Three-dimensional numerical analysis as a tool for optimization of acoustophoretic separation in polymeric chips.

Polymeric separators have been developed since 2010 to produce acoustophoretic separation of particles or cells in suspension with high efficiency. They rely on three-dimensional (3D) resonances of their whole structure actuated by ultrasounds. In this paper, a numerical 3D analysis is presented and validated as the only tool for optimization of these polymeric chips to perform efficient separation applications. In contrast to conventional acoustophoretic techniques based on the establishment of standing waves in the liquid phase of the channel (requiring rigid chip materials, such as silicon or glass), whole-structure resonances of the chip allow the use of materials that are acoustically soft and of low acoustic impedance, which is close to that of the liquid samples hosted. The resonance requirement is not restricted to the liquid phase in the polymeric chips, but it extends to the 3D whole structure, allowing any material. It provides significant advantages in the design and manufacture of our chips, allowing the use of low-cost materials and cheap manufacturing processes and even printing of devices. The extraordinary complexity of their multiple resonances requires theoretical approaches to optimize their acoustophoretic performance. Hence, the importance of 3D numerical analyses, which are capable of predicting the acoustic behavior of these chips, is to perform acoustophretica separation in suspensions.

Infrared laser threshold magnetometry with a NV doped diamond intracavity etalon.

We propose a hybrid laser system consisting of a semiconductor external cavity laser associated to an intra-cavity diamond etalon doped with nitrogen-vacancy color centers. We consider laser emission tuned to the infrared absorption line that is enhanced under the magnetic field dependent nitrogen-vacancy electron spin resonance and show that this architecture leads to a compact solid-state magnetometer that can be operated at room-temperature. The sensitivity to the magnetic field limited by the photonshot-noise of the output laser beam is estimated to be less than 1 pT/Hz. Unlike usual NV center infrared magnetometry, this method would not require an external frequency stabilized laser. Since the proposed system relies on the competition between the laser threshold and an intracavity absorption, such laser-based optical sensor could be easily adapted to a broad variety of sensing applications based on absorption spectroscopy.

Overview of the role of kinetoplastid surface carbohydrates in infection and host cell invasion: prospects for therapeutic intervention.

Kinetoplastid parasites are responsible for serious diseases in humans and livestock such as Chagas disease and sleeping sickness (caused by Trypanosoma cruzi and Trypanosoma brucei, respectively), and the different forms of cutaneous, mucocutaneous and visceral leishmaniasis (produced by Leishmania spp). The limited number of antiparasitic drugs available together with the emergence of resistance underscores the need for new therapeutic agents with novel mechanisms of action. The use of agents binding to surface glycans has been recently suggested as a new approach to antitrypanosomal design and a series of peptidic and non-peptidic carbohydrate-binding agents have been identified as antiparasitics showing efficacy in animal models of sleeping sickness. Here we provide an overview of the nature of surface glycans in three kinetoplastid parasites, T. cruzi, T. brucei and Leishmania. Their role in virulence and host cell invasion is highlighted with the aim of identifying specific glycan-lectin interactions and carbohydrate functions that may be the target of novel carbohydrate-binding agents with therapeutic applications.

Carbohydrate-Binding Non-Peptidic Pradimicins for the Treatment of Acute Sleeping Sickness in Murine Models.

Current treatments available for African sleeping sickness or human African trypanosomiasis (HAT) are limited, with poor efficacy and unacceptable safety profiles. Here, we report a new approach to address treatment of this disease based on the use of compounds that bind to parasite surface glycans leading to rapid killing of trypanosomes. Pradimicin and its derivatives are non-peptidic carbohydrate-binding agents that adhere to the carbohydrate moiety of the parasite surface glycoproteins inducing parasite lysis in vitro. Notably, pradimicin S has good pharmaceutical properties and enables cure of an acute form of the disease in mice. By inducing resistance in vitro we have established that the composition of the sugars attached to the variant surface glycoproteins are critical to the mode of action of pradimicins and play an important role in infectivity. The compounds identified represent a novel approach to develop drugs to treat HAT.

Cell cycle regulation and novel structural features of thymidine kinase, an essential enzyme in Trypanosoma brucei.

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.

Carbohydrate-binding agents act as potent trypanocidals that elicit modifications in VSG glycosylation and reduced virulence in Trypanosoma brucei.

The surface of Trypanosoma brucei is covered by a dense coat of glycosylphosphatidylinositol-anchored glycoproteins. The major component is the variant surface glycoprotein (VSG) which is glycosylated by both paucimannose and oligomannose N-glycans. Surface glycans are poorly accessible and killing mediated by peptide lectin-VSG complexes is hindered by active endocytosis. However, contrary to previous observations, here we show that high-affinity carbohydrate binding agents bind to surface glycoproteins and abrogate growth of T. brucei bloodstream forms. Specifically, binding of the mannose-specific Hippeastrum hybrid agglutinin (HHA) resulted in profound perturbations in endocytosis and parasite lysis. Prolonged exposure to HHA led to the loss of triantennary oligomannose structures in surface glycoproteins as a result of genetic rearrangements that abolished expression of the oligosaccharyltransferase TbSTT3B gene and yielded novel chimeric enzymes. Mutant parasites exhibited markedly reduced infectivity thus demonstrating the importance of specific glycosylation patterns in parasite virulence.

The MAK16 gene of Entamoeba histolytica and its identification in isolates from patients.

To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher's exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient's symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.

Super-Resolution Diamond Magnetic Microscopy of Superparamagnetic Nanoparticles.

Scanning-probe and wide-field magnetic microscopes based on nitrogen-vacancy (NV) centers in diamond have enabled advances in the study of biology and materials, but each method has drawbacks. Here, we implement an alternative method for nanoscale magnetic microscopy based on optical control of the charge state of NV centers in a dense layer near the diamond surface. By combining a donut-beam super-resolution technique with optically detected magnetic resonance spectroscopy, we imaged the magnetic fields produced by single 30 nm iron-oxide nanoparticles. The magnetic microscope has a lateral spatial resolution of ∼100 nm, and it resolves the individual magnetic dipole features from clusters of nanoparticles with interparticle spacings down to ∼190 nm. The magnetic feature amplitudes are more than an order of magnitude larger than those obtained by confocal magnetic microscopy due to the narrower optical point-spread function and the shallow depth of NV centers. We analyze the magnetic nanoparticle images and sensitivity as a function of the microscope's spatial resolution and show that the signal-to-noise ratio for nanoparticle detection does not degrade as the spatial resolution improves. We identify sources of background fluorescence that limit the present performance, including diamond second-order Raman emission and imperfect NV charge state control. Our method, which uses <10 mW laser power and can be parallelized by patterned illumination, introduces a promising format for nanoscale magnetic imaging.

High-sensitivity magnetometry based on quantum beats in diamond nitrogen-vacancy centers.

We demonstrate an absolute magnetometer based on quantum beats in the ground state of nitrogen-vacancy centers in diamond. We show that, by eliminating the dependence of spin evolution on the zero-field splitting D, the magnetometer is immune to temperature fluctuation and strain inhomogeneity. We apply this technique to measure low-frequency magnetic field noise by using a single nitrogen-vacancy center located within 500 nm of the surface of an isotopically pure (99.99% 12C) diamond. The photon-shot-noise limited sensitivity achieves 38 nT/sqrt[Hz] for 4.45 s acquisition time, a factor of sqrt[2] better than the implementation which uses only two spin levels. For long acquisition times (>10 s), we realize up to a factor of 15 improvement in magnetic sensitivity, which demonstrates the robustness of our technique against thermal drifts. Applying our technique to nitrogen-vacancy center ensembles, we eliminate dephasing from longitudinal strain inhomogeneity, resulting in a factor of 2.3 improvement in sensitivity.

Culture of human bone marrow-derived mesenchymal stem cells on of poly(L-lactic acid) scaffolds: potential application for the tissue engineering of cartilage.

PURPOSE: Due to the attractive properties of poly(L-lactic acid) (PLLA) for tissue engineering, the aim was to determine the growth and differentiation capacity of mesenchymal stromal cells (MSCs) in PLLA scaffolds and their potential use in the treatment of cartilage diseases. METHODS: MSCs were cultured in PLLA films and thin porous membranes to study adherence and proliferation. Permeability and porosity were determined for the different scaffolds employed. The optimal conditions for cell seeding were first determined, as well as cell density and distribution inside the PLLA. Scaffolds were then maintained in expansion or chondrogenic differentiation media for 21 days. Apoptosis, proliferation and chondrogenic differentiation was assessed after 21 days in culture by immunohistochemistry. Mechanical characteristics of scaffolds were determined before and after cell seeding. RESULTS: MSCs uniformly adhered to PLLA films as well as to porous membranes. Proliferation was detected only in monolayers of pure PLLA, but was no longer detected after 10 days. Mechanical characterization of PLLA scaffolds showed differences in the apparent compression elastic modulus for the two sizes used. After determining high efficiencies of seeding, the production of extracellular matrix (ECM) was determined and contained aggrecan and collagens type I and X. ECM produced by the cells induced a twofold increase in the apparent elastic modulus of the composite. CONCLUSIONS: Biocompatible PLLA scaffolds have been developed that can be efficiently loaded with MSCs. The scaffold supports chondrogenic differentiation and ECM deposition that improves the mechanics of the scaffold. Although this improvement does not met the expectations of a hyaline-like cartilage ECM, in part due to the lack of a mechanical stimulation, their potential use in the treatment of cartilage pathologies encourages to improve the mechanical component.

Optical and spin coherence properties of nitrogen-vacancy centers placed in a 100 nm thick isotopically purified diamond layer.

We have studied optical and spin properties of near-surface nitrogen-vacancy (NV) centers incorporated during chemical vapor phase growth of isotopically purified (12)C single-crystal diamond layers. The spectral diffusion-limited line width of zero-phonon luminescence from the NV centers is 1.2 ± 0.5 GHz, a considerable improvement over that of NV centers formed by ion implantation and annealing. Enhanced spin dephasing times (T(2)* ≈ 90 µs, T(2) ≈ 1.7 ms) due to the reduction of (13)C nuclear spins persist even for NV centers placed within 100 nm of the surface.

Super-resolution diamond magnetic microscopy of superparamagnetic nanoparticles.

Scanning-probe and wide-field magnetic microscopes based on Nitrogen-Vacancy (NV) centers in diamond have enabled remarkable advances in the study of biology and materials, but each method has drawbacks. Here, we implement an alternative method for nanoscale magnetic microscopy based on optical control of the charge state of NV centers in a dense layer near the diamond surface. By combining a donut-beam super-resolution technique with optically detected magnetic resonance spectroscopy, we imaged the magnetic fields produced by single 30-nm iron-oxide nanoparticles. The magnetic microscope has a lateral spatial resolution of ~100 nm, and it resolves the individual magnetic dipole features from clusters of nanoparticles with interparticle spacings down to ~190 nm. The magnetic feature amplitudes are more than an order of magnitude larger than those obtained by confocal magnetic microscopy due to the smaller characteristic NV-nanoparticle distance within nearby sensing voxels. We analyze the magnetic point-spread function and sensitivity as a function of the microscope's spatial resolution and identify sources of background fluorescence that limit the present performance, including diamond second-order Raman emission and imperfect NV charge-state control. Our method, which uses less than 10 mW laser power and can be parallelized by patterned illumination, introduces a new format for nanoscale magnetic imaging.

Temperature Sensitivity of 14N-V and 15N-V Ground-State Manifolds.

We measure electron- and nuclear-spin transition frequencies in the ground state of nitrogen-vacancy (N-V) centers in diamond for two nitrogen isotopes (14N-V and 15N-V) over temperatures ranging from 77 to 400 K. Measurements are performed using Ramsey interferometry and direct optical readout of the nuclear and electron spins. We extract coupling parameters Q (for 14N-V), D, A‖, A⊥, and γe/γn, and their temperature dependences for both isotopes. The temperature dependences of the nuclear-spin transitions within the ms=0 spin manifold near room temperature are found to be 0.52(1) ppm/K for 14N-V(|mI = -1⟩ ↔ |mI = +1⟩) and -1.1(1) ppm/K for 15N-V(|mI = -1/2⟩ ↔ |mI = +1/2⟩). An isotopic shift in the zero-field splitting parameter D between 14N-V and 15N-V is measured to be ~ 120 kHz. Residual transverse magnetic fields are observed to shift the nuclear-spin transition frequencies, especially for 15N-V. We have precisely determined the set of parameters relevant for the development of nuclear-spin-based diamond quantum sensors with greatly reduced sensitivity to environmental factors.

Nuclear quadrupole resonance spectroscopy with a femtotesla diamond magnetometer.

Radio frequency (RF) magnetometers based on nitrogen vacancy centers in diamond are predicted to offer femtotesla sensitivity, but previous experiments were limited to the picotesla level. We demonstrate a femtotesla RF magnetometer using a diamond membrane inserted between ferrite flux concentrators. The device provides ~300-fold amplitude enhancement for RF magnetic fields from 70 kHz to 3.6 MHz, and the sensitivity reaches ~70 fT√s at 0.35 MHz. The sensor detected the 3.6-MHz nuclear quadrupole resonance (NQR) of room-temperature sodium nitrite powder. The sensor's recovery time after an RF pulse is ~35 µs, limited by the excitation coil's ring-down time. The sodium-nitrite NQR frequency shifts with temperature as -1.00±0.02 kHz/K, the magnetization dephasing time is T2*=887±51 µs, and multipulse sequences extend the signal lifetime to 332±23 ms, all consistent with coil-based studies. Our results expand the sensitivity frontier of diamond magnetometers to the femtotesla range, with potential applications in security, medical imaging, and materials science.

A nuclear orthologue of the dNTP triphosphohydrolase SAMHD1 controls dNTP homeostasis and genomic stability in Trypanosoma brucei.

Maintenance of dNTPs pools in Trypanosoma brucei is dependent on both biosynthetic and degradation pathways that together ensure correct cellular homeostasis throughout the cell cycle which is essential for the preservation of genomic stability. Both the salvage and de novo pathways participate in the provision of pyrimidine dNTPs while purine dNTPs are made available solely through salvage. In order to identify enzymes involved in degradation here we have characterized the role of a trypanosomal SAMHD1 orthologue denominated TbHD82. Our results show that TbHD82 is a nuclear enzyme in both procyclic and bloodstream forms of T. brucei. Knockout forms exhibit a hypermutator phenotype, cell cycle perturbations and an activation of the DNA repair response. Furthermore, dNTP quantification of TbHD82 null mutant cells revealed perturbations in nucleotide metabolism with a substantial accumulation of dATP, dCTP and dTTP. We propose that this HD domain-containing protein present in kinetoplastids plays an essential role acting as a sentinel of genomic fidelity by modulating the unnecessary and detrimental accumulation of dNTPs.

Coupling of nitrogen-vacancy centers to photonic crystal cavities in monocrystalline diamond.

The zero-phonon transition rate of a nitrogen-vacancy center is enhanced by a factor of ∼70 by coupling to a photonic crystal resonator fabricated in monocrystalline diamond using standard semiconductor fabrication techniques. Photon correlation measurements on the spectrally filtered zero-phonon line show antibunching, a signature that the collected photoluminescence is emitted primarily by a single nitrogen-vacancy center. The linewidth of the coupled nitrogen-vacancy center and the spectral diffusion are characterized using high-resolution photoluminescence and photoluminescence excitation spectroscopy.

Trypanosoma cruzi Antigenic Proteins Shared with Acute Lymphoblastic Leukemia and Neuroblastoma.

Background. Research studies indicate that immunization with protein extracts of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, prevents the appearance of tumors in 60% of mice injected with the murine lung carcinoma tumor line. The molecular basis of this process is unknown, although the presence of specific antigens in tumor cells and on the surface of T. cruzi suggests an antiparasitic immune response, with an effective cross-reaction against cancer cells, hence the importance to identify the antigens involved and determine their potential as target cells in anticancer therapy. Aim. This study aimed to determine the presence of antigenic proteins of T. cruzi shared with acute lymphoblastic leukemia and neuroblastoma cells. Material and methods. To achieve this, polyclonal antibodies against T. cruzi were developed in rabbits, and reactivity was determined with protein extracts of acute lymphoblastic leukemia cells and neuroblastoma. The immunodetection of five different strains of T. cruzi against anti-T. cruzi polyclonal antibodies was also performed. Conclusion. The study allows the knowledge of the immunological interactions between cancer and parasites to be expanded and, therefore, contributes to the design of more and better projects that improve the therapeutic strategies applied in oncology.

Identification of a residue critical for the excision of 3'-blocking ends in apurinic/apyrimidinic endonucleases of the Xth family.

DNA single-strand breaks containing 3'-blocking groups are generated from attack of the sugar backbone by reactive oxygen species or after base excision by DNA glycosylase/apurinic/apyrimidinic (AP) lyases. In human cells, APE1 excises sugar fragments that block the 3'-ends thus facilitating DNA repair synthesis. In Leishmania major, the causal agent of leishmaniasis, the APE1 homolog is the class II AP endonuclease LMAP. Expression of LMAP but not of APE1 reverts the hypersensitivity of a xth nfo repair-deficient Escherichia coli strain to the oxidative compound hydrogen peroxide (H(2)O(2)). To identify the residues specifically involved in the repair of oxidative DNA damage, we generated random mutations in the ape1 gene and selected those variants that conferred protection against H(2)O(2). Among the resistant clones, we isolated a mutant in the nuclease domain of APE1 (D70A) with an increased capacity to remove 3'-blocking ends in vitro. D70 of APE1 aligns with A138 of LMAP and mutation of the latter to aspartate significantly reduces its 3'-phosphodiesterase activity. Kinetic analysis shows a novel role of residue D70 in the excision rate of 3'-blocking ends. The functional and structural differences between the parasite and human enzymes probably reflect a divergent molecular evolution of their DNA repair responses to oxidative damage.

Demonstration of diamond nuclear spin gyroscope.

We demonstrate the operation of a rotation sensor based on the nitrogen-14 (14N) nuclear spins intrinsic to nitrogen-vacancy (NV) color centers in diamond. The sensor uses optical polarization and readout of the nuclei and a radio-frequency double-quantum pulse protocol that monitors 14N nuclear spin precession. This measurement protocol suppresses the sensitivity to temperature variations in the 14N quadrupole splitting, and it does not require microwave pulses resonant with the NV electron spin transitions. The device was tested on a rotation platform and demonstrated a sensitivity of 4.7°/s (13 mHz/Hz), with a bias stability of 0.4 °/s (1.1 mHz).

A Mitochondrial Orthologue of the dNTP Triphosphohydrolase SAMHD1 Is Essential and Controls Pyrimidine Homeostasis in Trypanosoma brucei.

The maintenance of deoxyribonucleotide triphosphate (dNTP) homeostasis through synthesis and degradation is critical for accurate genomic and mitochondrial DNA replication fidelity. Trypanosoma brucei makes use of both the salvage and de novo pathways for the provision of pyrimidine dNTPs. In this respect, the sterile α motif and histidine-aspartate domain-containing protein 1 (SAMHD1) appears to be the most relevant dNTPase controlling dNTP/deoxynucleoside homeostasis in mammalian cells. Here, we have characterized the role of a unique trypanosomal SAMHD1 orthologue denominated TbHD52. Our results show that TbHD52 is a mitochondrial enzyme essential in bloodstream forms of T. brucei. Knockout cells are pyrimidine auxotrophs that exhibit strong defects in genomic integrity, cell cycle progression, and nuclear DNA and kinetoplast segregation in the absence of extracellular thymidine. The lack of TbHD52 can be counteracted by the overexpression of human dCMP deaminase, an enzyme that is directly involved in dUMP formation yet absent in trypanosomes. Furthermore, the cellular dNTP quantification and metabolomic analysis of TbHD52 null mutants revealed perturbations in the nucleotide metabolism with a substantial accumulation of dCTP and cytosine-derived metabolites while dTTP formation was significantly reduced. We propose that this HD-domain-containing protein unique to kinetoplastids plays an essential role in pyrimidine dNTP homeostasis and contributes to the provision of deoxycytidine required for cellular dTTP biosynthesis.