Stretching-Induced Conformational Transition of [3-13C]Ser- and [3-13C]Tyr-Antheraea yamamai Silk Fibroin before Spinning Investigated with 13C Solid-State NMR Spectroscopy.

The conformational transition of [3-13C]Ser- and [3-13C]Tyr-Antheraea yamamai silk fibroin before spinning induced by stretching was investigated with 13C CP/MAS NMR spectroscopy. The α-helix content of the silk fibroin before stretching was found to be 31.6% based on the Ala and Ser peaks. With increasing stretching ratio, the α-helix and the random coil Ala Cß peaks decreased gradually, while the ß-sheet peak was observed at a stretching ratio of ×5 and increased rapidly upon further stretching. For Ser residue, the α-helix peak decreased monotonically with increasing stretching ratio, but the random coil peak increased slightly till the stretching ratio of ×5 and then decreased. A small ß-sheet peak was observed before stretching and then increased rapidly starting from the stretching ratio of ×7. In contrast, a gradual decrease of random coil peak and an increase of ß-sheet peak were observed for the Tyr residue. The results of this investigation may be helpful for further studies of fiber formation mechanism in A. yamamai and in the future design of artificial silk materials.

Single-cell transcriptome analyses reveal heterogeneity in suspension cultures and clonal markers of CHO-K1 cells.

Cell-to-cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum-free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle-dependent subpopulations and cell clusters, which typically appear in other single-cell transcriptome analyses, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle-dependent heterogeneity. Whole mitochondrial single-cell genome sequencing showed cell-to-cell mitochondrial genome variation and heteroplasmy within cells. The mitochondrial genome sequencing method developed here is potentially useful for the validation of cell clonality. The culture time-dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose.

Packing Structure of Antiparallel ß-Sheet Polyalanine Region in a Sequential Model Peptide of Nephila clavipes Dragline Silk Studied Using 13C Solid-State NMR and MD Simulation.

Packing structures of polyalanine regions, which are considered to be the reason for the extremely high strength of spider dragline silks, were studied using a series of sequential peptides: (Glu)4GlyGlyLeuGlyGlyGlnGlyAlaGly(Ala)nGlyGlyAlaGlyGlnGlyGlyTyrGlyGly(Glu)4 (n = 3-8) using 13C solid-state NMR spectroscopy. The conformations of (Ala)n in the freeze-dried peptides changed gradually with increasing n from random coils to α-helices with partial antiparallel ß-sheet (AP-ß) structures. Conversely, all the insolubilized peptides, n = 6-8 after low-pH treatment and n = 4-8 after formic acid/methanol treatment, formed AP-ß structures with significant amounts of staggered packing arrangements. These results are different from previously obtained results for pure alanine oligopeptides, that is, AP-ß (Ala)n formed rectangular packing for less than n = 6 but staggered packings for n ≥ 7. The 13C-labeled peptides were also used to confirm the staggered packing arrangements from NMR dynamics. Furthermore, a MD simulation supported the observed results.

Emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers.

The conditions required for the emergence of supercontraction in regenerated silkworm (Bombyx mori) silk fibers are assessed through an experimental approach that combines the spinning of regenerated fibers with controlled properties and their characterization by 13C solid-state nuclear magnetic resonance (NMR). Both supercontracting and non-supercontracting regenerated fibers are produced using the straining flow spinning (SFS) technique from 13C labeled cocoons. The short-range microstructure of the fibers is assessed through 13C CP/MAS in air and 13C DD/MAS in water, and the main microstructural features are identified and quantified. The mechanical properties of the regenerated fibers and their microstructures are compared with those of natural silkworm silk. The combined analysis highlights two possible key elements as responsible for the emergence of supercontraction: (1) the existence of an upper and a lower limit of the amorphous phase compatible with supercontraction, and (2) the existence of two ordered phases, ß-sheet A and B, which correspond to different packing arrangements of the protein chains.

Modulating proactive cognitive control by reward: differential anticipatory effects of performance-contingent and non-contingent rewards.

The present study investigated the influences of two different forms of reward presentation in modulating cognitive control. In three experiments, participants performed a flanker task for which one-third of trials were precued for a chance of obtaining a reward (reward trials). In Experiment 1, a reward was provided if participants made the correct response on reward trials, but a penalty was given if they made an incorrect response on these trials. The anticipation of this performance-contingent reward increased response speed and reduced the flanker effect, but had little influence on the sequential modulation of the flanker effect after incompatible trials. In Experiment 2, participants obtained a reward randomly on two-thirds of the precued reward trials and were given a penalty on the remaining one-third, regardless of their performance. The anticipation of this non-contingent reward had little influence on the overall response speed or flanker effect, but reduced the sequential modulation of the flanker effect after incompatible trials. Experiment 3 also used performance non-contingent rewards, but participants were randomly penalized more often than they were rewarded; non-contingent penalty had little influence on the sequential modulation of the flanker effect. None of the three experiments showed a reliable influence of the actual acquisition of rewards on task performance. These results indicate anticipatory effects of performance-contingent and non-contingent rewards on cognitive control with little evidence of aftereffects.

Unusual Dynamics of Alanine Residues in Polyalanine Regions with Staggered Packing Structure of Samia cynthia ricini Silk Fiber in Dry and Hydrated States Studied by 13C Solid-State NMR and Molecular Dynamics Simulation.

Recently, the wild silkworm and spider dragline silks have been paid considerable attention as potentially valuable biomedical materials. Samia cynthia ricini is one of the wild silkworms and the primary structure of the silk fibroin (SF) consists of tandemly repeated polyalanine (poly-A:(A)12,13). Here, we report the unusual dynamical character observed in Ala Cß groups in the poly-A region which forms an antiparallel-ß-sheet structure with a staggered packing arrangement. The 13C spin-lattice relaxation ( T1's) and spin-spin relaxation times ( T2's) of Ala Cß peaks in S. c. ricini SF fibers were observed in dry and hydrated states. The lowest field peak in Ala Cß of the poly-A region showed 2 times longer T1 value and shorter correlation time than the other Ala Cß peaks of the staggered packing structure, suggesting unusually fast hopping in methyl groups. Molecular dynamics simulations indicated that two of the Ala Cß carbons out of eight existing in the unit cell of the staggered packing structure exhibited the fastest hopping motion in spite of the shortest Cß-Cß distance, indicating a geared hopping motion. T2 values of the hydrated and dry Ala Cß peaks showed a similar value, indicating that the backbone motion of S. c. ricini SF fiber is not significantly affected by hydration.

Effect of Water on the Structure and Dynamics of Regenerated [3-13C] Ser, [3-13C] , and [3-13C] Ala-Bombyx mori Silk Fibroin Studied with 13C Solid-State Nuclear Magnetic Resonance.

The effects of water on the structure and dynamics of natural and regenerated silk fibroin (SF) samples were studied using 13C solid-state nuclear magnetic resonance (NMR) spectroscopy. We prepared different types of SF materials, sponges, and fibers with different preparation methods and compared their NMR spectra in the dry and hydrated states. Three kinds of 13C NMR techniques, r-INEPT, CP/MAS, and DD/MAS, coupled with 13C isotope labeling of Ser, Tyr, and Ala residues were used. In the hydrated sponges, several conformations, that is, Silk I* and two kinds of ß-sheets, A and B, random coil, and highly mobile hydrated random coil were observed, and the fractions were determined. The fractions were remarkably different among the three sponges but with only small differences among the regenerated and native fibers. The increase in the fraction of ß-sheet B might be one of the structural factors for preparing stronger regenerated SF fiber.

Packing Arrangements and Intersheet Interaction of Alanine Oligopeptides As Revealed by Relaxation Parameters Obtained from High-Resolution 13C Solid-State NMR.

Alanine oligopeptides provide a key structure of the crystalline domains of the silks from spiders and wild silkworm and also the sequences included in proteins such as antifreeze proteins and amyloids. In this paper, the local dynamics of alanine oligopeptides, (Ala)3, (Ala)4, and (Ala)6 were examined by high-resolution 13C solid-state NMR. The 13C spin-lattice relaxation times (T1's) for the Cß4 carbons of antiparallel (AP)-ß-sheet (Ala)4 significantly prolonged and the correlation time was estimated as 3.6 × 10-11 s which was shorter than those of other carbons in the AP-ß-sheet (Ala)4 (2.8 × 10-10 s). The T1 values for the Cß carbons of (Ala)6 showed significantly longer correlation time (8.8 × 10-9 s) than those of AP-ß-sheet (Ala)4. It is thus revealed that AP-ß-sheet (Ala)6 exhibited stronger intersheet interaction than those of AP-ß-sheet (Ala)4. The 13C spin-spin relaxation times (T2's) for the Cß4 carbons showed longer than those of the other Cß1-3 carbons of AP-ß-sheet (Ala)4. T2 values of Cß carbons reflect the slow time-scale (∼70 kHz) backbone motions. The C-terminal forms strong hydrogen bonds with water molecules and thus the backbone motion is slower than ∼70 kHz, while the central backbone motions are faster than ∼70 kHz in the AP-ß-sheet (Ala)4.

Distinct solvent- and temperature-dependent packing arrangements of anti-parallel ß-sheet polyalanines studied with solid-state 13C NMR and MD simulation.

Polyalanine (polyA) sequences are well known as the simplest sequence that naturally forms anti-parallel ß-sheets and constitute a key element in the structure of spider and wild silkworm silk fibers. We have carried out a systematic analysis of the packing of anti-parallel ß-sheets for (Ala)n, n = 5, 6, 7 and 12, using primarily 13C solid-state NMR and MD simulation. HFIP and TFA are frequently used as the dope solvents for recombinant silks, and polyA was solidified from both HFIP and TFA solutions by drying. An analysis of Ala Cß peaks in the 13C CP/MAS NMR spectra indicated that polyA from HFIP was mainly rectangular but polyA from TFA was mainly staggered. The transition from the rectangular to the staggered arrangement in (Ala)6 was observed for the first time from the change in the Ala Cß peak through heat treatment at 200 °C for 4 h. The removal of the bound water was confirmed by thermal analysis. This transition could be reproduced by MD simulation of (Ala)6 molecules at 200 °C after removal of the bound water molecules. In this way, the origin of the stability of the different packing arrangements of polyA was clarified.

Refined Crystal Structure of Samia cynthia ricini Silk Fibroin Revealed by Solid-State NMR Investigations.

Samia cynthia ricini is one of the wild silkworms and its silk fibroin (SF) consists of alternatively repeating poly-l-alanine (PLA) sequences as crystalline domain and glycine-rich sequences as noncrystalline domain; the structure is similar to those of spider silk and other wild silkworm silks. In this paper, we proposed a new staggered model for the packing arrangement of the PLA sequence through the use of the Cambridge Serial Total Energy Package program and a comparison of the observed and calculated chemical shifts of the PLA sequence with the Gauge Including Projector Augmented Wave method. The new model was supported by the interatomic distance information from the cross peaks of Ala Cß dipolar-assisted rotational resonance (DARR) spectrum of the PLA sequences in S. c. ricini SF fiber. In addition, three 13C NMR peaks observed in the ß-sheet region were assigned to the carbons with different environments in the same model, but not assigned to different ß-sheet structures.

Pointing hand stimuli induce spatial compatibility effects and effector priming.

The present study investigated the automatic influence of perceiving a picture that indicates other's action on one's own task performance in terms of spatial compatibility and effector priming. Participants pressed left and right buttons with their left and right hands respectively, depending on the color of a central dot target. Preceding the target, a left or right hand stimulus (pointing either to the left or right with the index or little finger) was presented. In Experiment 1, with brief presentation of the pointing hand, a spatial compatibility effect was observed: responses were faster when the direction of the pointed finger and the response position were spatially congruent than when incongruent. The spatial compatibility effect was larger for the pointing index finger stimulus compared to the pointing little finger stimulus. Experiment 2 employed longer duration of the pointing hand stimuli. In addition to the spatial compatibility effect for the pointing index finger, the effector priming effect was observed: responses were faster when the anatomical left/right identity of the pointing and response hands matched than when the pointing and response hands differed in left/right identity. The results indicate that with sufficient processing time, both spatial/symbolic and anatomical features of a static body part implying another's action simultaneously influence different aspects of the perceiver's own action. Hierarchical coding, according to which an anatomical code is used only when a spatial code is unavailable, may not be applicable if stimuli as well as responses contain anatomical features.

Effects of visual cue and response assignment on spatial stimulus coding in stimulus-response compatibility.

Tlauka and McKenna ( 2000 ) reported a reversal of the traditional stimulus-response compatibility (SRC) effect (faster responding to a stimulus presented on the same side than to one on the opposite side) when the stimulus appearing on one side of a display is a member of a superordinate unit that is largely on the opposite side. We investigated the effects of a visual cue that explicitly shows a superordinate unit, and of assignment of multiple stimuli within each superordinate unit to one response, on the SRC effect based on superordinate unit position. Three experiments revealed that stimulus-response assignment is critical, while the visual cue plays a minor role, in eliciting the SRC effect based on the superordinate unit position. Findings suggest bidirectional interaction between perception and action and simultaneous spatial stimulus coding according to multiple frames of reference, with contribution of each coding to the SRC effect flexibly varying with task situations.

Visual and auditory accessory stimulus offset and the Simon effect.

We investigated the effect on the right and left responses of the disappearance of a task-irrelevant stimulus located on the right or left side. Participants pressed a right or left response key on the basis of the color of a centrally located visual target. Visual (Experiment 1) or auditory (Experiment 2) task-irrelevant accessory stimuli appeared or disappeared at locations to the right or left of the central target. In Experiment 1, responses were faster when onset or offset of the visual accessory stimulus was spatially congruent with the response. In Experiment 2, responses were again faster when onset of the auditory accessory stimulus and the response were on the same side. However, responses were slightly slower when offset of the auditory accessory stimulus and the response were on the same side than when they were on opposite sides. These findings indicate that transient change information is crucial for a visual Simon effect, whereas sustained stimulation from an ongoing stimulus also contributes to an auditory Simon effect.

Response-specifying cue for action interferes with perception of feature-sharing stimuli.

Perceiving a visual stimulus is more difficult when a to-be-executed action is compatible with that stimulus, which is known as blindness to response-compatible stimuli. The present study explored how the factors constituting the action event (i.e., response-specifying cue, response intention, and response feature) affect the occurrence of this blindness effect. The response-specifying cue varied along the horizontal and vertical dimensions, while the response buttons were arranged diagonally. Participants responded based on one dimension randomly determined in a trial-by-trial manner. The response intention varied along a single dimension, whereas the response location and the response-specifying cue varied within both vertical and horizontal dimensions simultaneously. Moreover, the compatibility between the visual stimulus and the response location and the compatibility between that stimulus and the response-specifying cue was separately determined. The blindness effect emerged exclusively based on the feature correspondence between the response-specifying cue of the action task and the visual target of the perceptual task. The size of this stimulus-stimulus (S-S) blindness effect did not differ significantly across conditions, showing no effect of response intention and response location. This finding emphasizes the effect of stimulus factors, rather than response factors, of the action event as a source of the blindness to response-compatible stimuli.

Effectiveness of communication campaign on iron deficiency anemia in Kyzyl-Orda region, Kazakhstan: a pilot study.

BACKGROUND: In 2004, wheat flour fortification (WFF) with iron was implemented in Kazakhstan as a public health strategy to increase the iron intake of all women of childbearing age and of children. In 2003, before starting the flour fortification program, a communication campaign on health education took place in a region with a high prevalence of iron deficiency anemia (IDA). The present study aimed to evaluate the prevalence of anemia, iron deficiency and IDA before and after the campaign. In addition, knowledge about IDA and its prevention, as well as awareness about fortified wheat flour, was assessed. METHODS: The subjects of the study were women aged 15-49 years and children aged 2-14 years. The study was carried out in urban and rural areas of Kyzyl-Orda region in 2003 before (March) and after (December) the campaign. Blood samples were collected in order to measure hemoglobin and serum ferritin. In March 80 women and 57 children in the urban area, and 41 women and 41 children in the rural area, participated in the IDA testing. The corresponding participants in December numbered 62, 52, 52, and 57, respectively. The impacts of the communications and information received by participants during the campaign was surveyed with a questionnaire for 195 women in March and 198 women in December including some who participated in the IDA testing. RESULTS: In March, the prevalence of anemia was 52.0% among 121 women and 58.1% among 98 children, and those with low iron reserve were 63.6%, 49.1% and IDA 40.5%, 11.0%, respectively. In December, the prevalence of anemia had significantly decreased among rural women (from 65.9% to 48.0%, p < 0.05) and among urban children (from 63.1% to 11.5%, p < 0.001). The prevalence of iron deficiency was significantly reduced among the children (from 51.1% to 24.8%, p < 0.001). IDA prevalence was meaningfully decreased among women in urban and combined areas (from 37.5% to 15.0% and 40.5 to 14.8%, respectively, p < 0.001) and among urban children (from 7.1% to 2.1%, p < 0.05). The surveys found that most women knew about IDA and its prevention and that the numbers were similar both in March and in December. The knowledge of the anti-anemic effect of wheat fortified flour improved significantly over the period of the campaign among women both in urban (from 48.5% to 80.9%, p < 0.001) and rural (from 69.8% to 88.6%, p < 0.001) areas. CONCLUSION: The study demonstrated that the communication campaign before implementation of WFF program was effectively carried out, giving a biological impact on hematological indices.

Effector identity and orthogonal stimulus-response compatibility in blindness to response-compatible stimuli.

Perceiving a visual stimulus is hampered when the stimulus is compatible with simultaneously prepared or executed action (blindness effect). We explored the roles of the effector identity of the responding hand and of orthogonal compatibility (above-right/below-left correspondence) in the blindness effect. In Experiment 1, participants conducted bimanual key presses with vertically arranged responses while perceiving a brief presentation of rightward or leftward arrowheads. A blindness effect based on the effector identity did emerge, but only with the above-right/below-left key-hand arrangement. An orthogonal blindness effect was not found in Experiment 2 with a horizontal key-press action task and a vertical arrowhead perception task. We concluded that the anatomical identity of the responding hand was not integrated into the action plan with an orthogonally incompatible key-hand arrangement. The findings are discussed in terms of the generality and limits of the blindness effect, and hierarchical response coding.

Effects of laterality and pitch height of an auditory accessory stimulus on horizontal response selection: the Simon effect and the SMARC effect.

In the present article, we investigated the effects of pitch height and the presented ear (laterality) of an auditory stimulus, irrelevant to the ongoing visual task, on horizontal response selection. Performance was better when the response and the stimulated ear spatially corresponded (Simon effect), and when the spatial-musical association of response codes (SMARC) correspondence was maintained-that is, right (left) response with a high-pitched (low-pitched) tone. These findings reveal an automatic activation of spatially and musically associated responses by task-irrelevant auditory accessory stimuli. Pitch height is strong enough to influence the horizontal responses despite modality differences with task target.

Healthcare waste management in the capital city of Mongolia.

Inconsistencies are present in the management options for healthcare wastes in Mongolia. One of the first critical steps in the process of developing a reliable waste management plan requires the performance of a waste characterization analysis. The objectives of this study were an assessment of the current situation of healthcare waste management (HCWM) and characterization of healthcare wastes generated in Ulaanbaatar. A total about 2.65 tonnes of healthcare wastes are produced each day in Ulaanbaatar (0.78 tons of medical wastes and 1.87 tons of general wastes). The medical waste generation rate per kg/patient-day in the inpatient services of public healthcare facilities was 1.4-3.0 times higher than in the outpatient services (P<0.01). The waste generation rate in the healthcare facilities of Ulaanbaatar was lower than in some other countries; however, the percentage of medical wastes in the total waste stream was comparatively high, ranging from 12.5% to 69.3%, which indicated poor waste handling practices. Despite the efforts for the management of wastes, the current system of healthcare waste management in Ulaanbaatar city of Mongolia is under development and is in dire need of immediate attention and improvement. It is essential to develop a national policy and implement a comprehensive action plan for HCWM providing environmentally sound technological measures to improve HCWM in Mongolia.

Preparation for horizontal or vertical dimensions affects the right-left prevalence effect.

When stimulus and response simultaneously vary in both horizontal and vertical dimensions, the stimulus-response compatibility effect is often larger for the horizontal dimension. We investigated the role of preparation for each dimension in this right-left prevalence. In Experiment 1, tasks based on horizontal and vertical dimensions were mixed in random order, and the relevant dimension in each trial was cued with a variable cue-target stimulus onset asynchrony (SOA). A right-left prevalence effect was observed only when participants prepared for the upcoming task. Experiment 2 replicated the absence of the prevalence effect for the simultaneous presentation of cue and target using a fixed SOA of 0 msec. In Experiment 3, the right-left prevalence emerged with a 0-msec SOA when participants prepared for e achdimension basedon its frequency. These resultssuggest that participants' internal set can be greater for the horizontal dimension, leading to the right-left prevalence effect.

Symptom dimensions and needs of care among patients with schizophrenia in hospital and the community.

The purpose of the present paper was to examine the psychiatric symptom dimensions related to needs of care among patients with schizophrenia in hospital and in the community. Subjects were 217 patients with F2 ICD-10 diagnoses. Hospital patients included 102 inpatients (47.0%) in psychiatric long-term care units. Community patients included 115 outpatients (53.0%) living in their homes or residential facilities. Psychiatrists used the Brief Psychiatric Rating Scale (BPRS) to assess patients' psychiatric symptoms. Formal care providers assessed needs of care using a scale developed by the Committee on Case Management Guidelines for People with Mental Disabilities in Japan. Instrumental Activities of Daily Living (IADL) self-performance and difficulty were also measured using a scale from the Minimum Data Set-Home Care (MDS-HC). Multiple regression analyses were applied, using the symptom dimensions as dependent variables and needs of care as independent variables. Patient group (hospital or community) was also used as an independent variable. Hospital patients demonstrated more severe psychiatric symptoms and greater needs of care than community patients. Multiple regression analyses showed that the total needs of care were greater among male patients (B = 0.142, P = 0.005), hospital patients (B = 0.310, P < 0.001), patients with poor IADL self-performance (B = 0.217, P = 0.047), and patients with severe negative symptoms (B = 0.240, P = 0.002; R(2) = 0.515). The present results suggested an association between negative symptoms and needs of care in schizophrenia. Hospital patients had greater needs of care, even though their psychiatric symptoms were controlled for. Further research should examine the relationships between psychiatric symptoms and needs of care in a cohort study following patients when hospitalized and when living in the community.