The objective of this paper is to offer experimental evidence which shows that the process of excitation in the nerve is accompanied by a transient change in optical properties of the nervous tissue. The optical properties examined include fluorescence, turbidity, and birefringence.Changes in fluorescence were examined after nervous tissues were stained with the dye 8-anilinonaphthalene-1-sulfonic acid (ANS).1 Our search for fluorescence under these conditions was prompted by the work of Aronson, Detert, and Morales,2 who demonstrated that the fluorescence of ANS is extremely sensitive to conformational changes of various macromolecules.Our attempt to measure turbidity changes of the nerve during excitation was made with a view to extending the work reported by Cohen and Keynes3 by the use of monochromatic light.The observation by Inoue and Sato4 of rapid changes in the mitotic figure of the Pectinaria obcyte under a polarizing microscope aroused our interest in the birefringent properties of the nerve during excitation. Our experiments along this line were greatly accelerated when we encountered a very significant paper by Cohen et al.5 on this subject in the early stages of our investigation.Changes in the optical properties of the nerve during excitation are very small. In fluorescence and turbidity studies, optical signs of nerve excitation could not be measured without the use of a computer to average multiple responses. However, in birefringence studies, it was possible to record optical signs of nerve excitation directly on an oscillograph screen.

The protozoan parasite responsible for malaria affects over 500 million people each year. Current antimalarials have experienced decreased efficacy due to the development of drug-resistant strains of Plasmodium spp., resulting in a critical need for the discovery of new antimalarials. Hemozoin, a crystalline by-product of heme detoxification that is necessary for parasite survival, serves as an important drug target. The quinoline antimalarials, including amodiaquine and chloroquine, act by inhibiting the formation of hemozoin. The formation of this crystal does not occur spontaneously, and recent evidence suggests crystallization occurs in the presence of neutral lipid particles located in the acidic digestive vacuole of the parasite. To mimic these conditions, the lipophilic detergent NP-40 has previously been shown to successfully mediate the formation of ␤-hematin, synthetic hemozoin. Here, an NP-40 detergent-based assay was successfully adapted for use as a high-throughput screen to identify inhibitors of ␤-hematin formation. The resulting assay exhibited a favorable Z of 0.82 and maximal drift of less than 4%. The assay was used in a pilot screen of 38,400 diverse compounds at a screening concentration of 19.3 M, resulting in the identification of 161 previously unreported ␤-hematin inhibitors. Of these, 48 also exhibited >90% inhibition of parasitemia in a Plasmodium falciparum whole-cell assay at a screening concentration of 23 M. Eight of these compounds were identified to have nanomolar 50% inhibitory concentration values near that of chloroquine in this assay.

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 β-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.

Graphical abstract

The growing drug resistance of Plasmodia spp. to current antimalarial agents in the quinine and artemisinin families further asserts the need for novel drug classes to combat malaria infection. One approach to the discovery of new antimalarials is the screening of natural product extracts for activity against the formation of hemozoin, a biomineral essential to parasite survival. By mimicking the in vivo lipid-water interface at which native hemozoin is found, hemozoin can be synthesized outside the parasite. In this work, a variety of lipophilic mediators was used to determine the optimal platform for in vitro hemozoin formation and then tested for efficacy in preliminary screens containing crude natural product extracts. The complete optimization and validation of a NP-40 detergent-mediated assay provide a screening template with an expedited 4-hour incubation time and identical IC50 values to those measured from the parasite’s native lipid component.

Precise rare-cell technologies require the blood to be processed immediately or be stabilized with fixatives. Such restrictions limit the translation of circulating tumor cell (CTC)-based liquid biopsy assays that provide accurate molecular data in guiding clinical decisions. Here we describe a method to preserve whole blood in its minimally altered state by combining hypothermic preservation with targeted strategies that counter cooling-induced platelet activation. Using this method, whole blood preserved for up to 72 h can be readily processed for microfluidic sorting without compromising CTC yield and viability. The tumor cells retain high-quality intact RNA suitable for single-cell RT-qPCR as well as RNA-Seq, enabling the reliable detection of cancer-specific transcripts including the androgen-receptor splice variant 7 in a cohort of prostate cancer patients with an overall concordance of 92% between fresh and preserved blood. This work will serve as a springboard for the dissemination of diverse blood-based diagnostics.

Cryptosporidiosis in an enteric infection caused by Cryptosporidium parasites and is a major cause of acute infant diarrhea in the developing world. A major bottleneck to research progress is the lack of methods to cryopreserve Cryptosporidium oocysts, thus requiring routine propagation in laboratory animals. Here, we report a method to cryopreserve C. parvum oocysts by ultra-fast cooling. Cryopreserved oocysts exhibit high viability and robust in vitro excystation, and are infectious to interferon-γ knockout mice. The course of the infection is comparable to what we observe with unfrozen oocysts. Oocyst viability and infectivity is not visibly changed after several weeks of cryogenic storage. Cryopreservation will facilitate the sharing of oocysts from well-characterized isolates and transgenic strains among different laboratories.

The rapid degradation of blood ex vivo imposes logistical limitations on the utilization of blood-borne cells in medical diagnostics and scientific investigations. A fundamental but overlooked aspect in the storage of this fluid tissue is blood settling, which induces physical stress and compaction, aggregates blood cells, and causes collateral damage due to leukocyte activation. Here we show that the polymer Ficoll 70 kDa stabilized blood samples and prevented blood settling over the course of 72 hours, primarily by inhibiting depletion-mediated red blood cell aggregation. Physical stabilization decreased echinocyte formation, improved leukocyte viability, and inhibited the release of neutrophil elastase—a marker of neutrophil extracellular trap formation. In addition, Ficoll-stabilized blood was compatible with common leukocyte enrichment techniques including red blood cell lysis and immunomagnetic purification. This study showed for the first time that blood settling can be prevented using polymers and has implications in diagnostics.