Studying the rigidity of red blood cells induced by Plasmodium falciparum infection.

We study the effect of different chemical moieties on the rigidity of red blood cells (RBCs) induced by Plasmodium falciparum infection, and the bystander effect previously found. The infected cells are obtained from a culture of parasite-infected RBCs grown in the laboratory. The rigidity of RBCs is measured by looking at the Brownian fluctuations of individual cells in an optical-tweezers trap. The results point towards increased intracellular cyclic adenosine monophosphate (cAMP) levels as being responsible for the increase in rigidity.

Tuning of the Hanle effect from EIT to EIA using spatially separated probe and control beams.

We demonstrate a technique for continuous tuning of the Hanle effect from electromagnetically induced transparency (EIT) to electromagnetically induced absorption (EIA) by changing the polarization ellipticity of a control beam. In contrast to previous work in this field, we use spatially separated probe and control beams. The experiments are done using magnetic sublevels of the Fg = 4 → Fe = 5 closed hyperfine transition in the 852 nm D2 line of 133Cs. The atoms are contained in a room temperature vapor cell with anti-relaxation (paraffin) coating on the walls. The paraffin coating is necessary for the atomic coherence to be transported between the beams. The experimental results are supported by a density-matrix analysis of the system, which also explains the observed amplitude and zero-crossing of the resonances. Such continuous tuning of the sign of a resonance has important applications in quantum memory and other precision measurements.

Precise measurement of hyperfine structure in the 3S1/2 state of 7Li.

We report a precise measurement of hyperfine structure in the 3S1/2 state of the odd isotope of Li, namely 7Li. The state is excited from the ground 2S1/2 state (which has the same parity) using two single-photon transitions via the intermediate 2P3/2 state. The value of the hyperfine constant we measure is A = 93.095(52) MHz, which resolves two discrepant values reported in the literature measured using other techniques. Our value is also consistent with theoretical calculations.

Diagnosis of malarial infection using change in properties of optically trapped red blood cells.

BACKGROUND: In previous work studying the properties of red blood cells (RBCs) held in an optical tweezers trap, we observed an increase in the spectrum of Brownian fluctuations for RBCs from a Plasmodium falciparum culture-due to increased rigidity of the cells-compared to normal RBCs. We wanted to extend the study to patient samples, since the earlier work was done with cultures grown in the lab. METHODS: Individual RBCs were held in an optical-tweezers trap. Its position fluctuations were measured and the power spectrum determined. The corner frequency (fc) of the spectrum gave a quantitative measurement of the spectrum. RESULTS: The value of fc was 25 Hz for normal cells, which increased to 29 Hz for infected cells-both for P. falciparum and Plasmodium vivax infections. CONCLUSION: The technique of measuring fc can be used as a screening tool for malaria in patients with fever, since RBCs not carrying the parasite will also show the change due to the bystander effect, irrespective of whether it is caused by P. falciparum or P. vivax.

Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes.

Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes.

Homology-Based Prediction of Potential Protein-Protein Interactions between Human Erythrocytes and Plasmodium falciparum.

Plasmodium falciparum, a causative agent of malaria, is a well-characterized obligate intracellular parasite known for its ability to remodel host cells, particularly erythrocytes, to successfully persist in the host environment. However, the current levels of understanding from the laboratory experiments on the host-parasite interactions and the strategies pursued by the parasite to remodel host erythrocytes are modest. Several computational means developed in the recent past to predict host-parasite/pathogen interactions have generated testable hypotheses on feasible protein-protein interactions. We demonstrate the utility of protein structure-based protocol in the recognition of potential interacting proteins across P. falciparum and host erythrocytes. In concert with the information on the expression and subcellular localization of host and parasite proteins, we have identified 208 biologically feasible interactions potentially brought about by 59 P. falciparum and 30 host erythrocyte proteins. For selected cases, we have evaluated the physicochemical viability of the predicted interactions in terms of surface complementarity, electrostatic complementarity, and interaction energies at protein interface regions. Such careful inspection of molecular and mechanistic details generates high confidence on the predicted host-parasite protein-protein interactions. The predicted host-parasite interactions generate many experimentally testable hypotheses that can contribute to the understanding of possible mechanisms undertaken by the parasite in host erythrocyte remodeling. Thus, the key protein players recognized in P. falciparum can be explored for their usefulness as targets for chemotherapeutic intervention.

The bystander effect in optically trapped red blood cells due to Plasmodium falciparum infection.

BACKGROUND: In a previous study of the properties of red blood cells (RBC) trapped in an optical tweezers trap, an increase in the spectrum of Brownian fluctuations for RBCs from a Plasmodium falciparum culture (due to increased rigidity) compared with normal RBCs was measured. A bystander effect was observed, whereby RBCs actually hosting the parasite had an effect on the physical properties of remaining non-hosting RBCs. METHODS: The distribution of corner frequency (fc) in the power spectrum of single RBCs held in an optical tweezers trap was studied. Two tests were done to confirm the bystander effect. In the first, RBCs from an infected culture were separated into hosting and non-hosting RBCs. In the second, all RBCs were removed from the infected culture, and normal RBCs were incubated in the spent medium. The trapping environment was the same for all measurements so only changes in the properties of RBCs were measured. RESULTS: In the first experiment, a similar and statistically significant increase was measured both for hosting and non-hosting RBCs. In the second experiment, normal RBCs incubated in spent medium started to become rigid after a few hours and showed complete changes (comparable with RBCs from the infected culture) after 24 h. CONCLUSION: These experiments provide direct evidence of medium-induced changes in the properties of RBCs in an infected culture, regardless of whether the RBCs actually host the parasite.

Change in spectrum of Brownian fluctuations of optically trapped red blood cells due to malarial infection.

We study the properties of single red blood cells (RBCs) held in an optical-tweezers trap. We observe a change in the spectrum of Brownian fluctuations between RBCs from normal and malaria-infected samples. The change, caused by infection-induced structural changes in the cell, appears as a statistical increase in the mean (by 25%) and standard deviation (by 200%) of the corner frequency measured over approximately 100 cells. The increase is observed even though the ensemble of cells being measured consists mostly of cells that do not actually host the parasite, but are from an infected pool. This bystander effect appears to vindicate other observations that infected cells can affect the biomechanical properties of uninfected cells. The change is also observed to be independent of the stage of infection and its duration, highlighting its potential for disease detection.

Absolute frequency determination of the 5P3/2-->7S1/2 transition in 87Rb.

We report the absolute frequency of the important 5S(1/2)-->7S(1/2) two-photon transition in (87)Rb. We access the upper state using two dipole-allowed transitions via the intermediate 5P(3/2) state. This allows us to use much lower laser intensities compared to directly driving the two-photon transition, thereby avoiding potential errors due to the AC Stark shift. Collisional shifts are also minimized because the atomic density required is several orders of magnitude smaller. Our values are consistent with earlier frequency-comb measurements.

Observation of persistent flow of a Bose-Einstein condensate in a toroidal trap.

We have observed the persistent flow of Bose-condensed atoms in a toroidal trap. The flow persists without decay for up to 10 s, limited only by experimental factors such as drift and trap lifetime. The quantized rotation was initiated by transferring one unit variant Planck's over 2pi of the orbital angular momentum from Laguerre-Gaussian photons to each atom. Stable flow was only possible when the trap was multiply connected, and was observed with a Bose-Einstein condensate fraction as small as 20%. We also created flow with two units of angular momentum and observed its splitting into two singly charged vortices when the trap geometry was changed from multiply to simply connected.

Quantized rotation of atoms from photons with orbital angular momentum.

We demonstrate the coherent transfer of the orbital angular momentum of a photon to an atom in quantized units of variant Planck's over 2pi, using a 2-photon stimulated Raman process with Laguerre-Gaussian beams to generate an atomic vortex state in a Bose-Einstein condensate of sodium atoms. We show that the process is coherent by creating superpositions of different vortex states, where the relative phase between the states is determined by the relative phases of the optical fields. Furthermore, we create vortices of charge 2 by transferring to each atom the orbital angular momentum of two photons.

Direct measurement of the oscillation frequency in an optical-tweezers trap by parametric excitation.

We demonstrate a novel technique for direct measurement of the oscillation frequency in an optical-tweezers trap. The technique uses the phenomenon of parametric resonance in an oscillator when the stiffness of the trapping potential is modulated. The trapped particle is a strongly damped oscillator; hence, the signature of parametric resonance is not an increase in the amplitude but an increase in the size of Brownian fluctuations. The trap frequency is measured with an accuracy of 0.1%, which is better than previous techniques and thus opens up new possibilities in experiments with optical tweezers.

Frequency locking of tunable diode lasers to a rubidium-stabilized ring-cavity resonator.

We demonstrate a technique for locking the frequency of a tunable diode laser to a ring-cavity resonator. The resonator is stabilized to a diode laser that is in turn locked to an atomic transition in rubidium, thus giving it absolute frequency calibration. The principal advantage of the ring-cavity design is that there is no feedback destabilization of the laser. The cavity has a free-spectral range of 1.3 GHz and Q of approximately 35, which provides robust locking of the laser. The locked laser is able to track large scans of the cavity.

Saturated-absorption spectroscopy: eliminating crossover resonances by use of copropagating beams.

We demonstrate a new technique for saturated-absorption spectroscopy by use of copropagating beams that does not have the problem of crossover resonances. The pump beam is locked to a transition, and its absorption signal is monitored while the probe beam is scanned. As the probe comes into resonance with another transition, the pump absorption is reduced and the signal shows a Doppler-free dip. We use this technique to measure hyperfine intervals in the D2 line of 85Rb with a precision of 70 kHz and to resolve hyperfine levels in the D2 line of 39K that are less than 10 MHz apart.

Precise frequency measurements of atomic transitions by use of a Rb-stabilized resonator.

We demonstrate a technique for frequency measurements of atomic transitions with a precision of 30 kHz. The frequency is measured with a ring-cavity resonator whose length is calibrated against a reference laser locked to the D2 line of 87Rb, the frequency of which is known with 10-kHz accuracy. We have used this method to measure the hyperfine structure in the 5P(3/2) state of 85Rb. We obtain precise values for the hyperfine constants, A = 25.041(6) MHz and B = 26.013(25) MHz, and a value of 77.992(20) MHz for the isotope shift in the D2 line.