Monocyte-driven atypical cytokine storm and aberrant neutrophil activation as key mediators of COVID-19 disease severity.

Epidemiological and clinical reports indicate that SARS-CoV-2 virulence hinges upon the triggering of an aberrant host immune response, more so than on direct virus-induced cellular damage. To elucidate the immunopathology underlying COVID-19 severity, we perform cytokine and multiplex immune profiling in COVID-19 patients. We show that hypercytokinemia in COVID-19 differs from the interferon-gamma-driven cytokine storm in macrophage activation syndrome, and is more pronounced in critical versus mild-moderate COVID-19. Systems modelling of cytokine levels paired with deep-immune profiling shows that classical monocytes drive this hyper-inflammatory phenotype and that a reduction in T-lymphocytes correlates with disease severity, with CD8+ cells being disproportionately affected. Antigen presenting machinery expression is also reduced in critical disease. Furthermore, we report that neutrophils contribute to disease severity and local tissue damage by amplification of hypercytokinemia and the formation of neutrophil extracellular traps. Together our findings suggest a myeloid-driven immunopathology, in which hyperactivated neutrophils and an ineffective adaptive immune system act as mediators of COVID-19 disease severity.

Evidence for long-term sensitization of the bowel in patients with post-infectious-IBS.

Post-infectious irritable bowel syndrome (PI-IBS) is a common gastrointestinal disorder characterized by persistent abdominal pain despite recovery from acute gastroenteritis. The underlying mechanisms are unclear, although long-term changes in neuronal function, and low grade inflammation of the bowel have been hypothesized. We investigated the presence and mechanism of neuronal sensitization in a unique cohort of individuals who developed PI-IBS following exposure to contaminated drinking water 7 years ago. We provide direct evidence of ongoing sensitization of neuronal signaling in the bowel of patients with PI-IBS. These changes occur in the absence of any detectable tissue inflammation, and instead appear to be driven by pro-nociceptive changes in the gut micro-environment. This is evidenced by the activation of murine colonic afferents, and sensitization responses to capsaicin in dorsal root ganglia (DRGs) following application of supernatants generated from tissue biopsy of patients with PI-IBS. We demonstrate that neuronal signaling within the bowel of PI-IBS patients is sensitized 2 years after the initial infection has resolved. This sensitization appears to be mediated by a persistent pro-nociceptive change in the gut micro-environment, that has the capacity to stimulate visceral afferents and facilitate neuronal TRPV1 signaling.

An orthologous non-MHC locus in rats and mice is linked to CD4+ and CD8+ T-cell proportion.

CD4+ and CD8+ T cells have a central role in the immune system due to their ability to protect against infection and cancer development without targeting self. Consequently, changes in CD4+ and CD8+ T-cell homeostasis can be indicative of an array of serious illnesses, ranging from viral infections to autoimmune diseases. In addition to environmental influences, there is evidence for a genetic component regulating the proportion of CD4+ and CD8+ T cells in lymphoid organs. Indeed, identifying the genetic determinants defining the frequency of the T-cell subsets is critical as it may reveal a targetable genetic pathway to modulate CD4+ and CD8+ T-cell numbers, which could be of clinical relevance for multiple disease settings. In this study, we aim to uncover non-MHC genetic factors regulating the proportion of CD4+ and CD8+ T cells in lymphoid tissues. By investigating linkage analyses on three independent F2 cohorts, namely a rat F2 (BBDP × ACI.1U.LYP) cohort, a mouse 3A9 TCR transgenic F2 (B10.BR × NOD.H2k) cohort and a mouse F2 (C57BL/6 × FVB/N) cohort, we uncover an orthologous non-MHC locus on rat chromosome 1 and mouse chromosome 7 that is linked to T-cell proportion amongst total lymphocytes.

Programmed cell death-1 expression correlates with disease severity and IL-5 in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Programmed cell death-1 (PD-1) is a negative regulator of T-cell responses. Expression of PD-1 and its ligands PD-L1 and PD-L2 in chronic rhinosinusitis with nasal polyps (CRSwNP) is poorly studied. METHODS: Expression of PD-1, PD-L1, PD-L2, TGF-ß, IL-5, and IL-10 mRNA was measured by real-time quantitative PCR on tissue homogenates of patients with CRSwNP (n = 21) and healthy controls (n = 21) and on primary epithelial cells. Disease severity was scored using the Lund-Mackay scores of maxillofacial computed tomography (CT) scans. Expression of PD-1 and PD-L1/L2 was evaluated at the cellular and tissue levels (n = 6) by flow cytometry and immunohistochemistry. RESULTS: Programmed cell death-1 mRNA expression was increased in tissue homogenates from patients with CRSwNP compared with controls, irrespective of the atopy status. Importantly, expression of PD-1 correlated with the total CT scan scores (r = 0.5, P = 0.02). Additionally, a significant association was found between PD-1 mRNA and expression of IL-5 mRNA in control nasal tissue (r = 0.95, P < 0.0001) and in CRSwNP (r = 0.63, P = 0.002). PD-1 was expressed on different subsets of T cells and CD11b- dendritic cells. Both PD-1 and its ligands were expressed on primary epithelial cells from control nasal tissue and nasal polyp tissue. CONCLUSIONS: Higher PD-1 expression was found in CRSwNP than in nasal tissue from controls. This was associated with disease severity and tissue IL-5 expression but unrelated to the patients' atopy status.

C-kit is important for SOD1(G93A) mouse survival independent of mast cells.

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease leading to progressive and lethal paralysis. The disease process is multi-factorial and is characterized by selective motor neuron degeneration. Previous work demonstrated that the local concentration of various growth factors can influence motor neuron survival and disease progression. A potential role for c-kit, a growth factor receptor present in the spinal cord, in ALS is unknown. To dissect the role of c-kit in ALS we interbred SOD1(G93A) mice with kit(w-sh/w-sh) mice, which have a 70% decrease in c-kit expression in the spinal cord. kit(w-sh/w-sh) SOD1(G93A) mice have a reduced survival compared to SOD1(G93A) mice, while the amount of motor neurons at end stage is similar. By means of grip strength and nerve conductance analysis we show that kit(w-sh/w-sh) mice have diminished strength and slightly impaired compound muscle action potential latency, although the number of neurons is similar across genotypes. Decreasing kit gene expression in SOD1(G93A) mice is detrimental and our results imply that this effect is independent of mast cells, as tested by ketotifen administration. To conclude, our data expand on the protective role of growth factors in ALS, as decreasing c-kit by approximately 70% is detrimental in SOD1(G93A) mice.

Idd13 is involved in determining immunoregulatory DN T-cell number in NOD mice.

Immunoregulatory T cells have been identified as key modulators of peripheral tolerance and participate in preventing autoimmune diseases. CD4(-)CD8(-) (double negative, DN) T cells compose one of these immunoregulatory T-cell subsets, where the injection of DN T cells confers protection from autoimmune diabetes progression. Interestingly, genetic loci defining the function and number of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) coincide with at least some autoimmune disease susceptibility loci. Herein, we investigate the impact of major insulin-dependent diabetes (Idd) loci in defining the number of DN T cells. We demonstrate that although Idd3, Idd5 and Idd9 loci do not regulate DN T-cell number, NOD mice congenic for diabetes resistance alleles at the Idd13 locus show a partial restoration in DN T-cell number. Moreover, competitive and non-competitive bone marrow chimera experiments reveal that DN T-cell number is defined by a bone marrow-intrinsic, but DN T-cell-extrinsic, factor. This suggests that non-autonomous candidate genes define DN T-cell number in secondary lymphoid organs. Together, our results show that the regulation of DN T-cell number in NOD mice is at least partially conferred by alleles at the Idd13 locus.

Anti-CD4 treatment inhibits autoimmunity in scurfy mice through the attenuation of co-stimulatory signals.

A major concept in autoimmunity is that disruption of Foxp3(+) regulatory T cells (Tregs) predisposes to breach of tolerance. This is exemplified by the Foxp3-linked disorder termed IPEX (immunodysregulation, polyendocrinopathy, enteropathy, X-linked) which affects newborn children. There has been considerable clinical interest in the role of non-depleting anti-CD4 antibodies as a means of upregulating the function of Foxp3(+) Tregs in order to control detrimental inflammatory responses such as transplant rejection. However, according to the paradigm of a Treg-dependent mechanism of action, the effectiveness of anti-CD4 antibodies as a therapy for human autoimmune diseases is unclear considering that Treg function might be intrinsically impaired. Specifically, anti-CD4 therapy is expected to fail in patients suffering from the IPEX syndrome due to the lack of functional Foxp3(+) Tregs. Taking advantage of natural Foxp3 mutant scurfy (sf) mice closely resembling the IPEX syndrome, and genetically engineered mice depleted of Foxp3(+) Tregs, we report here that anti-CD4 treatment induces tolerance independent of Foxp3(+) Tregs. This so far undefined mechanism is dependent on the recessive non-infectious tolerization of autoreactive T cells. Treg-independent tolerance alone is powerful enough to suppress both the onset and severity of autoimmunity and reduces clinically relevant autoantibody levels and liver fibrosis. Mechanistically, tolerance induction requires the concomitant activation of autoreactive T cells and is associated with the down-regulation of the co-stimulatory TNF-receptor superfamily members OX40 and CD30 sustaining CD4(+) T cell survival. In the light of ongoing clinical trials, our results highlight an unexpected potency of anti-CD4 antibodies for the treatment of autoimmune diseases. Particularly, CD4 blockade might represent a novel therapeutic option for the human IPEX syndrome.

Sex-determining chromosomes and sexual dimorphism: insights from genetic mapping of sex expression in a natural hybrid Fragaria × ananassa subsp. cuneifolia.

We studied the natural hybrid (Fragaria × ananassa subsp. cuneifolia) between two sexually dimorphic octoploid strawberry species (Fragaria virginiana and Fragaria chiloensis) to gain insight into the dynamics of sex chromosomes and the genesis of sexual dimorphism. Male sterility is dominant in both the parental species and thus will be inherited maternally, but the chromosome that houses the sex-determining region differs. Thus, we asked whether (1) the cytotypic composition of hybrid populations represents one or both maternal species, (2) the sex-determining chromosome of the hybrid reflects the location of male sterility within the maternal donor species and (3) crosses from the hybrid species show less sexual dimorphism than the parental species. We found that F. × ananassa subsp. cuneifolia populations consisted of both parental cytotypes but one predominated within each population. Genetic linkage mapping of two crosses showed dominance of male sterility similar to the parental species, however, the map location of male sterility reflected the maternal donor in one cross, but not the other. Moreover, female function mapped to a single region in the first cross, but to two regions in the second cross. Aside from components of female function (fruit set and seed set), other traits that have been found to be significantly sexually dimorphic in the pure species were either not dimorphic or were dimorphic in the opposite direction to the parental species. These results suggest that hybrids experience some disruption of dimorphism in secondary sexual traits, as well as novel location and number of quantitative trait locus (QTL) affecting sex function.

Finding a (pine) needle in a haystack: chloroplast genome sequence divergence in rare and widespread pines.

Critical to conservation efforts and other investigations at low taxonomic levels, DNA sequence data offer important insights into the distinctiveness, biogeographic partitioning and evolutionary histories of species. The resolving power of DNA sequences is often limited by insufficient variability at the intraspecific level. This is particularly true of studies involving plant organelles, as the conservative mutation rate of chloroplasts and mitochondria makes it difficult to detect polymorphisms necessary to track genealogical relationships among individuals, populations and closely related taxa, through space and time. Massively parallel sequencing (MPS) makes it possible to acquire entire organelle genome sequences to identify cryptic variation that would be difficult to detect otherwise. We are using MPS to evaluate intraspecific chloroplast-level divergence across biogeographic boundaries in narrowly endemic and widespread species of Pinus. We focus on one of the world's rarest pines - Torrey pine (Pinus torreyana) - due to its conservation interest and because it provides a marked contrast to more widespread pine species. Detailed analysis of nearly 90% ( approximately 105 000 bp each) of these chloroplast genomes shows that mainland and island populations of Torrey pine differ at five sites in their plastome, with the differences fixed between populations. This is an exceptionally low level of divergence (1 polymorphism/ approximately 21 kb), yet it is comparable to intraspecific divergence present in widespread pine species and species complexes. Population-level organelle genome sequencing offers new vistas into the timing and magnitude of divergence within species, and is certain to provide greater insight into pollen dispersal, migration patterns and evolutionary dynamics in plants.

Integridade cutâneo-mucosa: implicações para a família no cuidado domiciliário ao doente com câncer / Integridad cutáneo-mucosa: implicaciones para la familia en el cuidado domiciliar para los pacientes con cáncer / Skin and mucous membrane Integrity: implications for the family in home care of cancer patients

Avaliar o comprometimento cutâneo-mucoso apresentado por doentes em decorrência do câncer e seu tratamento e conhecer as ações preventivas e restauradoras das lesões, adotadas pela família no cuidado domiciliário, foram os objetivos deste trabalho que utilizou abordagem descritiva exploratória. A coleta de dados foi efetivada durante visita domiciliária, no período de março a maio de 2007, com díades ou tríades de 14 famílias de clientes cadastrados no projeto assistência de enfermagem ao doente com câncer, do Departamento de Enfermagem de uma universidade do interior de São Paulo que estavam sendo cuidados pela família no domicílio e seguindo instrumento utilizado na clínica. Os resultados apontaram comprometimento cutâneo-mucoso oriundo de quimioterapia, cirurgia, radioterapia, imobilidade no leito e dificuldades para o cuidado domiciliário de cunho instrumental técnico, financeiro e emocional. Conclui-se que muitas são as dificuldades da família nos cuidados aos clientes para manter/restaurar sua integridade cutâneo-mucosa. O profissional deve conhecer as reais necessidades da família, orientando-a e ajudando-a no cuidado domiciliário ao doente.

This exploratory descriptive study aimed to evaluate skin and mucous membrane impairment in patients due to cancer and its treatment and to learn what preventive and lesion restoration actions were taken by families in home care. Data was collected during home visits from March to May 2007, with dyads or triads from 14 families of clients enrolled in the cancer nursing care project of a university nursing department in São Paulo State, who were being cared for at home by their families, following guidelines used in the clinic. The results showed skin and mucous membrane impairment from chemotherapy, surgery, radiotherapy, immobility in bed, as well as technical, financial and emotional difficulties in home care. It was concluded that families encounter great difficulties in care to maintain or restore clients’ skin and mucous membrane integrity. Nursing professionals must learn the family’s real needs, and guide and help them in home care for the patient.

Evaluar el comprometimiento cutáneo-mucoso presentado por pacientes debido al cáncer y su tratamiento y conocer las medidas preventivas y curativas de las lesiones, adoptadas por la familia en el cuidado domiciliario, fueron los objetivos de este trabajo que utilizó enfoque descriptivo exploratorio. Los datos fueron recogidos en visitas a domicilios en el período de marzo a mayo de 2007, con díadas o tríadas de 14 familias registradas en el proyecto de asistencia de enfermería al enfermo con cáncer del Departamiento de Enfermería de una Universidad del interior de São Paulo-Brasil, que estaban siendo cuidados por la familia, en casa pero siguiendo el instrumento utilizado en la clínica. Los resultados mostraron comprometimiento cutáneo-mucoso proveniente de quimioterapia, cirugía, radioterapia, inmovilidad en la cama dificultades para el cuidado domiciliario de cuño instrumental técnico, financiero y emocional, evidenciando la necesidad de que el profesional de enfermería se encuentre con la familia para conocer sus reales necesidades, y así pueda ofrecerles orientación y ayuda en el cuidado domiciliario al paciente.

Enfermagem de família: um enfoque em oncologia / Family nursing: a focus on oncology

Há um desafio especial nos cuidados de enfermagem em cancerologia, pois os doentes oncológicos não representam uma unidade apenas quanto ao cuidado, mas constituem num complexo doente-família. Buscou-se implementar um modelo de atenção que permita compreender o significado da doença e sua influência sobre o bem-estar da família, identificando as condições de vulnerabilidade e os recursos potenciais para o enfrentamento da doença. O estudo, orientado pelos pressupostos teóricos da Family Nursing, ocorreu em 2003 e 2004. A pesquisa, mediante abordagem compreensiva da análise da realidade, abrangeu 10 famílias de uma cidade do interior do Estado de São Paulo com membros portadores de câncer. Como resultado, a assistência a esses doentes oferece suporte não só àqueles submetidos a tratamentos médico-hospitalares, mas a todo núcleo familiar que sofre as conseqüências da doença. A Family Nursing possibilitou compreender a vivência com o câncer e perceber a importância da família para reabilitação e manutenção de cada membro em particular e da família como um todo.

Modelling cardiac signal as a confound in EEG-fMRI and its application in focal epilepsy studies.

Cardiac noise has been shown to reduce the sensitivity of functional Magnetic Resonance Imaging (fMRI) to an experimental effect due to its confounding presence in the blood oxygenation level-dependent (BOLD) signal. Its effect is most severe in particular regions of the brain and a method is yet to take it into account in routine fMRI analysis. This paper reports the development of a general and robust technique to improve the reliability of EEG-fMRI studies to BOLD signal correlated with interictal epileptiform discharges (IEDs). In these studies, ECG is routinely recorded, enabling cardiac effects to be modelled, as effects of no interest. Our model is based on an over-complete basis set covering a linear relationship between cardiac-related MR signal and the phase of the cardiac cycle or time after pulse (TAP). This method showed that, on average, 24.6 +/- 10.9% of grey matter voxels contained significant cardiac effects and 22.3 +/- 24.1% of those voxels exhibiting significantly IED-correlated BOLD signal also contained significant cardiac effects. We quantified the improvement of the TAP model over the original model, without cardiac effects, by evaluating changes in efficiency, with respect to estimating the contrast of the effects of interest. Over voxels containing significant, cardiac-related signal, efficiency was improved by 18.5 +/- 4.8%. Over the remaining voxels, no improvement was demonstrated. This suggests that, while improving sensitivity in particular regions of the brain, there is no risk that the TAP model will reduce sensitivity elsewhere.

fMRI temporal clustering analysis in patients with frequent interictal epileptiform discharges: comparison with EEG-driven analysis.

Temporal clustering analysis (TCA) is an exploratory data-driven technique that has been proposed for the analysis of resting fMRI to localise epileptiform activity without need for simultaneous EEG. Conventionally, fMRI of epileptic activity has been limited to those patients with subtle clinical events or frequent interictal epileptiform EEG discharges, requiring simultaneous EEG recording, from which a linear model is derived to make valid statistical inferences from the fMRI data. We sought to evaluate TCA by comparing the results with those of EEG correlated fMRI in eight selected cases. Cases were selected with clear epileptogenic localisation or lateralisation on the basis of concordant EEG and structural MRI findings, in addition to concordant activations seen on EEG-derived fMRI analyses. In three, areas of activation were seen with TCA but none corresponding to the electro-clinical localisation or activations obtained with EEG driven analysis. Temporal clusters were closely coincident with times of maximal head motion. We feel this is a serious confound to this approach and recommend that interpretation of TCA that does not address motion and physiological noise be treated with caution. New techniques to localise epileptogenic activity with fMRI alone require validation with an appropriate independent measure. In the investigation of interictal epileptiform activity, this is best done with simultaneous EEG recording.

The effect of layers in imaging brain function using electrical impedance tomograghy.

Electrical impedance tomography (EIT) has promise for imaging brain function with rings of scalp electrodes, but hitherto human images have been collected and reconstructed using a simple algorithm in which the head was modelled as a homogeneous sphere. The purpose of this work was to assess the improvement in image quality which could be achieved by adding layers to represent the cerebro-spinal fluid (CSF), skull and scalp in the forward model employed by the reconstruction algorithm. Solutions to the forward model were produced analytically and using the linear finite element method (FEM). This was undertaken for computer simulated data when a spherical conductivity change of 10%, radius 5 mm, was moved through 29 positions within a head modelled as four concentric spheres of radius 80-92 mm in order to verify the accuracy of the linear FEM by comparison with the analytical method. Test data were also recorded in a 93.5 mm, spherical, saline-filled tank in which the skull was simulated by a hollow sphere of plaster of Paris, 5 mm thick and a 20 x 20 mm right-cylindrical Perspex object, a 100% conductivity decrease, was moved through 39 positions. The best images were achieved by reconstruction with a four- or three-shell analytical model, giving a spatial accuracy of 5.8 +/- 2.2 mm for computer simulated or 14.0 +/- 5.8 mm for tank data. Mean FWHM was 57 mm and 91 mm in the XY-plane and along the z-axis, respectively. Reconstruction with a homogeneous analytical model gave localization errors greater by about 50-300%, but a reduction in FWHM of about 5% of the image diameter. Unexpectedly, reconstruction with FEM models gave poorer results similar to the analytical homogeneous case. This confirms that addition of shells to the forward model improves image quality as expected with an analytical model for reconstruction, but that the FEM method employed, which used a medium mesh and a linear element computation, requires improvement in order to yield the expected benefits.

Neuromagnetic field strength outside the human head due to impedance changes from neuronal depolarization.

The holy grail of neuroimaging would be to have an imaging system, which could image neuronal electrical activity over milliseconds. One way to do this would be by imaging the impedance changes associated with ion channels opening in neuronal membranes in the brain during activity. In principle, we could measure this change by using electrical impedance tomography (EIT) but it is close to its threshold of detectability. With the inherent limitation in the use of electrodes, we propose a new scheme based on recording the magnetic field resulting from an injected current with superconducting quantum interference devices (SQUIDs), used in magnetoencephalography (MEG). We have performed a feasibility study using computer simulation. The head was modelled as concentric spheres to mimic the scalp, skull, cerebrospinal fluid and brain using the finite element method. The magnetic field 1 cm away from the scalp was estimated. An impedance change of 1% in a 2 cm radius volume in the brain was modelled as the region of depolarization. A constant current of 100 microA was injected into the head from diametrically opposite electrodes. The model predicts that the standing magnetic field is about 10 pT and changed by about 3 fT (0.03%) on depolarization. The spectral noise density in a typical MEG system in the frequency band 1-100 Hz is about 7 fT, so this places the change at the limit of detectability. This is similar to electrical recording, as in conventional EIT systems, but there may be advantages to MEG in that the magnetic field directly traverses the skull and instrumentation errors from the electrode-skin interface will be obviated.

The relative importance of sexual reproduction versus clonal spread in an aridland bunchgrass.

Festuca idahoensis (Idaho fescue) is a perennial caespitose grass, common in semi-arid rangelands of the Intermountain West. To determine how individuals are recruited into a population, we studied two long-term monitoring plots that were established in 1937 at the Northern Great Basin Experimental Range in southeastern Oregon. The plots measured 3.05x3.05 m, and were located approximately 30 m apart. One plot was ungrazed, the other was subject to moderate levels of cattle grazing. The number of F. idahoensis plants in both plots increased ten-fold between 1937 and 1996, but whether this was due primarily to reproduction by seed or clonal fragmentation was unknown. In 1996, we mapped and sampled 160 plants of F. idahoensis. We used dominant inter-simple sequence repeat (ISSR) markers and codominant allozyme markers in order to identify genetic individuals and measure genetic diversity. Both plots were characterized by high levels of genetic and clonal diversity. When information from ISSRs, allozymes and sample location were combined, 126 genets were recognized, each consisting of one to four samples (ramets). By measuring the diameter of clones surrounding plants that were present in 1937, we estimated that clonal spread occurred at a rate of approximately 3.7 cm per decade, and thus was of secondary importance in the maintenance and increase of F. idahoensis stands. Sexual reproduction, rather than clonal fragmentation, accounted for most of the recruitment of new plants into these plots. The grazed plot had fewer ramets, genotypes, and clones than the ungrazed plot, but the ramets were significantly larger. Levels of genetic diversity did not differ in the grazed and ungrazed plots, but there was some evidence for a small, but significant level of genetic differentiation between the two. The results also indicate that F. idahoensis has the potential to be a long-lived species with some individuals persisting in excess of 60 years. This study demonstrates how long-term monitoring can be supplemented by genetic analysis to obtain detailed information on the population dynamics of plants. In the case of this community dominant species, this provides essential information for understanding succession and developing management and restoration strategies.

A multi-shell algorithm to reconstruct EIT images of brain function.

Electrical impedance tomography (EIT) may be used to image brain function, but an important consideration is the effect of the highly resistive skull and other extracerebral layers on the flow of injected current. We describe a new reconstruction algorithm, based on a forward solution which models the head as four concentric, spherical shells, with conductivities of the brain, cerebrospinal fluid, skull and scalp. The model predicted that the mean current travelling in the brain in the diametric plane for current injection from polar electrodes was 5.6 times less than if the head was modelled as a homogeneous sphere; this suggests that an algorithm based on this should be more accurate than one based on a homogeneous sphere model. In images reconstructed from computer-simulated data or data from a realistic saline-filled tank containing a real skull, a Perspex rod was localized to within 17% or 20% of the tank diameter of its true position, respectively. Contrary to expectation, the tank images were less accurate than those obtained with a reconstruction algorithm based on a homogeneous sphere. It is not yet clear if the theoretical advantages of this algorithm will yield practical advantages for head EIT imaging; it may be necessary to proceed to more complex algorithms based on numerical models which incorporate realistic head geometry. If so, this analytical forward model and algorithm may be used to validate numerical solutions.

Variation in the nrDNA ITS of Pinus subsection Cembroides: implications for molecular systematic studies of pine species complexes.

The pinyon pines (Pinus subsection Cembroides), distributed in semiarid regions of the western United States and Mexico, include a mixture of relictual and more recently evolved taxa. To investigate relationships among the pinyons, we screened and partially sequenced 3000-bp clones of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 16 taxa from subsect. Cembroides and nine representatives from four other subsections of subgenus Strobus. Restriction digests of clones reveal within-individual heterogeneity, suggesting that concerted evolution is operating slowly on the ITS in pine species. Two ITS clones were identified as pseudogenes. Tandem subrepeats in the ITS1 form stem loops comparable to those in other genera of Pinaceae and may be promoting recombination between rDNA repeats, resulting in ITS1 chimeras. Within the pinyon clade, phylogenetic structure is present, but different clones from the same (or different) individuals of a species are polyphyletic, indicating that coalescence of ITS copies within individual genomes predates evolutionary divergence in the group. At the level of subsection and above, the ITS region corresponds well with morphological and cpDNA evidence. Except for P. nelsonii, the pinyons are monophyletic, with both subsect. Cembroides and P. nelsonii forming a clade with the foxtail and bristlecone pines (subsect. Balfourianae) of western North America.

Population structure and genetic diversity of Botrychium pumicola (Ophioglossaceae) based on inter-simple sequence repeats (ISSR).

Species of Botrychium reproduce by spores that form subterranean gametophytes and a few, like B. pumicola, also reproduce asexually with subterranean sporophytic gemmae. The goal of this study was to examine the genetic diversity of B. pumicola populations and to better understand the role of gemmae. Ninety-nine individuals from three monitored populations were sampled. The technique of inter-simple sequence repeats (ISSR) produced 15 polymorphic loci and identified 71 ISSR genotypes. Sixteen of the ISSR genotypes were shared by more that one individual in a population, representing potential clones. Ten of the 16 shared genotypes were not limited to clusters of plants (groups of plants growing from the same point). The ten potential clones were disjunct (separated by other genotypes) and not in patches as might be expected for an underground propagule. There is a high probability that these shared genotypes arose from independent sexual events suggesting they were not clones. These results suggest that the long-distance dispersal of gemmae is at best a rare event.