Getting the right cells to the array: Gene expression microarray analysis of cell mixtures and sorted cells.

BACKGROUND: Most biological samples are cell mixtures. Some basic questions are still unanswered about analyzing these heterogeneous samples using gene expression microarray technology (MAT). How meaningful is a cell mixture's overall gene expression profile (GEP)? Is it necessary to purify the cells of interest before microarray analysis, and how much purity is needed? How much does the purification itself distort the GEP, and how well can the GEP of a small cell subset be recovered? METHODS: Model cell mixtures with different cell ratios were analyzed by both spotted and Affymetrix MAT. GEP distortion during cell purification and GEPs of purified cells were studied. CD34+ cord blood cells were purified and analyzed by MAT. RESULTS: GEPs for mixed cell populations were found to mirror the cell ratios in the mixture. Over 75% pure samples were indistinguishable from pure cells by their overall GEP. Cell purification preserved the GEP. The GEPs of small cell subsets could be accurately recovered by cell sorting both from model cell mixtures and from cord blood. CONCLUSIONS: Purification of small cell subsets from a mixture prior to MAT is necessary for meaningful results. Even completely hidden GEPs of small cell subpopulations can be recovered by cell sorting.     

Trajectories of shifting of dipole sources of visual evoked potentials over the human brain

Dynamic study of 3D localization of the equivalent current dipole (ECD) sources of visual evoked potentials (EP) in the human brain was performed in 18 healthy subjects using a two-dipole model. Dipole tracing was performed for relatively early EP components (N1, P1, and N2) with 1-ms step. The analysis confirmed localization of these ECDs mainly in the right occipital cortex and revealed their successive shift over this area in the anterior-medial direction and then backwards in all subjects during generation of the EP components. Typically, some successive arch-like trajectories of the shift were revealed (75.8%); their duration was relatively standard (about 25 ms) and did not depend on the stimulus shape and EP phase. Between the 1st and the 2nd trajectories (110-120 ms after the stimulus onset) a jump in ECD coordinates in the medial direction was found in 85% of cases. Possible significance of the findings for the insight into dynamic topography of the visual feature processing in the human brain is discussed.     

Quantifying difference in gene expression profile between bovine blastocysts derived by in vitro fertilization and somatic cell nuclear transfer

Epigenetic reprogramming intensely occurs in somatic-cell nuclear transfer (SCNT) embryos, which highlights the importance of proper expressions of reprogramming-related genes in SCNT embryos. We here assessed gene expression profiles (GEPs) difference between bovine blastocyst groups derived by in-vitro fertilization (IVF) or SCNT; in SCNT, cumulus cells and ear skin fibroblasts were used for cSCNT and fSCNT blastocysts, respectively. We obtained GEPs of 15 reprogramming-related genes in single blastocysts using multiplex PCR and found a broad range of variations in their GEPs. Weighted root-mean-square deviation (wRMSD) analysis, which calculates the deviation of SCNT blastocysts' GEPs from IVF blastocysts' mean GEP, found a significant difference between IVF and fSCNT and between cSCNT and fSCNT blastocysts (p < 0.001) but not between IVF and cSCNT. Since the fibroblasts' GEP was more distant from the IVF blastocysts' than the cumulus cells', it might partly explain the less similarity of fSCNT blastocysts' GEPs to the IVF's mean GEP. Our wRMSD method succeeds in expressing in figures how different two comparable embryo groups of different derivations are in GEP, which would be useful to select a better embryo derivation protocol among the candidates prior to field applications.     

Functional MRI detection of activation in the primary gustatory cortices in humans

Magnetoencephalography (MEG) has recently revealed that the transitions between the parietal operculum (Pop) and the insula (area G) and the ventral end of the central sulcus (cs) were activated with the shortest latency by instrumental gustatory stimulation, which suggests that the location of the primary gustatory area is in these two regions. However, studies using other noninvasive brain-imaging methods such as positron-emission tomography or functional magnetic resonance imaging (fMRI) with manual application of tastants into the mouth have been unable to confirm this. The present study examined cortical activation by repetitive stimulation of the tongue tip with 1 M NaCl with a computer-controlled stimulator and used fMRI to detect it. In individual brains, activations were detected with multiple comparisons (false discovery rate) across the whole brain corrected (threshold at P < 0.05) at both area G and frontal operculum (Fop) in 8 of 11 subjects and at the rolandic operculum (Rop) in 7 subjects. Activations were also found at the ventral end of the cs (n = 3). Group analysis with random-effect models (multiple comparison using familywise error in regions of interest, P < 0.02) revealed activation at area G in both hemispheres and in the Fop, Rop, and ventral end of the cs on the left side. The present study revealed no activation on the gyrus of the external cerebral surface except for the Rop. Taking MEG findings into consideration, the present findings strongly indicate that the primary gustatory area is present at both the transition between the Pop and insula and the Rop including the gray matter within a ventral part of the cs.     

Rom1p and Rom2p are GDP/GTP exchange proteins (GEPs) for the Rho1p small GTP binding protein in Saccharomyces cerevisiae.

The RHO1 gene encodes a homolog of the mammalian RhoA small GTP binding protein in the yeast Saccharomyces cerevisiae. Rho1p is localized at the growth site and is required for bud formation. Multicopy suppressors of a temperature-sensitive, dominant negative mutant allele of RHO1, RHO1(G22S, D125N), were isolated and named ROM (RHO1 multicopy suppressor). Rom1p and Rom2p were found to contain a DH (Dbl homologous) domain and a PH (pleckstrin homologous) domain, both of which are conserved among the GDP/GTP exchange proteins (GEPs) for the Rho family small GTP binding proteins. Disruption of ROM2 resulted in a temperature-sensitive growth phenotype, whereas disruption of both ROM1 and ROM2 resulted in lethality. The phenotypes of deltarom1deltarom2 cells were similar to those of deltarho1 cells, including growth arrest with a small bud and cell lysis. Moreover, the temperature-sensitive growth phenotype of deltarom2 was suppressed by overexpression of RHO1 or RHO2, but not of CDC42. The glutathione-S-transferase (GST) fusion protein containing the DH domain of Rom2p showed the lipid-modified Rholp-specific GDP/GTP exchange activity which was sensitive to Rho GDP dissociation inhibitor. These results indicate that Rom1p and Rom2p are GEPs that activate Rho1p in S.cerevisiae.     

Similarities in function and gene structure of cytohesin-4 and cytohesin-1, guanine nucleotide-exchange proteins for ADP-ribosylation factors

Activation of ADP-ribosylation factors (ARFs), approximately 20-kDa GTPases that are inactive in the GDP-bound form, depends on guanine nucleotide-exchange proteins (GEPs) to accelerate GTP binding. A novel ARF GEP, designated cytohesin-4, was cloned from a human brain cDNA library. Deduced amino acid sequence of the 47-kDa protein contains the same structural components present in cytohesin -1, -2, and -3, including an approximately 200-amino acid Sec7 domain with an approximately 100-residue pleckstrin homology domain near the C terminus. The Sec7 domain sequence is 77% identical to those of other cytohesins. Structures of the cytohesin-4 and cytohesin-1 genes were remarkably similar, except for an extra 3-base pair (GAG) exon present in cytohesin-1. Two mRNAs with and without the 3-base pair sequence were found in brain in different ratios for cytohesin-1, -2, and -3 but not cytohesin-4. Recombinant cytohesin-4 stimulated guanosine 5'-3-O-(thio)triphosphate binding by human ARF1 and ARF5 but not ARF6. Like other cytohesins and unlike the approximately 200-kDa ARF GEPs, it was not inhibited by brefeldin A. A cytohesin-4 mRNA of approximately 3.7 kilobases, abundant in leukocytes, was not detected in most tissues. Among separated populations of blood cells, approximately 90% of CD33(+) (monocytes), 80% of CD2(+) (NK/T), and 10-20% of CD19(+) (B) cells contained cytohesin-4 mRNA by in situ hybridization. Thus, in gene structure and brefeldin A-insensitive GEP activity, cytohesin-4 resembles other cytohesins, but its tissue distribution differs considerably, consistent with a different specific function.     

Activation of toxin ADP-ribosyltransferases by eukaryotic ADP-ribosylation factors

ADP-ribosylation factors (ARFs) are members of a multigene family of 20-kDa guanine nucleotide-binding proteins that ate regulatory components in several pathways of intracellular vesicular trafficking. The relatively small (~180-amino acids) ARF proteins interact with a variety of molecules (in addition to GTP/GDP, of course). Cholera toxin was the first to be recognized, hence the name. Later it was shown that ARF also activates phospholipase D. Different parts of the molecule are responsible for activation of the two enzymes. In vesicular trafficking, ARF must interact with coatomer to recruit it to a membrane and thereby initiate vesicle budding. ARF function requires that it alternate between GTP- and GDP-bound forms, which involves interaction with regulatory proteins. Inactivation of ARF-GTP depends on a GTPase-activating protein or GAP. A guanine nucleotide-exchange protein or GEP accelerates release of bound GDP from inactive ARF-GDP to permit GTP binding. Inhibition of GEP by brefeldin A (BFA) blocks ARF activation and thereby vesicular transport. In cells, it causes apparent disintegration of Golgi structure. Both BFA-sensitive and insensitive GEPs are known. Sequences of peptides from a BFA-sensitive GEP purified in our laboratory revealed the presence of a Sec7 domain, a sequence of ~200 amino acids that resembles a region in the yeast Sec7 gene product, which is involved in Golgi vesicular transport. Other proteins of unknown function also contain Sec7 domains, among them a lymphocyte protein called cytohesin-1. To determine whether it had GEP activity, recombinant cytohesin-1 was synthesized in E. coli. It preferentially activated class I ARFs 1 and 3 and was not inhibited by BFA but failed to activate ARF5 (class II). There are now five Sec7 domain proteins known to have GEP activity toward class I ARFs. It remains to be determined whether there are other Sec7 domain proteins that are GEPs for ARFs 4, 5, or 6.     

Prediction of future gene expression profile by analyzing its past variation pattern

A number of initial Hematopoietic Stem Cells (HSC) are considered in a container that are able to divide into HSCs or differentiate into various types of descendant cells. In this paper, a method is designed to predict an approximate gene expression profile (GEP) for future descendant cells resulted from HSC division/differentiation. First, the GEP prediction problem is modeled into a multivariate time series prediction problem. A novel method called EHSCP (Extended Hematopoietic Stem Cell Prediction) is introduced which is an artificial neural machine to solve the problem. EHSCP accepts the initial sequence of measured GEPs as input and predicts GEPs of future descendant cells. This prediction can be performed for multiple stages of cell division/differentiation. EHSCP considers the GEP sequence as time series and computes correlation between input time series. Two novel artificial neural units called PLSTM (Parametric Long Short Term Memory) and MILSTM (Multi-Input LSTM) are designed. PLSTM makes EHSCP able to consider this correlation in output prediction. Since there exist thousands of time series in GEP prediction, a hierarchical encoder is proposed that computes this correlation using 101 MILSTMs. EHSCP is trained using 155 datasets and is evaluated on 39 test datasets. These evaluations show that EHSCP surpasses existing methods in terms of prediction accuracy and number of correctly-predicted division/differentiation stages. In these evaluations, number of correctly-predicted stages in EHSCP was 128 when as many as 8 initial stages were given.     

Electrophysiological correlates preceding and following the movement onset in man

The cortical potential changes associated with unilateral voluntary self-paced hand movements were detected over the surface of the scalp by the summation method of EEG activity in 20 young subjects. A typical complex wave form of average movement potential (AMP): N1, P1, N2, P2, were discerned in all subjects in our records. This paper presents the results of the topographical distribution of the second potential of the AMP (Premotion Positivity, P1) and the last potential of the AMP (Positive Postmovement onset Potential, P2). Our results indicate a bilateral symmetrical presence of both positive components precentrally and parietally. They also indicate that both these potentials are bilaterally large posterior to the rolandic fissure, and laterality effects in amplitudes occurred only in the second positive wave parietally during right-hand responses in right handers.     

Differences in semantic event-related potentials in learning-disabled, normal, and gifted children.

Cortical event-related potentials (ERP) were recorded over FZ, CZ, and PZ scalp sites in 15 learning-disabled (LD), 14 gifted (G), and 13 normal control (N) children of ages 8-12. The common stimulus consisted of nouns presented 80 percent of the time; the target stimulus of animal names presented 20 per cent of the time. ERPs were averaged over subjects from 180 msec pre-stimulus to 900 msec post-stimulus. Principal components analysis was used to determine if there were amplitude differences at different post-stimulus latencies as a function of condition. Differences in ERP's between groups (LD, gifted, and controls), scalp locations, and common versus target stimuli were analyzed by ANOVAs. P3, Late, P2, and N1 components represented by four factors were identified. Significant differences between G and LD and the N and LD groups were found target stimulus at all central locations for the P3 component. Differences were found centrally between G and LD, G and N, and N and LD groups for the P2 component centrally. Other differences were found for the N1 and late components. These differences could be interpreted as a deficit in either attentional mechanisms or information processing for the LD group.     

A taste receptor required for the caffeine response in vivo

Caffeine is a methylxanthine present in the coffee tree, tea plant, and other naturally occurring sources and is among the most commonly consumed drugs worldwide. Whereas the pharmacological action of caffeine has been studied extensively, relatively little is known concerning the molecular mechanism through which this substance is detected as a bitter compound. Unlike most tastants, which are detected through cell-surface G protein-coupled receptors, it has been proposed that caffeine and related methylxanthines activate taste-receptor cells through inhibition of a cyclic nucleotide phosphodiesterase (PDE) . Here, we show that the gustatory receptor Gr66a is expressed in the dendrites of Drosophila gustatory receptor neurons and is essential for the caffeine response. In a behavioral assay, the aversion to caffeine was specifically disrupted in flies missing Gr66a. Caffeine-induced action potentials were also eliminated, as was the response to theophylline, the methylxanthine in tea. The Gr66a mutant exhibited normal tastant-induced action potentials upon presentation of theobromine, a methylxanthine in cocoa. Given that theobromine and caffeine inhibit PDEs with equal potencies , these data further support the role of Gr66a rather than a PDE in mediating the caffeine response. Gr66a is the first gustatory receptor shown to be essential for caffeine-induced behavior and activity of gustatory receptor cells in vivo.     

Scalp topography and dipolar source modelling of potentials evoked by CO2 laser stimulation of the hand

CO₂ laser evoked potentials to hand stimulation recorded using a scalp 19-channel montage in 11 normal subjects consistently showed early N1/P1 dipolar field distribution peaking at a mean latency of 159 ms. The N1 negativity was distributed in the temporoparietal region contralateral to stimulation and the P1 positivity in the frontal region. The N1/P1 response was followed by 3 distinct components: (1) N2a reaching its maximal amplitude at the vertex and ipsilaterally to the stimulated hand, (2) N2b mostly distributed in the frontal region, and (3) P2 with a mid-central topography. Brain electrical source analysis showed that this sequence was explained, with a residual variance below 5%, by a model including two dipoles in the upper bank of the Sylvian fissure of each hemisphere, a frontal dipole close to the midline, and two anterior medial temporal dipoles, thus suggesting a sequential activation of the two second somatosensory areas, anterior cingulate gyrus and the amygdalar nuclei or the hippocampal formations, respectively. This model fitted well with the scalp field topography of grand average responses to stimulation of left and right hand obtained across all subjects as well as when applied to individual data. Our findings suggest that the second somatosensory area contralateral to the stimulation is the first involved in the building of pain-related responses, followed by ipsilateral second somatosensory area and limbic areas receiving noxious inputs from the periphery.     

Visual evoked potentials in response to dichoptic presentation of sinusoidal grating and noise background

Dichoptic stimulation was used in comparison of visual evoked potentials (VEPs) with those obtained with monocular stimulation (recordings made from the occipital area). 16 subjects viewed sinusoidal gratings with the right eye while a visual noise was added via a mirror for the left eye. In presence of the noise, amplitude of the early VEP components' N1, P1b, and the late component P2 decreased, P1a is not changed in presence of the noise, and the late negative wave N2 increased for all spatial frequencies. The effect of noise on the amplitude of VEPs obtained for monocular and dichoptic stimulation was similar. The data suggest that external noise is filtered by the V1 cortical neurons--matched filters for the gratings.     

基于生态系统生产总值核算的生态保护成效评估——以赣南地区为例

开展自然生态系统生产总值的评估,对于生态保护政策效益的量化考核,推动生态效益纳入社会经济体系具有重要的意义。赣南地区地处典型的南方丘陵山地带,森林覆盖率高,是赣江、东江上游的重要生态屏障。以赣南为研究区域,基于2000-2018年间3期遥感影像,并运用遥感、地理信息技术的方法,构建了生态系统生产总值的评估指标体系,探讨了将生态系统生产总值（GEP）核算应用于生态保护成效的评估。结果表明：（1）赣南地区2018年生态系统生产总值为10963.26亿元,约为当年GDP的3.9倍;气候调节、水源涵养和洪水调蓄是赣南地区生态系统的核心服务功能,这3项服务价值总和占当年GEP的75.62%;重点生态功能区县的人均GEP高于非重点生态功能区县,而单位面积GEP低于非重点生态功能区县;（2）2000年、2010年和2018年绿金指数（GEP/GDP）分别为5.20、4.19、3.91,呈现出逐年下降的趋势;（3）2000-2018年,赣南地区GEP以可比价计算增幅为21.75%,其中2000-2010年、2010-2018年的年平均增幅分别为1.46%、0.78%,GEP增幅有所减缓,但生态保护成效总体上呈现向好趋势。研究表明,GEP核算结果能很好地反映赣南地区一定时期的生态环境状态和变化,体现生态保护建设工程综合实施效果,为赣南地区后续生态保护建设提供科学依据。     

Somatosensory evoked magnetic fields and potentials following passive toe movement in humans

The somatosensory evoked magnetic fields (SEFs) and evoked potentials (SEPs) following passive toe movement were studied in 10 normal subjects. Five main components were identified in SEFs recorded around the vertex around the foot area of the primary sensory cortex (SI). The first and second components, 1M and 2M, were identified at approximately 35 and 46 ms. Equivalent current dipoles (ECDs) of both 1M and 2M were estimated around SI in the hemisphere contralateral to the movement toe, and were probably generated in area 3a or area 2, which mainly receive inputs ascending through muscle and joint afferents. The large inter-individual difference of 1M and 2M in terms of ECD orientation was probably due to a large anatomical variance of the foot area of SI. The third and fourth components, 3M and 4M, were identified at approximately 62 ms and 87 ms, respectively. They appeared to be a single large long-duration component with two peaks. Since the 3M and 4M components were significantly larger than the 1M and 2M components in amplitude and their ECD location was significantly superior to that of 1M and 2M, we suspected that they were generated in different sites from those of 1M and 2M, probably area 3b or area 4. Four components, 1E, 2E, 3E and 4E, were identified in SEPs, which appeared to correspond to 1M, 2M, 3M and 4M, respectively. The variation observed in the scalp distribution of the primary component, 1E, could be accounted for by the variation of the orientation of ECD of the 1M component. There was a large difference in the waveform of the long-latency component (longer than 100 ms) between SEFs and SEPs. The 5E of SEPs was a large amplitude component, but the 5M of SEFs was small or absent. We speculate that this long-latency component was generated by multiple generators.     

Olfactory event-related potentials in young and elderly adults: evaluation of tracking task versus eyes open/closed recording.

The purpose of the present study was to evaluate olfactory event-related potentials (OERPs) elicited by amyl acetate from subjects performing a visuomotor tracking task compared with the no-task conditions of eyes open and eyes closed. Task condition did not produce any reliable effects for any amplitude measure. Task type weakly influenced only P2 latency. Elder adults evinced smaller P2 and N1/P2 amplitudes and longer N1 and P2 latencies than young adults. The results suggest that tracking task performance is not necessary to obtain robust OERPs from normal subjects of a wide age range.     

Effects of localized nitrogen supply treatments on growth and root parameters in Pinus massoniana families under phosphorus deficiency

Aims A heterogeneous spatially distribution of nutrients in natural soil may affect plant growth. The objective of this study was to determine the effects of localized nitrogen (N) supply treatments on growth traits and root parameters among different families in Pinus massoniana.Methods Five families of P. massoniana seedlings from full-sib progenies were used as test materials (1, 25, 49, 52, and 57). This study included two conditions, (i.e. homogeneous phosphorus (P) deficiency vs. heterogeneous P efficiency) among soil layers in combination with four N supply treatments in a one-year pot experiment. These N supply treatments were: (1) Homogeneously high N along the soil profile (HHH); (2) high N-high N-low N (HHL); (3) low N-low N-high N (LLH); (4) low N-low N-on side with N addition and the other side without N supply (LLH/L).Important findings This study indicated that localized N supply treatment did enhance the growth of P. massoniana, and this enhancement mainly happened in the pattern of N applied to deep soil. The results showed: 1) Compared to the homogeneous low P condition, there were increase in the growth traits and root parameters of P. massoniana under heterogeneous low P condition. Particularly, the root length and root surface area under the heterogeneous P deficiency condition were 1.95 times and 2.11 times higher than that subjected to the homogeneous P deficiency. 2) Localized N supply treatment affected seedling growth, and there was a significant interaction among N supply pattern and P condition. In compared with homogeneous N supply treatment, the height, basal diameter and dry weight of seedlings increased significantly by localized N supply treatments (LLH and/or LLH/L) under both two P deficiency conditions. But when the seedlings parameters were enhanced under homogeneous P deficiency, they were inhibited under heterogeneous P deficiency subjected to HHL. 3) Within the two P conditions, LLH and LLH/L stimulated root proliferation significantly, and root parameters were significantly enhanced under the heterogeneous P deficiency condition. Specifically, the root length and root surface area subjected to LLH/L rather than HHH were significantly enhanced by 29.2% and 32.3%, respectively. However, the length and surface area of the roots were suppressed by HHL treatment. 4). There were significant differences in response to different N supply treatments among P. massoniana families Seedlings in the families of 49, 52, and 57 responded to the localized N supply treatments with increased root proliferation, which enhanced seedling dry mass. On the other hand, the seedling growth in the family of 25 were stimulated by N and (or) P concentration, while the response of seedlings in the family of 1 to local nitrogen supply was relatively slow and exhibited growth retardation.     

Role of Rab3 GDP/GTP exchange protein in synaptic vesicle trafficking at the mouse neuromuscular junction

The Rab3 small G protein family consists of four members, Rab3A, -3B, -3C, and -3D. Of these members, Rab3A regulates Ca(2+)-dependent neurotransmitter release. These small G proteins are activated by Rab3 GDP/GTP exchange protein (Rab3 GEP). To determine the function of Rab3 GEP during neurotransmitter release, we have knocked out Rab3 GEP in mice. Rab3 GEP-/- mice developed normally but died immediately after birth. Embryos at E18.5 showed no evoked action potentials of the diaphragm and gastrocnemius muscles in response to electrical stimulation of the phrenic and sciatic nerves, respectively. In contrast, axonal conduction of the spinal cord and the phrenic nerve was not impaired. Total numbers of synaptic vesicles, especially those docked at the presynaptic plasma membrane, were reduced at the neuromuscular junction approximately 10-fold compared with controls, whereas postsynaptic structures and functions appeared normal. Thus, Rab3 GEP is essential for neurotransmitter release and probably for formation and trafficking of the synaptic vesicles.     

Cooperative function of rho GDS and rho GDI to regulate rho p21 activation in smooth muscle.

The GDP/GTP exchange reaction of rho p21, a member of ras p21-related small GTP-binding protein superfamily, is regulated by two stimulatory GDP/GTP exchange proteins (GEPs), named smg GDS and rho GDS, and by one inhibitory GEP, named rho GDI. In bovine aortic smooth muscle, rho GDS and rho GDI were major GEPs for rho p21, and the rho GDI activity on the GDP/GTP exchange reaction of rho p21 was stronger than the rho GDS activity in their simultaneous presence. Moreover, in the crude cytosol, the GDP-bound form of rho p21 was complexed with rho GDI but not with rho GDS. These results, together with our recent finding that rho p21 is involved in the vasoconstrictor-induced Ca2+ sensitization of smooth muscle contraction, suggest that there is some mechanism to release the inhibitory action of rho GDI and to make rho p21 sensitive to the stimulatory action of rho GDS, eventually leading to the rho p21 activation, in the signaling pathways of the vasoconstrictor receptors in smooth muscle.