Equilibrium binding energies from fluctuation theorems and force spectroscopy simulations.

Brownian dynamics simulations are used to study the detachment of a particle from a substrate. Although the model is simple and generic, we attempt to map its energy, length and time scales onto a specific experimental system, namely a bead that is weakly bound to a cell and then removed by an optical tweezer. The external driving force arises from the combined optical tweezer and substrate potentials, and thermal fluctuations are taken into account by a Brownian force. The Jarzynski equality and Crooks fluctuation theorem are applied to obtain the equilibrium free energy difference between the final and initial states. To this end, we sample non-equilibrium work trajectories for various tweezer pulling rates. We argue that this methodology should also be feasible experimentally for the envisioned system. Furthermore, we outline how the measurement of a whole free energy profile would allow the experimentalist to retrieve the unknown substrate potential by means of a suitable deconvolution. The influence of the pulling rate on the accuracy of the results is investigated, and umbrella sampling is used to obtain the equilibrium probability of particle escape for a variety of trap potentials.

Adhesion of Plasmodium falciparum-infected erythrocytes to hyaluronic acid in placental malaria.

Infection with Plasmodium falciparum during pregnancy leads to the accumulation of parasite-infected erythrocytes in the placenta, and is associated with excess perinatal mortality, premature delivery and intrauterine growth retardation in the infant, as well as increased maternal mortality and morbidity. P. falciparum can adhere to specific receptors on host cells, an important virulence factor enabling parasites to accumulate in various organs. We report here that most P. falciparum isolates from infected placentae can bind to hyaluronic acid, a newly discovered receptor for parasite adhesion that is present on the placental lining. In laboratory isolates selected for specific high-level adhesion, binding to hyaluronic acid could be inhibited by dodecamer or larger oligosaccharide fragments or polysaccharides, treatment of immobilized receptor with hyaluronidase, or treatment of infected erythrocytes with trypsin. In vitro flow-based assays demonstrated that high levels of adhesion occurred at low wall shear stress, conditions thought to prevail in the placenta. Our findings indicate that adhesion to hyaluronic acid is involved in mediating placental parasite accumulation, thus changing the present understanding of the mechanisms of placental infection, with implications for the development of therapeutic and preventative interventions.

Comparison of Babesia rossi and Babesia canis isolates with emphasis on effects of vaccination with soluble parasite antigens: a review.

Babesia canis and B. rossi are large Babesia species that infect dogs and cause clinical disease. The spectrum of disease is highly diverse with either parasite, but upon evaluation of field cases it has been suggested that in general B. rossi is more virulent than B. canis. This difference was also found in experimental infections using B. canis and B. rossi isolates and appeared to be related to a difference in parasitaemia. Whether this difference reflects the essential difference between B. canis and B. rossi species in general, or merely reflects the variability in virulence of individual isolates cannot be discerned. Comparative in vitro and in vivo studies revealed a number of qualitative differences between the B. canis and B. rossi isolates studied; however, more research is required to determine any causal relationship between in vitro and in vivo characteristics. Vaccination with a bivalent vaccine (containing soluble parasite antigen [SPA] from supernatants of in vitro cultures of B. canis and B. rossi) induced protection against clinical babesiosis upon challenge infection with either parasite. The dynamics of parasitaemia upon challenge infection of vaccinated animals indicated a biological difference between the B. canis and B. rossi isolates studied. Vaccinated dogs that were challenged with B. rossi parasites (2 isolates tested) effectively controlled parasitaemia. By contrast, in vaccinated dogs that were challenged with B. canis isolates (2 isolates tested) there was little or no effect on parasitaemia but levels of SPA in plasma were reduced. Apparently the nature of vaccine-induced immunity differs with respect to the challenge species.

Relationship between the fungal complex causing Fusarium head blight of wheat and environmental conditions.

ABSTRACT Over 4 years, the environmental conditions and the causal agents of Fusarium head blight (FHB) disease of wheat were determined in field sites in four European countries: Hungary, Ireland, Italy, and the United Kingdom. Polymerase chain reaction-based methods were used to detect each species causing FHB and quantify its DNA (as a measurement of fungal abundance) in the samples. Canonical correspondence analysis (CCA) was used to determine the relationship of the incidence and abundance of each species with weather variables. CCA indicated that little variability in the species prevalence data was explained by the weather variables. In contrast, a greater proportion of variability in abundance data was accounted for by the weather variables. Most samples contained two or more species and statistical analysis suggested that these species tended to coexist at field sites. CCA also indicated that there were differences in the relationships of the prevalence and abundance of the six FHB species with environmental variables. Fusarium poae was associated with relatively drier and warmer conditions, whereas F. graminearum was associated with warmer/humid conditions. F. avenaceum and F. culmorum were both associated with niches of cooler/wet/humid conditions. Two Microdochium species were associated with regions of relatively cool/moderate temperatures and frequent rainfalls of short duration. The results also suggested that environmental conditions differentially affect the infection and colonization processes, and the comparative abundance of the six species.

Advances in the application of genetic manipulation methods to apicomplexan parasites.

Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly impact animal health globally, and improved, cost-effective measures to control them are urgently required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Major gaps in our understanding of the biology of these relatively poorly characterised parasites and the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell modification, and asexual and sexual reproduction, and explore the potential of the application of relatively well-established or newly emerging genetic manipulation methods, such as classical transfection or gene editing, respectively, for closing important gaps in our knowledge of the function of specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation methods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biology of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of veterinary interest will ultimately lead to the development of novel and more efficient methods for disease control.

Steroid-dependent plasticity in the medial amygdala.

Behavioral sex differences have traditionally been thought to arise from gonadal steroids during a neonatal sensitive period. However, it is possible to sex-reverse certain behaviors by reversing the levels of circulating androgen in adult males and females. These results suggest that the sexually dimorphic substrates of sex behavior are subject to a high degree of plasticity, even in adulthood. I have found that circulating androgen exerts a trophic effect on the Nissl-stained morphology of an important nucleus in the control of sex behavior, namely, the posterodorsal subnucleus of the medial amygdala. First, sex-reversing the level of circulating androgen reversed the sex difference in soma size and regional volume of the posterodorsal subnucleus of the medial amygdala in adult rats. Interestingly, activation of both androgen and estrogen receptors was necessary for the post-castration maintenance of a masculine phenotype in terms of posterodorsal subnucleus of the medial amygdala cell size, whereas only estrogen receptor activity was necessary to maintain a masculine posterodorsal subnucleus of the medial amygdala volume. Then, we showed that seasonal variation in androgen was correlated with morphologic plasticity in the posterodorsal subnucleus of the medial amygdala of the Siberian hamster. However, if the experimental males were housed with females, their posterodorsal subnucleus of the medial amygdalas failed to regress in response to winter-like short daylengths. Furthermore, when male hamsters were castrated and treated with testosterone, the posterodorsal subnucleus of the medial amygdala responded to the hormone only if the animals were in summer-like photoperiods. Overall, these findings indicate that circulating androgens are critical for the maintenance of greater posterodorsal subnucleus of the medial amygdala regional volumes and soma sizes, and that environmental variables can regulate testosterone secretion and responsiveness.

Evaluation of Components of Fusarium Head Blight Resistance in Soft Red Winter Wheat Germ Plasm Using a Detached Leaf Assay.

A large environmental influence on phenotypic estimates of disease resistance and the complex polygenic nature of Fusarium head blight (FHB) resistance in wheat (Triticum aestivum) are impediments to developing resistant cultivars. The objective of this research was to investigate the utility of a detached leaf assay, inoculated using inoculum from isolates of Microdochium nivale var. majus, to identify components of FHB resistance among 30 entries of U.S. soft red winter wheat in the 2002 Uniform Southern FHB Nursery (USFHBN). Whole plant FHB resistance of the USFHBN entries was evaluated in replicated, mist-irrigated field trials at 10 locations in eight states during the 2001-2002 season. Incubation period (days from inoculation to the first appearance of a dull gray-green water-soaked lesion) was the only detached leaf variable significantly correlated across all FHB resistance parameters accounting for 45% of the variation in FHB incidence, 27% of FHB severity, 30% of Fusarium damaged kernels, and 26% of the variation in grain deoxynivalenol (DON) concentration. The results for incubation period contrasted with previous studies of moderately resistant European cultivars, in that longer incubation period was correlated with greater FHB susceptibility, but agreed with previous findings for the Chinese cultivar Sumai 3 and CIMMYT germ plasm containing diverse sources of FHB resistance. The results support the view that the detached leaf assay method has potential for use to distinguish between specific sources of FHB resistance when combined with data on FHB reaction and pedigree information. For example, entry 28, a di-haploid line from the cross between the moderately resistant U.S. cultivar Roane and the resistant Chinese line W14, exhibited detached leaf parameters that suggested a combination of both sources of FHB resistance. The USFHBN represents the combination of adapted and exotic germ plasm, but four moderately resistant U.S. commercial cultivars (Roane, McCormick, NC-Neuse, and Pat) had long incubation and latent periods and short lesion lengths in the detached leaf assay as observed in moderately FHB resistant European cultivars. The dichotomy in the relationship between incubation period and FHB resistance indicates that this may need to be considered to effectively combine exotic and existing/adapted sources of FHB resistance.

The malaria-infected red blood cell: structural and functional changes.

The asexual stage of malaria parasites of the genus Plasmodium invade red blood cells of various species including humans. After parasite invasion, red blood cells progressively acquire a new set of properties and are converted into more typical, although still simpler, eukaryotic cells by the appearance of new structures in the red blood cell cytoplasm, and new proteins at the red blood cell membrane skeleton. The red blood cell undergoes striking morphological alterations and its rheological properties are considerably altered, manifesting as red blood cells with increased membrane rigidity, reduced deformability and increased adhesiveness for a number of other cells including the vascular endothelium. Elucidation of the structural changes in the red blood cell induced by parasite invasion and maturation and an understanding of the accompanying functional alterations have the ability to considerably extend our knowledge of structure-function relationships in the normal red blood cell. Furthermore, interference with these interactions may lead to previously unsuspected means of reducing parasite virulence and may lead to the development of novel antimalarial therapeutics.

Automated measurement of plasma viscosity by capillary viscometer.

Plasma viscosity has several advantages over the erythrocyte sedimentation rate as a measurement of an acute phase response of more than 24 hours' duration. A new capillary viscometer (Coulter Viscometer II), which gives an automated measurement of plasma viscosity, was compared with the selected manual method (Harkness viscometer) of the International Committee for Standardization in Haematology. Automated measurement of plasma viscosity at 25 degrees C showed close correlation (r = 0.979, p less than 0.002) with the selected method for 160 specimens of plasma. Satisfactory precision both within batch and between batch (coefficients of variation of 1.7% or less) was obtained at viscosity values up to 5.7 mPa.s. There was no detectable carry over between samples and viscosity values were corrected adequately for ambient temperature for the range 15-32 degrees C. Careful daily cleaning was required to prevent accumulation of protein within the automatic sampling valve of the instrument. Automated measurement of plasma viscosity is an attractive alternative to measurement of the erythrocyte sedimentation rate.

Mechanisms of cytoadhesion of flowing, parasitized red blood cells from Gambian children with falciparum malaria.

Adhesion of parasitized red blood cells to vascular endothelium is considered to be a major factor in the pathophysiology of falciparum malaria, and so the molecular mechanisms and rheologic characteristics of this interaction are of profound importance. We have investigated the adhesive behavior of wild-type parasite isolates cultured from the blood of Gambian children with falciparum malaria and allowed to flow over surfaces coated with formaldehyde-fixed human umbilical vein endothelial cells (HUVEC) or platelets. Parasitized cells were able to attach to HUVEC and/or to platelets, and studies with monoclonal antibodies showed that intercellular adhesion molecule-1 (ICAM-1) and CD36 antigen were the major mediators of adhesion for the two surfaces, respectively. The levels of adhesion to HUVEC and to platelets were highly variable but did not correlate with each other, so that different isolates express independently variable capacities to bind to the two receptors. Adhesion was stationary for platelets and generally at a higher level compared with binding to HUVEC, which was predominantly (about 60%) of a rolling type. The stationary component of adhesion to HUVEC represented a greater proportion of adhesion for the wild isolates than for laboratory-adapted strains, and this form of adhesion was relatively insensitive to antibody to ICAM-1. This suggests the existence of an additional endothelial cell-expressed receptor for the wild isolates. These studies show wide variation in the ability of wild isolates of Plasmodium falciparum to adhere to ICAM-1, CD36 antigen, and possibly other receptors in the presence of physiologically relevant flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Post-weaning social isolation of male rats reduces the volume of the medial amygdala and leads to deficits in adult sexual behavior.

At 21 days of age, gonadally intact male Long Evans rats were weaned and placed into standard laboratory conditions (three per cage) or housed singly. They were tested for noncontact erections and sexual performance at 90 and 220 days of age. Rats raised in isolation displayed significantly fewer noncontact erections in response to sensory cues from an estrous female and fewer intromissions when allowed to mate with a female than did males raised in groups. The volume of the posterodorsal component of the medial amygdala (MePD) and the size of neurons within the MePD were significantly smaller in the isolated males than in socially housed males. Similarly, neurons in the sexually dimorphic nucleus of the preoptic area (SDN-POA) were smaller in isolate animals than in controls. As both MePD volume and SDN-POA soma size are responsive to sex steroids, these differences could result if the isolates experienced lower testosterone levels. Finally, the volume of the overall medial amygdala (MeA) correlated significantly with the number of noncontact erections, a relationship that was not explained by housing condition. These findings highlight the role of social experience as a factor in the sexual differentiation of the brain and suggest a positive relationship between the volume of a brain structure and the display of sexual behaviors.

Sex difference and laterality in the volume of mouse dentate gyrus granule cell layer.

Sex differences in spatial learning have been reported in both humans and rodents. Correspondingly, there have been reports of sexual dimorphism in the morphology of the hippocampal formation (HF), a brain structure implicated in spatial cognition. In Experiment 1, we confirmed earlier reports that the overall volume of the granule cell layer (GCL) of the dentate gyrus (DG) of A/J mice is larger in males than in females. We also found that male A/J mice have a larger GCL volume in the right hemisphere than the left. Female A/J mice displayed no such laterality. A similar pattern of laterality, favoring the right HF, had been reported previously in male, but not female, rats. In Experiment 2, we examined mice with a defective structural gene for androgen receptors (testicular feminization mutant, or tfm mice) on a C57/BL6J background. The C57/J strain had not previously been examined for hippocampal sexual dimorphism. We found no sexual dimorphism in the left, right, or total volume of the GCL in C57/BL6J mice whether they were wildtype or tfm. However, the right GCL volume was greater than the left in wildtype C57/BL6J mice of either sex. No lateralization of GCL volume was found in the androgen-insensitive tfm-affected males or the partially androgen-insensitive tfm-carrier females. These findings confirm earlier reports that sexual dimorphism in mouse HF is found in some inbred strains but not others, and indicate for the first time that mouse HF structures are lateralized. The absence of lateralization in partially or wholly androgen-insensitive mice suggests that androgen receptors may play a role in development of laterality in the GCL independently of any sexual dimorphism in this structure.

Photoperiod and social cues influence the medial amygdala but not the bed nucleus of the stria terminalis in the Siberian hamster.

We investigated whether the posterodorsal nucleus of the medial amygdala (MePD) and the posteromedial nucleus of the bed nucleus of the stria terminalis (BSTpm) undergo structural changes in response to photoperiod or social environment in the Siberian hamster, a seasonally breeding rodent. Adult male hamsters were either kept in long days (LD; 15:9 h light:dark) from birth or were transferred at 12-16 weeks of age to short days (SD; 8:16) and housed with a male conspecific for 11 weeks. Other males were transferred to SD but were housed with an unrelated female conspecific from LD. Males transferred to SD without a female cagemate displayed testicular regression, but males transferred to SD with a female cagemate did not. The regional volume and average soma size of the BSTpm and the MePD were estimated using Nissl-stained brain sections. Neither photoperiod nor social condition modified either of the BSTpm measures. Among males housed in same-sex groups, the average soma size in the MePD was significantly smaller in SD males than in LD males. Cohabitation with a female resulted in MePD volumes indistinguishable from LD males. These results indicate that the MePD, a nucleus implicated in socio-sexual behavior, can respond to photoperiodic as well as to social cues.

Synaptic reorganisation of the medial amygdala during puberty.

The medial amygdala (MeA) is an important site for the gonadal hormone control of several socio-sexual behaviours that emerge during puberty, including aggression, mating and parental behaviour. We have previously shown that rising levels of pubertal androgens increase the regional volume and mean soma size of neurones in the posterodorsal subnucleus of the MeA, the MePD. The present study aimed to determine some of the constituents of pubertal volumetric growth. Using computer-guided unbiased stereology, we compared the regional volume, mean somal volume and the overall number of neurones and glia in 45-day-old male Siberian hamsters (Phodopus sungorus). Half of the hamsters had completed puberty, whereas the remainder were prepubertal as a result of photoinhibition of the hypothalamic-pituitary-gonadal axis. Puberty significantly increased MePD regional volume and mean somal volume, as previously observed. We also compared the number of puncta immunoreactive for vesicular glutamate transporter-2 (vGlut2) and post-synaptic density 95 (PSD-95), which are both markers of glutamatergic pre- and post-synaptic specialisations, as well as glutamic acid decarboxylase 65 (GAD-65), which is a marker of GABAergic terminals. Puberty increased the number of vGlut2 and PSD-95 immunoreactive puncta by two- and three-fold, respectively, whereas the number of GAD-65 immunoreactive puncta was unchanged. These results suggest that numerous excitatory synapses are added to the MeA during puberty. More broadly, they show that the pubertal emergence of sexual behaviour is accompanied by synaptic reorganisation of a key network involved in the expression of sexual behaviour.

Molecular approaches to malaria: seeking the whole picture.

This year, Australia hosted its first major international conference on malaria - Molecular Approaches to Malaria in Lorne, Victoria, 2-5 February 2000 (MAM2000). The worldwide research effort toward a better understanding of the pathogenesis and control of malaria in the post-genomic era was discussed and debated by over 250 researchers from 18 countries during four days packed with molecular biology, cell biology, genomics, vaccines and pathogenic mechanisms. This special malaria edition of Parasitology Today is an attempt to capture and summarize the quality and breadth of work presented at the conference and place this in the context of the current global malaria research effort; eight of the nine Reviews in this issue have been written by session chairs or presenters at MAM2000.

Cytoadhesion and falciparum malaria: going with the flow.

Sequestration of parasitized red blood cells in the cerebral vasculature is the predisposing event to the development of cerebral malaria during infection with Plasmodium falciparum. The adhesive interaction between these cells and receptors on the endothelial cell (cytoadhesion) occurs in the dynamic environment of the microcirculation, but most studies have neglected this factor and have concentrated on measuring adhesion in static (no flow) assays. Such studies ignore the markedly different rheological properties of parasitized red blood cells that become apparent when adhesion is examined under dynamic, flow conditions that resemble those of the circulation in vivo. Here, Brian Cooke and Ross Coppel review a number of novel aspects of cytoadhesion that have been identified using flow-based assays, and discuss their relevance to the pathophysiology, investigation and clinical management of falciparum malaria.

Plasmodium falciparum: characterization of adhesion of flowing parasitized red blood cells to platelets.

Adhesion of parasitized red blood cells to vascular endothelium contributes to the ischaemic pathology of severe falciparum malaria. One of the endothelial cytoadhesion receptors, CD36, is also expressed by platelets. We have studied adhesion of flowing parasitized cells to a surface coated with immobilized, activated platelets, both as a model for CD36-mediated adhesion and because interaction with platelets might play a direct role in thrombotic complications of malaria. Parasitized cells were able to bind firmly to platelets over a range of shear stress (up to 0.3 Pa) close to those found in the microcirculation. The binding was largely abolished by treatment of platelets with antibody to CD36, with only a small effect by antibody to ICAM-1. Binding showed pH sensitivity consistent with previous reports of CD36-mediated cytoadhesion. Fixation of the platelet surface with formaldehyde preserved adhesion and its antibody sensitivity, while fixation with glutaraldehyde greatly reduced adhesion and increased the sensitivity to antibody against ICAM-1. Thus CD36-mediated binding is inhibited by glutaraldehyde--but not formaldehyde--fixation, while ICAM-1 can mediate adhesion after either form of fixation. We conclude that platelet-coated surfaces (with or without fixation) represent a practically simple model for studying malarial cytoadhesion and that platelets are likely to be able to bind parasitized cells in vivo and could thus promote vascular occlusion.