detection of Fe3+ in both free and protein-bound states in aqueous solution is achieved with 0.1 pM and 0.08 µM levels of detection limits, respectively. Furthermore, practical applicability of fabricated nanoprobe was validated by detection of free Fe3+ in pretreated living HeLa cells by direct insertion of a SERS active nanoprobe. Regarding the appropriate precision, good reproducibility (relative standard deviation, RSD 7.2-7.6%), and recyclability (retain good Raman intensity even after three renewing cycles) of the method, the developed sensing strategy on a nanopipette has potential benefits for label-free, qualitative and quantitative recognition of heavy metal ions within nanoliter volumes.

Hemozoin Pigment: An Important Tool for Low Parasitemic Malarial Diagnosis.

Low parasitemic condition in malaria remains a diagnostic challenge; as the available diagnostic methods failed to detect. Currently, hemozoin (Hz) pigment is gaining attention in the diagnosis of malaria. The major drawback is ease of detection of Hz in routine practice. A pilot study was conducted to evaluate the role of Hz pigment and to compare the performance of quantitative buffy coat assay (QBC) and PCR in such conditions. Clinically suspected cases of malaria were examined by both Giemsa stain and immunochromatographic test (ICT). Samples positive by ICT and negative by Giemsa stain were further examined by nested PCR targeting 18S rRNA and QBC for the presence of malaria parasites and pigments. Thirty blood samples fulfilled the inclusion criteria out of which 23 were Plasmodium vivax (Pv), 4 Plasmodium falciparum (Pf), and 3 mixed (Pv and Pf) by immunochromatographic test. Twenty-one out of 30 (70%) were positive by nested PCR in comparison to 25/30 (83%) by QBC. Samples containing both malaria parasites and Hz pigment by QBC completely showed concordance with the PCR result. However, 61% of total samples containing only Hz pigment were observed positive by PCR. Hz pigment remains an important tool for malaria diagnosis. Identification of leukocytes containing pigments by QBC not only indicates recent malarial infections but also puts light on severity of the disease. QBC assay is a rapid, highly sensitive, and cost-effective method to detect malaria parasites and Hz pigment especially in low parasitemic conditions.

Hemozoin detection may provide an inexpensive, sensitive, 1-minute malaria test that could revolutionize malaria screening.

Malaria remains widespread throughout the tropics and is a burden to the estimated 3.5 billion people who are exposed annually. The lack of a fast and accurate diagnostic method contributes to preventable malaria deaths and its continued transmission. In many areas diagnosis is made solely based on clinical presentation. Current methods for malaria diagnosis take more than 20 minutes from the time blood is drawn and are frequently inaccurate. The introduction of an accurate malaria diagnostic that can provide a result in less than 1 minute would allow for widespread screening and treatment of endemic populations, and enable regions that have gained a foothold against malaria to prevent its return. Using malaria parasites' waste product, hemozoin, as a biomarker for the presence of malaria could be the tool needed to develop this rapid test.