Model-free dynamic contrast-enhanced MRI analysis: differentiation between active tumor and necrotic tissue in patients with glioblastoma.

OBJECTIVE: Treatment response assessment in patients with high-grade gliomas (HGG) is heavily dependent on changes in lesion size on MRI. However, in conventional MRI, treatment-related changes can appear as enhancing tissue, with similar presentation to that of active tumor tissue. We propose a model-free data-driven method for differentiation between these tissues, based on dynamic contrast-enhanced (DCE) MRI. MATERIALS AND METHODS: The study included a total of 66 scans of patients with glioblastoma. Of these, 48 were acquired from 1 MRI vendor and 18 scans were acquired from a different MRI vendor and used as test data. Of the 48, 24 scans had biopsy results. Analysis included semi-automatic arterial input function (AIF) extraction, direct DCE pharmacokinetic-like feature extraction, and unsupervised clustering of the two tissue types. Validation was performed via (a) comparison to biopsy result (b) correlation to literature-based DCE curves for each tissue type, and (c) comparison to clinical outcome. RESULTS: Consistency between the model prediction and biopsy results was found in 20/24 cases. An average correlation of 82% for active tumor and 90% for treatment-related changes was found between the predicted component and population-based templates. An agreement between the predicted results and radiologist's assessment, based on RANO criteria, was found in 11/12 cases. CONCLUSION: The proposed method could serve as a non-invasive method for differentiation between lesion tissue and treatment-related changes.

Somatosensory pain is not reliably modulated by weak acoustic stimuli.

BACKGROUND: Pain induced by electrical stimuli has been found in previous research to be reduced by brief, weak electrical pulses, termed prepulses, presented 40 to 60ms prior to the painful electrical stimulus. METHODS: The present experiment investigated the generality of this effect by presenting weak acoustic stimuli simultaneously with, or 80 or 1000ms prior to, painful electric shocks. In the second half of the experimental session, each participant (N=119) was told that the acoustic stimuli would either increase or decrease the pain induced by the electric shock, to investigate automatic and controlled cognitive processes in the modulation of pain. RESULTS: Acoustic stimuli presented simultaneously with painful stimulation increased pain slightly (4mm on a 100mm scale). Acoustic stimuli presented 80 and 1000ms prior to painful stimuli had no effect on pain. Information that acoustic stimuli would increase pain did so in females, but only when the acoustic stimulus was presented 80ms prior to the painful stimulus. CONCLUSIONS: The effect of the acoustic stimuli and of information was weak. Failure to replicate previous findings of decreased pain by weak prepulses was most likely due to the sensory modality of the prepulse stimuli. It is recommended that further studies of pain modulation by brief stimulation use electrical and not acoustic prepulse stimuli.

Cardiopulmonary baroreceptors affect reflexive startle eye blink.

Baroafferent signals originating from the 'high pressure' arterial vascular system are known to impact reflexive startle eye blink responding. However, it is not known whether baroafferent feedback of the 'low pressure' cardiopulmonary system loading status exerts a similar effect. Lower Body Negative Pressure (LBNP) at gradients of 0, -10, -20, and -30mm Hg was applied to unload cardiopulmonary baroreceptors. Acoustic startle noise bursts were delivered 230 and 530ms after spontaneous R-waves, when arterial baroreceptors are either loaded or unloaded. Eye blink responses were measured by EMG, and psychomotor reaction time by button pushes to startle stimuli. The new finding of this study was that unloading of cardiopulmonary baroreceptors increases startle eye blink responsiveness. Furthermore, we replicated the effect of relative loading/unloading of arterial baroreceptors on startle eye blink responsiveness. Effects of either arterial or cardiopulmonary baroreceptor manipulations were not present for psychomotor reaction times. These results demonstrate that the loading status of cardiopulmonary baroreceptors has an impact on brainstem-based CNS processes.

Attentional modulation of blink startle at long, short, and very short lead intervals.

The present research investigated attentional blink startle modulation at lead intervals of 60, 240 and 3500 ms. Letters printed in Gothic or standard fonts, which differed in rated interest, but not valence, served as lead stimuli. Experiment 1 established that identifying letters as vowels/consonants took longer than reading the letters and that performance in both tasks was slower if letters were printed in Gothic font. In Experiment 2, acoustic blink eliciting stimuli were presented 60, 240 and 3500 ms after onset of the letters in Gothic and in standard font and during intertrial intervals. Half the participants (Group Task) were asked to identify the letters as vowels/consonants whereas the others (Group No-Task) did not perform a task. Relative to control responses, blinks during letters were facilitated at 60 and 3500 ms lead intervals and inhibited at the 240 ms lead interval for both conditions in Group Task. Differences in blink modulation across lead intervals were found in Group No-Task only during Gothic letters with blinks at the 3500 ms lead interval facilitated relative to control blinks. The present results confirm previous findings indicating that attentional processes can modulate startle at very short lead intervals.

A complex containing CstF-64 and the SL2 snRNP connects mRNA 3' end formation and trans-splicing in C. elegans operons.

Polycistronic pre-mRNAs from Caenorhabditis elegans are processed by 3' end formation of the upstream mRNA and SL2-specific trans-splicing of the downstream mRNA. These processes usually occur within an approximately 100-nucleotide region and are mechanistically coupled. In this paper, we report a complex in C. elegans extracts containing the 3' end formation protein CstF-64 and the SL2 snRNP. This complex, immunoprecipitated with alphaCstF-64 antibody, contains SL2 RNA, but not SL1 RNA or other U snRNAs. Using mutational analysis we have been able to uncouple SL2 snRNP function and identity. SL2 RNA with a mutation in stem/loop III is functional in vivo as a trans-splice donor, but fails to splice to SL2-accepting trans-splice sites, suggesting that it has lost its identity as an SL2 snRNP. Importantly, stem/loop III mutations prevent association of SL2 RNA with CstF-64. In contrast, a mutation in stem II that inactivates the SL2 snRNP still permits complex formation with CstF-64. Therefore, SL2 RNA stem/loop III is required for both SL2 identity and formation of a complex containing CstF-64, but not for trans-splicing. These results provide a molecular framework for the coupling of 3' end formation and trans-splicing in the processing of polycistronic pre-mRNAs from C. elegans operons.

Prepulses reduce the pain of cutaneous electrical shocks.

OBJECTIVE: Both the startle reflex elicited by an intense acoustic or tactile stimulus and the perceived intensity of that stimulus can be diminished by a weak "prepulse" that precedes the startling stimulus. The present study examined whether prepulses can also diminish the pain produced by an intense electrical stimulus similar to that used to treat life-threatening cardiac arrhythmias in conscious patients with implantable cardioverter/defibrillators or transcutaneous pacemakers. METHODS: Perceptual and pain thresholds for electrical shocks to the arm were determined in 20 adults. Participants then rated the painfulness of 25 electrical shocks that were 1.5 times the pain threshold (mean shock intensity, approximately 160 V) and either presented alone or preceded (at 40-60 ms) by weak electrical prepulses equal to or 25% above the perceptual threshold. RESULTS: Prepulses significantly reduced the pain produced by the intense shocks. Individuals with the lowest pain thresholds experienced the greatest pain reduction with prepulses. In these more sensitive individuals, the most effective prepulses reduced perceived pain by 26% across the entire test session and by 54% in the initial block of five shocks. CONCLUSIONS: Prepulses may be useful in diminishing the pain associated with the therapeutic electrical shocks used to treat cardiac arrhythmias.

Interplay between AAUAAA and the trans-splice site in processing of a Caenorhabditis elegans operon pre-mRNA.

About half of Caenorhabditis elegans genes have a 1-2 bp mismatch to the canonical AAUAAA hexamer that signals 3' end formation. One rare variant, AGUAAA, is found at the 3' end of the mai-1 gene, the first gene in an operon also containing gpd-2 and gpd-3. When we expressed this operon under heat shock control, 3' end formation dependent on the AGUAAA was very inefficient, but could be rescued by a single bp change to create a perfect AAUAAA. When AGUAAA was present, most 3' ends formed at a different site, 100 bp farther downstream, right at the gpd-2 trans-splice site. Surprisingly, 3' end formation at this site did not require any observable match to the AAUAAA consensus. It is possible that 3' end formation at this site occurs by a novel mechanism--trans-splicing-dependent cleavage--as deletion of the trans-splice site prevented 3' end formation here. Changing the AGUAAA to AAUAAA also influenced the trans-splicing process: with AGUAAA, most of the gpd-2 product was trans-spliced to SL1, rather than SL2, which is normally used at downstream operon trans-splice sites. However, with AAUAAA, SL2 trans-splicing of gpd-2 was increased. Our results imply that (1) the AAUAAA consensus controls 3' end formation frequency in C. elegans; (2) the AAUAAA is important in determining SL2 trans-splicing events more than 100 bp downstream; and (3) in some circumstances, 3' end formation may occur by a trans-splicing-dependent mechanism.

Intercistronic region required for polycistronic pre-mRNA processing in Caenorhabditis elegans.

In Caenorhabditis elegans, polycistronic pre-mRNAs are processed by cleavage and polyadenylation at the 3' ends of the upstream genes and trans splicing, generally to the specialized spliced leader SL2, at the 5' ends of the downstream genes. Previous studies have indicated a relationship between these two events in the processing of a heat shock-induced gpd-2-gpd-3 polycistronic pre-mRNA. Here, we report mutational analysis of the intercistronic region of this operon by linker scan analysis. Surprisingly, no sequences downstream of the 3' end were important for 3'-end formation. In contrast, a U-rich (Ur) element located 29 bp downstream of the site of 3'-end formation was shown to be important for downstream mRNA biosynthesis. This approximately 20-bp element is sufficient for SL2 trans splicing and mRNA accumulation when transplanted to a heterologous context. Furthermore, when the downstream gene was replaced by a gene from another organism, no loss of trans-splicing specificity was observed, suggesting that the Ur element may be the primary signal required for downstream mRNA processing.

trans splicing of polycistronic Caenorhabditis elegans pre-mRNAs: analysis of the SL2 RNA.

Genes in Caenorhabditis elegans operons are transcribed as polycistronic pre-mRNAs in which downstream gene products are trans spliced to a specialized spliced leader, SL2. SL2 is donated by a 110-nucleotide RNA, SL2 RNA, present in the cell as an Sm-bound snRNP. SL2 RNA can be conceptually folded into a phylogenetically conserved three-stem-loop secondary structure. Here we report an in vivo mutational analysis of the SL2 RNA. Some sequences can be changed without consequence, while other changes result in a substantial loss of trans splicing. Interestingly, the spliced leader itself can be dramatically altered, such that the first stem-loop cannot form, with only a relatively small loss in trans-splicing efficiency. However, the primary sequence of stem II is crucial for SL2 trans splicing. Similarly, the conserved primary sequence of the third stem-loop plays a key role in trans splicing. While mutations in stem-loop III allow snRNP formation, a single nucleotide substitution in the loop prevents trans splicing. In contrast, the analogous region of SL1 RNA is not highly conserved, and its mutation does not abrogate function. Thus, stem-loop III appears to confer a specific function to SL2 RNA. Finally, an upstream sequence, previously predicted to be a proximal sequence element, is shown to be required for SL2 RNA expression.

Functional characterization of five eIF4E isoforms in Caenorhabditis elegans.

Recognition of the 5'-cap structure of mRNA by eIF4E is a critical step in the recruitment of most mRNAs to the ribosome. In Caenorhabditis elegans, approximately 70% of mRNAs contain an unusual 2,2,7-trimethylguanosine cap structure as a result of trans-splicing onto the 5' end of the pre-mRNA. The characterization of three eIF4E isoforms in C. elegans (IFE-1, IFE-2, and IFE-3) was reported previously. The present study describes two more eIF4E isoforms expressed in C. elegans, IFE-4 and IFE-5. We analyzed the requirement of each isoform for viability by RNA interference. IFE-3, the most closely related to mammalian eIF4E-1, binds only 7-methylguanosine caps and is essential for viability. In contrast, three closely related isoforms (IFE-1, IFE-2, and IFE-5) bind 2,2, 7-trimethylguanosine caps and are partially redundant, but at least one functional isoform is required for viability. IFE-4, which binds only 7-methylguanosine caps, is most closely related to an unusual eIF4E isoform found in plants (nCBP) and mammals (4E-HP) and is not essential for viability in any combination of IFE knockout. ife-2, ife-3, ife-4, and ife-5 mRNAs are themselves trans-spliced to SL1 spliced leaders. ife-1 mRNA is trans-spliced to an SL2 leader, indicating that its gene resides in a downstream position of an operon.

Numerical fourier transform spectroscopy of EMG half-waves: fragmentary-decomposition-based approach to nonstationary signal analysis.

A nonstationary signal analysis technique is introduced, which regards an oscillatory physiological signal as a sum of its fragments, presented in the form of a fragmentary decomposition (FD). The virtue of FD is that it is free of the necessity to choose a priori the basis functions intended for signal analysis or synthesis. FD uses an unchanged signal fragment between adjacent zero-crossings, as a natural basis function called the half-wave function (HWF). To show that such a function is a physically meaningful object, Fourier transform methods were employed, supported by the similar basis function (SBF) algorithm, which provides the means for numerical Fourier transform spectroscopy of separate half-waves and their frequency domain description in terms of both amplitude and phase. The application of this method to parameter identification of 751 EMG half-waves from the eye blink EMG records of ten normal subjects showed that HWF's frequency domain image represents a Gaussian distribution, which applies over a defined range of relative frequencies. This empirical evidence shows that HWFs are produced by a specific system of first-order nonlinear differential equations, whose dependency on a number of random factors is characteristic of deterministic chaos. The particular form of solutions indicates that statistical regularities relevant to the central limit theorem are likely to underlie the genesis of the mass potentials studied. FD shows potential utility in a range of nonstationary physiological signals.

Caffeine-associated stimuli elicit conditioned responses: an experimental model of the placebo effect.

RATIONALE: A neutral stimulus repeatedly paired with administration of a drug may elicit a conditioned response. This process, termed pharmacological classical conditioning, may be important in the understanding of placebo effects. OBJECTIVE: The unconditioned response to caffeine is increased physiological and psychological arousal. The present study investigated whether stimuli associated with the use of caffeine, i.e. the smell and taste of coffee, elicited a conditioned increase in arousal. It was also investigated whether conditioned arousal modulated the unconditioned arousal induced by caffeine. METHODS: Twenty subjects who drank at least two cups of coffee per day were exposed to four conditions in a within-subjects design, where the subjects received coffee or orange juice crossed with placebo or 2 mg/kg caffeine. Dependent variables were skin conductance responses and startle reflexes to 85 dB noise bursts, skin conductance levels, blood pressure, heart rate, and subjective measures of arousal. RESULTS: Both caffeine (caffeinated juice) and caffeine-associated stimuli (decaffeinated coffee) increased subjective and physiological arousal. When caffeine and caffeine-associated stimuli were presented together (caffeinated coffee), a non-significant tendency towards an additive effect of the conditioned arousal on the unconditioned arousal to caffeine was seen in some dependent variables. CONCLUSIONS: Presentation of caffeine-associated stimuli to caffeine-users elicited conditioned responses similar to the unconditioned drug response. Thus, presentation of caffeine-associated stimuli could be used as an experimental model of placebo effects.

U2AF35 is encoded by an essential gene clustered in an operon with RRM/cyclophilin in Caenorhabditis elegans.

In most species the 3' splice site is recognized initially by an interaction between the two-subunit splicing factor U2AF with the polypyrimidine (poly(Y)) tract that results in recruitment of the U2 snRNP to the branch-point consensus just upstream. In contrast, in Caenorhabditis elegans, both the poly(Y) tract and the branch-point consensus sequences are missing, apparently replaced by the highly conserved U4CAG/R 3' splice site consensus. Nevertheless C. elegans U2AF65 is very similar to its mammalian and fly counterparts and may recognize the 3' splice site consensus. Here we report the cloning of the C. elegans U2AF35 gene, uaf-2. We show that it lacks an identifiable RS domain, which, in flies, has been shown to play a role in RNA binding, but it contains an extended glycine-rich stretch at its C-terminus. uaf-2 is in an operon with cyp-13, a gene that encodes a cyclophilin with an RRM domain at its N-terminus. We demonstrate by RNA interference that both U2AF genes, uaf-1 (which encodes U2AF65) and uaf-2, are required for viability, whereas cyp-13 is apparently not.

An exon that prevents transport of a mature mRNA.

In Caenorhabditis elegans, pre-mRNA for the essential splicing factor U2AF65 sometimes is spliced to produce an RNA that includes an extra 216-bp internal exon, exon 3. Inclusion of exon 3 inserts an in-frame stop codon, yet this RNA is not subject to SMG-mediated RNA surveillance. To test whether exon 3 causes RNA to remain nuclear and thereby escape decay, we inserted it into the 3' untranslated region of a gfp reporter gene. Although exon 3 did not affect accumulation or processing of the mRNA, it dramatically suppressed expression of green fluorescent protein (GFP). We showed by in situ hybridization that exon 3-containing gfp RNA is retained in the nucleus. Intriguingly, exon 3 contains 10 matches to the 8-bp 3' splice-site consensus. We hypothesized that U2AF might recognize this octamer and thereby prevent export. This idea is supported by RNA interference experiments in which reduced levels of U2AF resulted in a small burst of gfp expression.

Both subunits of U2AF recognize the 3' splice site in Caenorhabditis elegans.

Introns are defined by sequences that bind components of the splicing machinery. The branchpoint consensus, polypyrimidine (poly(Y)) tract, and AG at the splice boundary comprise the mammalian 3' splice site. Although the AG is crucial for the recognition of introns with relatively short poly(Y) tracts, which are termed 'AG-dependent introns', the molecule responsible for AG recognition has never been identified. A key player in 3' splice site definition is the essential heterodimeric splicing factor U2AF, which facilitates the interaction of the U2 small nuclear ribonucleoprotein particle with the branch point. The U2AF subunit with a relative molecular mass (Mr 65K) of 65,000 (U2AF65) binds to the poly(Y) tract, whereas the role of the 35K subunit (U2AF35) has not been clearly defined. It is not required for splicing in vitro but it plays a critical role in vivo. Caenorhabditis elegans introns have a highly conserved U4CAG/ R at their 3' splice sites instead of branch-point and poly(Y) consensus sequences. Nevertheless, C. elegans has U2AF, 12). Here we show that both U2AF subunits crosslink to the 3' splice site. Our results suggest that the U2AF65-U2AF35 complex identifies the U4CAG/R, with U2AF35 being responsible for recognition of the canonical AG.

SL1 trans splicing and 3'-end formation in a novel class of Caenorhabditis elegans operon.

Many Caenorhabditis elegans genes exist in operons in which polycistronic precursors are processed by cleavage at the 3' ends of upstream genes and trans splicing 100 to 400 nucleotides away, at the 5' ends of downstream genes, to generate monocistronic messages. Of the two spliced leaders, SL1 is trans spliced to the 5' ends of upstream genes, whereas SL2 is reserved for downstream genes in operons. However, there are isolated examples of what appears to be a different sort of operon, in which trans splicing is exclusively to SL1 and there is no intercistronic region; the polyadenylation signal is only a few base pairs upstream of the trans-splice site. We have analyzed the processing of an operon of this type by inserting the central part of mes-6/cks-1 into an SL2-type operon. In this novel context, cks-1 is trans spliced only to SL1, and mes-6 3'-end formation occurs normally, demonstrating that this unique mode of processing is indeed intrinsic to this kind of operon, which we herein designate "SL1-type." An exceptionally long polypyrimidine tract found in the 3' untranslated regions of the three known SL1-type operons is shown to be required for the accumulation of both upstream and downstream mRNAs. Mutations of the trans-splice and poly(A) signals indicate that the two processes are independent and in competition, presumably due to their close proximity, raising the possibility that production of upstream and downstream mRNAs is mutually exclusive.

A parametric study of the separate contributions of the tactile and acoustic components of airpuffs to the blink reflex.

Airpuffs are often used as blink reflex eliciting stimuli, but the airpuff makes an abrupt noise when it leaves the tube. The present study investigated the effects of the tactile and acoustic components of airpuffs on the blink reflex in 18 human subjects. Subjects received airpuffs to the temple at 10.3, 20.7 and 31.0 kPa in a within-subjects design. The airpuff-associated noise was attenuated (Airpuff + Attenuated noise) or airpuffs were presented without noise-attenuation (Airpuff+ Unattenuated noise) in two conditions. In the third condition, the airpuff was directed away from the temple to eliminate the tactile component of the airpuff (Noise alone). Reflexes were larger, more probable and faster to the Airpuff+ Unattenuated noise compared to the Airpuff+ Attenuated noise. This indicates that there is an excitatory influence from the acoustic component of the airpuff. The findings suggest that researchers should reduce the acoustic component of airpuffs in studies that use airpuffs as reflex eliciting stimuli.

Gene clusters and polycistronic transcription in eukaryotes.

Sometimes genes are arranged nonrandomly on the chromosomes of eukaryotes. This review considers instances of gene clusters in which two genes or more are expressed from a single promoter. This includes cases in which a polycistronic pre-mRNA is processed to make monocistronic mRNAs in nematodes, as well as isolated examples of polycistronic mRNAs found in mammals, flies, and perhaps plants.

Effects of caffeine and caffeine-associated stimuli on the human startle eyeblink reflex.

An experiment was performed (n = 19) that investigated the effect of caffeine and expectancy of caffeine on the eyeblink component of the startle reflex. Nineteen habitual caffeine users received caffeinated coffee, caffeinated juice, decaffeinated coffee, or decaffeinated juice in four sessions spaced 1 week apart. Twenty-five to 30 min after ingestion of the liquid, 30 acoustic startle stimuli were presented. The results showed that caffeine increased startle eyeblink amplitude. Startle reflex onset latency was significantly longer in the decaffeinated coffee condition than in the other three conditions. This may have been due to the activation of a compensatory slowing of the reflex by the anticipation of caffeine, a slowing that was then overridden by caffeine speeding the response.

The effects of caffeine and directed attention on acoustic startle habituation.

The present experiment tested the effects of caffeine on acoustic startle habituation during different attention tasks in which subjects either (a) attended to the acoustic startle stimulus (auditory attention; n = 9) (b) attended to a visual search task during presentation of acoustic startle stimuli (visual attention; n = 10), or (c) were given no specific instructions during acoustic startle testing (no attention; n = 9). Startle eyeblink responses were measured after subjects received either caffeine (1 mg/kg) or placebo. Caffeine significantly delayed response habituation in the no attention group and in the auditory attention group, but had no effect on habituation in the visual attention group. These data show that startle habituation can occur with minimal attention being directed to the acoustic startle stimulus, and that visual attention cancels the effects of caffeine on startle habituation.