Population genetic and biophysical evidences reveal that purifying selection shapes the genetic landscape of Plasmodium falciparum RH ligands in Chhattisgarh and West Bengal, India.

BACKGROUND: Reticulocyte binding protein-like homologs (RHs) are currently being evaluated as anti-erythrocytic stage vaccine targets against Plasmodium falciparum malaria. Present study explores the possible evolutionary drivers shaping the genetic organization of Pfrhs in Indian parasite population. It simultaneously evaluates a putative gain-of-function variant of PfRH5, a keystone member of PfRH family. METHODS: Receptor binding regions of Pfrh1, Pfrh2a/b, Pfrh4 and whole Pfrh5 were amplified using blood samples of P. falciparum malaria patients from Chhattisgarh and West Bengal and sequenced. Assembled sequences were analysed using MEGA7 and DnaSPv6. Binding affinities of recombinant PfRH5 proteins with basigin (BSG) were compared using in silico (CHARMM and AUTODOCK) and in vitro (Circular dichroism, fluorescence spectroscopy and isothermal titration calorimetry) methods. RESULTS: Pfrh1 (0.5), Pfrh2a/b (0.875), Pfrh4 (0.667) and Pfrh5 (0.778) sequence changes corresponded to low frequency (< 0.05) variants which resulted in an overall negative Tajima's D. Since mismatch distribution of none of the Pfrh loci corroborated with the model of demographic expansion, a possible role of natural selection formulating Pfrh sequence diversity was investigated. Among the 5 members, Pfrh5 displayed very high dN/dS (5.7) ratio. Nevertheless, the model of selective sweep due to presence of any advantageous substitutions could not be invoked as polymorphic nonsynonymous sites (17/18) for Pfrh5 exceeded significantly over the divergent (62/86) ones (p = 0.0436). The majority of extant PfRH5 sequences (52/83) differed from the reference Pf3D7 allele by a single amino acid mismatch (C203Y). This non-conservative alteration was predicted to lower the total interaction energy of that PfRH5variant with BSG, compared to PfRH53D7. Biophysical evidences validated the proposition that PfRH5variant formed a more stable complex with BSG. Thermodynamic association constant for interaction of BSG with PfRH5variant was also found to be higher (Kavariant = 3.63E6 ± 2.02E6 M-1 and Ka3D7 = 1.31E6 ± 1.21E6 M-1). CONCLUSIONS: Together, the study indicates that the genetic architecture of Pfrhs is principally shaped by purifying selection. The most abundant and ubiquitous PfRH5 variant harbouring 203Y, exhibits a greater affinity for BSG compared to PfRH53D7 possessing 203C allele. The study underscores the importance of selecting the functional allele that best represents circulating strains in natural parasite populations as vaccine targets.

Time-dependent enhancement of fluorescence from Rhodobacter capsulatus SB1003 and its critical dependence on concentration temperature and static magnetic field.

Continuous exposure of 395 nm light increases the fluorescence emission intensity of photosynthetic purple non-sulphur bacteria, Rhodobacter capsulatus (SB1003). We show that such an increase in fluorescence emission of extracellular pigment complexes (PC) from these photosynthetic bacteria depends on the concentration of the pigment and temperature and can also be modulated by the static magnetic field. The time-dependent enhanced emission disappears either at or below 300 K or below a threshold sample concentration (0.1 mg/ml). The enhanced emission reappears at this condition (T < 278 K) if a static magnetic field (395 mT) is introduced during fluorescence measurement. The time dependence of emission is expressed in terms of a first-order rate constant, k = dF/(Fdt). The sign of k shifts from positive to negative as PC concentration is lowered than a threshold value, implying onset of fluorescence decay (k < 0) rather than amplification (k > 0). At PC concentration higher than a threshold, k becomes negative if the temperature is lowered. But, surprisingly, at low temperature, a static magnetic field reverts the k value to positive. We explain the logical nature of k-switching and photo-dynamics of the aforesaid microbial fluorescence emission by aggregation of protoporphyrin rings present in the PC. While the simultaneous presence of decay in fluorescence and susceptibility to static magnetic field suggests the dominance of triplet states at low temperatures, the process is reversed by SMF-induced removal of spin degeneracy. At higher temperatures, the optical excitability and lack of magnetic response suggest the dominance of singlet states. We propose that the restructuring of the singlet-triplet distribution by intersystem crossing may be the basis of this logical behaviour. In context with microbial function, time-dependent enhancement of fluorescence also implies relay of red photons to the neighbouring microbes not directly exposed to the incident radiation, thus serving as an indirect photosynthetic regulator.

Real-time electro-diffusion method to discriminate carbon nanomaterials.

We report both the experimental and theoretical insights of differential electro-diffusion behavior of carbon nanomaterials (e.g. single wall, multiwall carbon nanotubes, and graphene). We thus discriminate one from the other in a soft gel system. The differential mobility of such material depends on their intrinsic properties, both extend and rate of migration bearing the discriminatory signature. The mobility analysis is made by a real time monitoring of the respective bands.

Design of heat shock-resistant surfaces to prevent protein aggregation: Enhanced chaperone activity of immobilized α-Crystallin.

α-Crystallin is a multimeric protein belonging to the family of small heat shock proteins, which function as molecular chaperones by resisting heat and oxidative stress induced aggregation of other proteins. We immobilized α-Crystallin on a self-assembled monolayer on glass surface and studied its activity in terms of the prevention of aggregation of aldolase. We discovered that playing with grafted protein density led to interesting variations in the chaperone activity of immobilized α-Crystallin. This result is in accordance with the hypothesis that dynamicity of subunits plays a vital role in the functioning of α-Crystallin and might be able to throw light on the structure-activity relationship. We showed that the chaperone activity of a certain number of immobilized α-Crystallins was superior compared to a solution containing an equivalent number of the protein and 10 times the number of the protein at temperatures >60 °C. The α-Crystallin grafted surfaces retained activity on reuse. This could also lead to the design of potent heat-shock resistant surfaces that can find wide applications in storage and shipping of protein based biopharmaceuticals.

Thermal fluctuation based study of aqueous deficient dry eyes by non-invasive thermal imaging.

In this paper we have studied the thermal fluctuation patterns occurring at the ocular surface of the left and right eyes for aqueous deficient dry eye (ADDE) patients and control subjects by thermal imaging. We conducted our experiment on 42 patients (84 eyes) with aqueous deficient dry eyes and compared with 36 healthy volunteers (72 eyes) without any history of ocular surface disorder. Schirmer's test, Tear Break-up Time, tear Meniscus height and fluorescein staining tests were conducted. Ocular surface temperature measurement was done, using an FL-IR thermal camera and thermal fluctuation in left and right eyes was calculated and analyzed using MATLAB. The time series containing the sum of squares of the temperature fluctuation on the ocular surface were compared for aqueous deficient dry eye and control subjects. Significant statistical difference between the fluctuation patterns for control and ADDE was observed (p < 0.001 at 95% confidence interval). Thermal fluctuations in left and right eyes are significantly correlated in controls but not in ADDE subjects. The possible origin of such correlation in control and lack of correlation in the ADDE subjects is discussed in the text.

Probiotics as cheater cells: parameter space clustering for individualized prescription.

Clinicians often perform infection management administering probiotics along with antibiotics. Such probiotics added to an infecting population showing antibiotic resistance can be compared to a dynamical system composed of cheaters and workers. The presence of cheater strains is known to modulate the fitness of the infecting population. We propose a model where probiotics as cheater strain re-establishes the susceptibility of a resistant population towards an antibiotic. Control parameters must assume optimal values in order to attain minimum worker number within a finite time-scale feasible in a clinical set-up. The problem is made non-trivial by the complicated interplay between parameters. The model is an extension of a logistic framework, where a pay-off function has been included to account for the effect of instantaneous worker number on death rates of each species. The outcomes for a randomized set of parameter values and initial conditions are utilized in partitioning the set and desired clusters were identified. For a test case, one can take random combinations of controllable parameters and combine them with fixed parameters and find out the closeness of the points to the desired cluster centroids. This process leads to the identification of optimum antibiotic versus probiotic dosage range leading to elimination or limited existence of the genetically resistant population.

Static magnetic field (SMF) sensing of the P(723)/P(689) photosynthetic complex.

Moderate intensity SMF have been shown to act as a controller of the protic potential in the coherent milieu of the thylakoid membranes. SMF of the order of 60-500 mT induces memory-like effect in photosystem I (PSI, P723) emission with a correlated oscillation of photosystem II (PSII, P689) fluorescence emission at a temperature of 77 K. The observed magnetic perturbation that affects the thylakoid photon capture circuitry was also found to be associated with the bio-energetic machinery of the thylakoid membranes. At normal pH, SMF causes an enhancement of PSI fluorescence emission intensity (P723/P689 > 1), followed by a slow relaxation on the removal of SMF. The enhancement of the PSI fluorescence intensity also occurs under no-field condition, if either the pH of the medium is lowered, or protonophores, such as carbonyl cyanide chlorophenylhydrazine or nigericin are added (P723/P689≥ 2). If SMF was applied under such a low pH condition or in the presence of protonophore, a reverse effect, particularly, a reduction of the enhanced PSI emission was observed. Because SMF is essentially equivalent to a spin perturbation, the observed effects can be explained in terms of spin re-organization, illustrating a memory effect via membrane re-alignment and assembly. The mimicry of conventional uncouplers by SMF is also notable; the essential difference being the reversibility and manoeuvrability of the latter (SMF). Finally, the effect implies numerous possibilities of externally regulating the photon capture and proton circulation in the thylakoid membranes using controlled SMF.

Chirality sensitive binding of tryptophan enantiomers with pristine single wall carbon nanotubes.

We report the differential binding nature of pristine single wall carbon nanotubes (SWNTs) with tryptophan enantiomers. The differential co-operative response between the pristine SWNTs (topologically chiral) and L- and D-tryptophan (geometrically chiral) provides the insight that geometrical chirality itself manifests with topological chirality in a complex way.

Corneal penetration of gold nanoparticles--therapeutic implications.

Gold nanoparticles can show anti-glycation activity thereby preventing the aggregation of proteins. As glycation is one of the leading causes of cataract formation, the finding is important in therapeutic management of ocular pathology that follows cataract formation (e.g., cortical changes often resulting in nuclear sclerosis). In the present study, we have successfully conducted in vivo experiments using guinea pig models. While the anti-glycation property of GNPs is known in vitro, the present work for the first time shows corneal penetration of GNPs. The therapeutic promise of using GNP as an anti-cataract agent thus seems imminent. GNPs traverse and get deposited into different layers of the cornea as examined by Transmission Electron Microscopy (TEM).

Drug delivery using platelet cancer cell interaction.

PURPOSE: To develop an efficient biocompatible and targeted drug delivery system in which platelets, an essential blood component having a natural affinity for cancer cells, are used as carrier of anticancer drug as delivery of drug to the targeted site is crucial for cancer treatment. METHODS: Doxorubicin hydrochloride, a potent anti cancer drug, was delivered in lung adenocarcinoma cell line (A549) using platelet as a delivery agent. This delivery mode was also tested in Ehrlich ascites carcinoma (EAC) bearing mice in presence and absence of platelets. RESULTS: The results show that platelets can uptake the drug and release the same upon activation. The efficiency of drug loaded platelets in inducing cytotoxicity was significantly higher in both in vitro and in vivo model, as compared to the free drug. CONCLUSIONS: The proposed drug delivery strategy may lead to clinical improvement in the management of cancer treatment as lower drug concentration can be used in a targeted mode. Additionally the method can be personalized as patient's own platelet can be used for deliver various drugs.

In vivo interaction of gold nanoparticles after acute and chronic exposures in experimental animal models.

With emerging use of gold nanopaticles (GNP) in biomedical science now concern lies upon the fact that how this nonanparticles interact with biological systems both in vivo and in vitro. In this study effects of GNP (50 nm) were investigated in animal models after acute and chronic exposure. For acute studies GNP was administered intravenously at three doses and urine and blood samples were collected for urinary and haematological analysis at regular time intervals. For chronic studies GNP was administered intra-peritoneally at two dose levels and urine, blood, serum and tissue samples were collected for urinary, haematological, serum biochemical and histo-pathological analysis at regular intervals. Acute exposure revealed significant increase in WBC count at all the three dose levels and significant dose-dependent increase in RBC count and Hb%. Chronic exposure at 2 mg/kg dose level showed high toxicity. Significant changes in physical, morphological, WBC count and Hb% were observed after chronic exposure for multiple days. Histo-pathological studies indicated detrimental tissue histological changes in chronic animal models. Therefore, the above studies indicate that both acute and chronic GNP exposure exhibits potential physiological changes within animal system.

Surface tunability of nanoparticles in modulating platelet functions.

Metallic nanoparticles are attractive candidates as MRI contrast agents and mediators for drug delivery, diagnostics, and therapy. Direct contact and exposure to blood circulation is common in many such applications. The consequent thrombotic response may therefore be important to study. The main objective of the present work was to study how platelet functions were changed in the presence of different nano-surface or surface capping, which may provide a measure for the safety of a nanoparticle, and also assess the use of such nanoparticles in platelet modulation. Aggregometry, ATP release reaction, flow cytometry and immune-blotting studies were performed to study platelet response to different nano-particles (iron oxide, gold and silver). For each nanoparticle surface conjugation (capping) was varied. It was found that citric acid functionalized iron oxide nanoparticles have anti-platelet activity, with a decrease in aggregation, tyrosine phosphorylation level, and granule release. On the other hand in other cases (e.g. gold nanoparticles) pro-aggregatory response was observed in the presence of nanoparticles and, in some cases, the nanoparticles behaved neutrally (e.g. for starch-coated iron oxide nanoparticles). Therefore, nanoparticles can induce antiplatelet or a pro-aggregatory response, or remain neutral depending on surface capping. A related observation is that antiplatelet drugs can be made more potent by nanoparticle conjugation.

Molecular discriminators using single wall carbon nanotubes.

The interaction between single wall carbon nanotubes (SWNTs) and amphiphilic molecules has been studied in a solid phase. SWNTs are allowed to interact with different amphiphilic probes (e.g. lipids) in a narrow capillary interface. Contact between strong hydrophobic and amphiphilic interfaces leads to a molecular restructuring of the lipids at the interface. The geometry of the diffusion front and the rate and the extent of diffusion of the interface are dependent on the structure of the lipid at the interface. Lecithin having a linear tail showed greater mobility of the interface as compared to a branched tail lipid like dipalmitoyl phosphatidylcholine, indicating the hydrophobic interaction between single wall carbon nanotube core and the hydrophobic tail of the lipid. Solid phase interactions between SWNT and lipids can thus become a very simple but efficient means of discriminating amphiphilic molecules in general and lipids in particular.

Cancer cell response to nanoparticles: criticality and optimality.

A stochastic variation in size and electrical parameters is common in nanoparticles. Synthesizing gold nanoparticles with a varying range of size and zeta potential, we show that there is clustering at certain regions of hydrodynamic diameter and zeta potentials that can be classified using k-clustering technique. A cluster boundary was observed around 50 nm, a size known for its optimal response to cells. However, neither size nor zeta potential alone determined the optimal cellular response (e.g., percentage cell survival) induced by such nanoparticles. A complex interplay prevails between size, zeta potential, nature of surface functionalization, and extent of adhesion of the cell to a solid matrix. However, it follows that the ratio of zeta potential to surface area, which scales as the electrical field (by Gaussian law), serves as an appropriate indicator for optimal cellular response. The phase plot spanned by fractional survival and effective electric field (charge density) indicates a positive correlation between mean cell survival and the magnitude of the electric field. The phase plot spanned by fractional survival and effective electric field (charge density) associated with the nanosurface shows a bifurcation behavior. Wide variation of cell survival response is observed at certain critical values of the surface charge density, whereas in other ranges the cellular response is well behaved and more predictable. Existence of phase points near the critical region corresponds to wide fluctuation of nanoparticle-induced response, for small changes in the nanosurface property. Smaller nanoparticles with low zeta potential (e.g., those conjugated with arginine) can have such an attribute (i.e., higher electrical field strength), and eventually they cause more cell death. The study may help in optimal design of nanodrugs.

Covalent immobilization of active lysozyme on Si/glass surface using alkoxy Fischer carbene complex on SAM.

A cross-metathesis reaction between an alkene terminated self-assembled monolayer (SAM) on glass/Si wafer and an alkene tethered Fischer carbene complex yielded a functionalized surface. Rapid aminolysis of the Fischer carbene moieties permit efficient anchoring of amine containing molecules on such a surface. Attachment of 1-pyrenemethylamine was thus monitored by ATR-IR spectroscopy and fluorescence microscopy. Similarly, BSA and lysozyme were individually grafted to such Fischer carbene modified surfaces using their pendant lysine residues. It has been demonstrated that the anchored lysozyme retains its bactericidal property.

Multistability in platelets and their response to gold nanoparticles.

The nanoparticle (NP) response of platelets is shown to be critically dependent on extent of preactivation of platelets by an agonist like ADP. A transition from de-aggregatory to aggregatory state is triggered in the presence of gold NPs (AuNP) only in such critical conditions. Adhered and suspended platelets respond differentially to NPs. Preactivation in the adhered state induced by shear force explains such observation. The NP effect is associated with enhanced release reaction, tyrosine phosphorylation and CD62P expression level. Unlike cancer cells, whose response is maximal when NP size is optimal (within the range 50 - 70 nm), the platelet response monotonically increases with reduction of the AuNP size. The uptake study, using quenching of quinacrine hydrochloride fluorescence by AuNP, indicates that accumulation 18 nm AuNP is several-fold higher than the 68 nm AuNP. It is further shown that AuNP response can provide a simple measure for thrombotic risk associated with nano-drugs. FROM THE CLINICAL EDITOR: Platelet aggregation can be triggered in the presence of gold nanoparticles (AuNP). Platelet response monotonically increases with reduction of the AuNP size. AuNP response can provide a simple measure for thrombotic risk associated with nano-drugs.

Nanoparticle induced conformational change in DNA and chirality of silver nanoclusters.

Nano-clusters formed on macromolecular templates carry the symmetry information of the template. Templates with broken symmetry thus lead to formation of asymmetric clusters. In response, such clusters induce a compensatory stress on the embedded template. Silver nanoparticles grown on a covalently closed negatively supercoiled plasmid DNA (pUC19) exhibit chiral behavior and as a reciprocal response, one observes alteration in DNA conformation. The inference was drawn using gel mobility-shift studies in which a silver nanoparticle (but not ions) induces a mobility shift implying a drift from supercoiled to relaxed state of the plasmid. Supporting evidences for such structural alterations were obtained from circular dichroism (CD) and Fourier transform infrared spectroscopy (FT-IR). Silver ion and silver nanoparticles induce differential FT-IR signals reflected in the fingerprint regions 1720, 1666, 1611, 1529 cm(-1) that respectively corresponds to binding in GT, ATGC, C, and AC (A, T, G, and C representing the four nucleotides). Existence of CD signal in the silver plasmon region (350-550 nm) suggests formation of a chiral clustering of nanoparticles. The reciprocal effect on the covalently closed circular (CCC) pUC19 DNA, namely the transition to a relaxed state, can be regarded as a mimicry of the topological enzyme acting on such CCC DNA.

Size dependent chaperon properties of gold nanoparticles.

Citrate synthase is a heat labile enzyme showing a loss of activity even in response to a modest shift of temperature (35 degrees-45 degrees C). Gold nanoparticle is shown to prevent the thermal aggregation of this enzyme. The chaperon like activity of the nanoparticle diminishes if the particle size is reduced from 40 nm to 20 nm keeping the atomic concentration of gold constant. This implies that the effect is not merely due to enhanced surface area offered by the nano-surfaces. The effect is reversible as the protein separated out from nanoparticles behaves similar to control. The observed coupling between chaperon activity of the nanoparticle and its cluster forming ability is illustrated in terms of a thermal cage model.

Label-free detection of thalassemia and other ROS impairing diseases.

Pathogenesis of different diseases showed that some of them, especially thalassemia (T) and rheumatoid arthritis (RA) have an implicit association with oxidative stress and altered levels of reactive oxygen species (ROS). Introducing ROS level and the balance between ROS and antioxidants as essential metrics, an attempt was made to classify T and RA from normal individuals (treated as controls)(C) using synchronous fluorescence spectroscopy (SFS) and Raman line intensity of water. This non-invasive and label-free approach was backed up by a categorization algorithm that helped in the prediction of disease types from serum samples. The predictive system constituted principal component analysis (PCA) with four parameters, namely spectral intensity ratios of reduced nicotinamide adenine dinucleotide (NADH) to tryptophan (Trp) (NADH/Trp), kynurenine (Kyn) to tryptophan (Kyn/Trp), kynurenine to NADH (Kyn/NADH), and logarithmic changes in Raman line intensity of water (Rline), with the index headers containing the disease types. Rline has a positive correlation with both Kyn/Trp and Kyn/NADH and a negative correlation with NADH/Trp ratio, implying its direct or indirect association with oxidative stress. In addition to the classification of T, RA, and C a sub-classification of T into beta major and E-beta in their post and pre-splenectomized surgical stages could also be realized. Furthermore, receiver operating characteristic (ROC) analysis was deployed to ascertain that the misclassification error (ME) was negligible for the disease types. Graphical Abstract A schematic representation of the workflow converging into the categorical classification of disease classes.

Non-synonymous amino acid alterations in PfEBA-175 modulate the merozoite ligand's ability to interact with host's Glycophorin A receptor.

The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins.