Bacteria under the physical constraints of periodic micro-nanofluidic junctions reveal morphological plasticity and dynamic shifting of Min patterns     

Jie-Pan Shen  Chia-Fu Chou 《Biomicrofluidics》2014,8(4)

Morphological plasticity is an important survival strategy for bacteria adapting to stressful environments in response to new physical constraints. Here, we demonstrate Escherichia coli morphological plasticity can be induced by switching stress levels through the physical constraints of periodic micro-nanofluidic junctions. Moreover, the generation of diverse morphological aberrancies requires the intact functions of the divisome- and elongasome-directed pathways. It is also intriguing that the altered morphologies are developed in bacteria undergoing morphological reversion as stresses are removed. Cell filamentation underlies the most dominant morphological phenotypes, in which transitions between the novel pattern formations by the spatial regulators of the divisome, i.e., the Min system, are observed, suggesting their potential linkage during morphological reversion.Most bacteria have evolved sophisticated systems to manage their characteristic morphology by orchestrating the spatiotemporal synthesis of the murein sacculus (peptidoglycan exoskeleton), which is known to be the stress-bearing component of cell wall and presides over de novo generation of cell shape.¹ Morphological plasticity is attributed to a bacterial survival strategy as responding to stressful environments such as innate immune effectors, antimicrobial therapy, quorum sensing, and protistan predation.² It comes of no surprise that stress-induced diversified morphology and mechanisms, ascribed to shape control and determination, have drawn great attention in both fundamental and clinical studies.^3–6 The molecular mechanism to form filamentous bacteria has been revealed that both β-lactam antibiotics³ and oxidative radicals produced by phagocytic cells⁵ trigger the SOS response, promoting cell elongation by inactivating cell division via the blockade of tubulin-like FtsZ, known as the divisome initiator. While apart from the scenario of length control by the divisome-directed filamentation, the elongasome assembled by proteins associated with actin-like MreB complex^1,7,8 helps the insertion of peptidoglycans into lateral cell wall, suggesting the role in the determination of cell diameter during cell elongation.Recently, additional mechanisms other than the divisome/elongasome-directed pathways of shape maintenance are discovered to regenerate normal morphology de novo from wall-less lysozyme-induced (LI) spheroplasts of E. coli via a plethora types of aberrant division intermediates.⁹ Similar morphological reversion from different aberrant bacterial shapes has been observed as squashed wild-type bacteria generated through sub-micron constrictions are released into connected microchambers.¹⁰ Previous work using the microfluidic approach focuses on the septation accuracy and robustness of constricted bacteria,¹¹ but the reversion process of stress-released bacteria is not well studied and analyzed. In particular, the aberrant bacterial shape is mainly branched-type with bent and curved variants in the reverting bacteria, analogous to the aberrant intermediate found in the morphological reversion of LI spheroplasts with PBP5-defective mutant.⁹ Since bacteria suffering from starvation¹² or confronting mechanical stresses exerted by phagocytosis and protistan grazing⁶ can induce morphological alterations, one could manipulate the stress levels of physical constraints by adopting repeated structures of sub-micron constricted channels (nanoslits) and microchambers,^10,11 to select and enrich bacteria converting to specified aberrant intermediates. The stress incurred by the nanoslit on bacteria is about the mechanical intervention over de novo synthesis of the cell wall, which is the major factor causing morphological aberrancy, while the second environmental stress comes from bacterial growth in the restricted space of microchamber as bacteria proliferate to full confluency, resulting in growth pressure of high population density, nutrient deficiency, and the size reduction of bacteria.Here, we report the selection of distinctive bacterial morphologies by size shrinkage in the outlet cross-section (W × H = 1.5 × 1.5 μm) of the terminal microchamber in the periodic structures of nanoslit-microchamber (Figs. 1(a) and 1(b)). The fluidic structures were micropatterned on fused silica wafers by photolithography, fabricated through reactive ion etching (RIE) and inductively coupled plasma (ICP) etching, and encapsulated by cover glasses coated with polydimethylsiloxane (PDMS) or polysilsesquioxane (PSQ) layer as described earlier.^13,14 Two days after the outgrowth of Escherichia coli (imp4213 [MC4100 ΔlamB106 imp4213]) loaded to the microfluidic device at 25 °C, bacteria started to penetrate into the nanoslit as they proliferated to full confluency in the first microchamber (Fig. 1(c)). It takes about 10 days for bacteria traversing 500 μm long (5 repeated nanoslit-microchamber units) via proliferations and being released from the outlet of the terminal microchamber. The narrowed outlet allows only bacteria with smaller diameters to be squeezed into the spacious and nutrient-rich region, thus it acts as a spatial filter to avoid the passage of branching bacteria with cross-sectional size larger than that of the outlet. The rationale of this design is to select aberrant bacteria prone to promote de novo shape regeneration other than the branched-type, which is the dominant morphology of reverting bacteria in the prior microfluidic constriction study.¹⁰ As anticipated, the stress-released bacteria through the narrowed outlet are therefore mostly filamentous (see statistical analysis for cell morphology in the supplementary material).¹⁵ However, it is noted that the aberrant morphology of lemon-like shape with tubular poles (Figs. 1(d-1), 1(d-3), and 1(d-11)) is developed about 3 h after the stress-released bacteria escaped through the outlet. Though the generation of the lemon-like aberrancy in bacteria has been reported in PBP5/7-defective E. coli mutant subjected to a high-level inhibition of both MreB and FtsZ, while the same mutant treated with low-level MreB inhibitor, together with antagonized-FtsZ, displays filamentous shape with varying diameters,¹⁶ these morphological aberrances can be observed in our system (Figs. 1(d-2) and 1(d-12)). Besides, a high-level inhibition of MreB in E. coli with an intact divisome function is known to cause round bacteria, resembling to the cell morphology of the bacteria shown in Fig. 1(d-4). Interestingly, parallel experiments using bacteria mutants carrying impaired regulatory functions in either the divisome (Min⁻) or the elongasome (MreB⁻) do not develop morphological plasticity (supplementary Fig. S1).¹⁵ Taken together, the filamentous and lemon-like variants selected from our microfluidic platform, while elaborating the morphological plasticity and reverting progression, require both the functional divisome/elongasome. Alternatively, the selection by the spatial filter does not fully exclude cells with aberrant shapes such as the branched-type with initial budding (Fig. 1(d-7)), cells with asymmetric cross-section perpendicular to the longitudinal axis (Figs. 1(d-2), 1(d-8), 1(d-9), 1(d-9′), and 1(d-10)), and those resembling to the morphological phenotypes of the division intermediates reported in the LI-spheroplasts carrying genetic defects on some non-cytoskeletal proteins (Figs. 1(d-5) and 1(d-6)). In particular, intracellular vesicles and cell autolysis are observed in some reverting bacteria (Figs. 1(d-5) and 1(d-6)), which are reminiscent to the phenomena reported in the division intermediates of the LI-spheroplasts lacking stress response system (Rcs) or some accessory proteins (PBP1B and LpoB). Unlike the bacteria grow with odd shapes under the stress of nanofluidic confinement only¹⁰ (Fig. 1(c)), all the morphological aberrancy reported here are developed in the reverting bacteria, which grow in the spacious and nutrition-rich environment and are free from physical constraints. Further investigations over the expression levels of the divisome/elongasome networks and the stress-response system in bacterial cells subjected to micro-nanofluidic junctions could be insightful in understanding their role in bacterial shape control.⁹Open in a separate windowFIG. 1.(a) Schematics of the microfluidic device used in this study with an H-shaped geometry (left upper panel), where repeated nanoslit (L×W×H = 50×10×0.4 μm)−microchamber (L×W×H = 50×50×1.5 μm) structures are bridged between two arms of the H-shaped microchannels (left lower panel and enlarged view in right panel). (b) Top-view layout of an individual channel in (a) with close view of the outlet in the terminal microchamber (orange: nanoslits; blue: microchambers). (c) Fluorescence micrograph of E. coli imp4213 penetrating a nanoslit (scale bar: 5 μm). (d) Bright-field micrographs for various cell morphology of the selected imp4213 released from the outlet (magenta arrows: cells with vesicles; scale bar: 5 μm). (e) Sequential bright-field micrographs of morphological reversion. T1–T3 indicate the time after bacteria escaping from the outlet. T1: 3 h; T2: 6 h; T3: 24 h. Scale bar: 10 μm.During the morphological reversion, the stress-released bacteria rapidly increase their size in the first 3 h after escaping from the terminal microchamber (T1 in Fig. 1(e)). Some filamentous bacteria even grow over 50 μm long, though such a morphological phenotype implicates the cessation of functional divisome. With active growth and proliferation, the progeny of stress-released bacteria increase their population but gradually reduce their size about 6 h after being released from the constriction stress (T2 in Fig. 1(e)). Fig. ​Fig.22 displays the marginal histograms for different shape factors, where Fig. 2(a) is the plot of the minimal Feret diameter (cell diameter) versus Feret diameter (cell length), i.e., the shortest versus the longest distance between any two points with parallel tangents along the cell peripheral, respectively, indicating that cell diameters are larger for reverting bacteria at T1 (mean ± S.E.M. = 1.89 ± 0.08 μm) with respect to T2 (1.51 ± 0.06 μm). Moreover, the histogram of Feret diameter depicts two major populations of the cell length for reverting bacteria at T1, which mostly resume to typical cell length at T2 (the median of Feret diameter = 3.33 μm; see statistical analysis for Fig. ​Fig.22 in the supplementary material).¹⁵ The shape factors of circularity (4π × [area]/[perimeter]²) and aspect ratio ([major axis]/[minor axis] for the cell geometry fitted to an ellipse) confirm the existence of dual populations for bacteria at T1 as well (Fig. 2(b)). About 24 h after escaping (T3 in Fig. 1(e)), almost all the progeny of stress-released bacteria regained the rod shape.Open in a separate windowFIG. 2.Marginal histograms for shape factors measured from the reverting imp4213 at T1 and T2. (a) Minimal Feret diameter (cell diameter) versus Feret diameter (cell length). (b) Circularity versus aspect ratio. N = 366 for T1 and N = 494 for T2.The bacterial size reduction of filamentous and lemon-like shape variants, though involving negative control of the divisome positioning by the spatial regulators of MinCDE system,¹⁷ is not completely understood as to how they coordinate in aberrant geometries. Besides, the filamentation of stress-released bacteria during the period of T1 to T2 implicates the inhibition of functional divisome. With minimal perturbation of the divisome by leaky expression of GFP-MinD and MinE (imp4213/P_lac-gfpmut2::minD minE), the patterning dynamics of GFP-MinD in different bacterial morphology were time-lapse imaged during morphological reversion. Intriguingly, more than the standing-wave-like pattern of MinD denoted in filamentous E. coli,¹⁸ we discovered bidirectional drifting of two standing-wave-like patterns of MinD occur in most reverting bacteria filaments (supplementary Figs. S2(a) and S2(b)).¹⁵ The bidirectional drifting in the longitudinal direction of the cells may be emanating from the cell poles (the blue upper panel of Fig. 3(a) and supplementary Fig. S2(c)¹⁵) and the cylinder region (the blue lower panel of Fig. 3(a) and supplementary Fig. S2(d)¹⁵). Furthermore, the MinD pattern transitions from the standing to traveling waves are occasionally observed (the lower panel of Figs. 3(a) and supplementary Fig. S2(e)¹⁵). Notably, the standing-wave-like MinD patterns exhibit bidirectional drifting along the cell longitudinal direction and intermittently change directions, implying the competition between coexisting MinD patterns can be supported under filamentous geometry. Despite there have been observations of multiple wave-packet of traveling waves in filamentous cells,¹⁹ the mixture of distinct wave-like MinD patterns have never been experimentally reported. While most intriguingly, multiple drifting movements of wave-like MinD patterns potentiate the mitigation of periodic minima in time-averaged Min gradient in the reverting filamentous bacteria, suggesting the disability of proper divisome positioning for recovering the typical rod shape. Apart from the wave-like movements, amoeba-like motion of Min proteins has been shown in vitro upon synthetic minimal system, but never been verified in vivo.²⁰ Strikingly, here amoeba-like motion of MinD is the dominant mode in lemon-like bacteria and the transitions between wave-like patterns and amoeba-like motion are supported even under filamentous geometry (Figs. 3(b) and 3(c), Multimedia view).Open in a separate windowFIG. 3.Kymographs for GFP-MinD dynamics in selected imp4213 cells during morphological reversion: (a) Mixture modes of standing wave packets and traveling wave. The left panel is the stacked fluorescence micrograph displaying cell shape (scale bar = 5 μm). The kymograph is derived from the filamentous cell indicated by the green arrow (scale bar: 120 s horizontal; 5 μm vertical), where the lower panel follows the upper panel in time. The yellow windows indicate bidirectional-drifting standing wave packets, while the green indicates traveling waves (see also supplementary Fig. S2).¹⁵ (b) Sequential fluorescence micrographs of GFP-MinD in lemon-shape imp4213 show amoeba-like motion, with the first left a bright-field image (scale bar: 10 μm). (c) Mixed modes of amoeba-like motion and waves in selected filamentous imp4213 cell indicated by the green arrow in the left panel (scale bar = 5 μm). The filamentous cells depicted in (a) and (c) locate at the top region while the lemon-shape cell in (b) at the central region of the movie (time stamp in min:s). (Multimedia view) [URL: http://dx.doi.org/10.1063/1.4892860.1]In summary, we have demonstrated that the development of bacterial morphological plasticity can be stress-induced by periodic physical constraints with intact functions of the divisome and elongasome-directed pathways. Through size exclusion, the constricted outlet structure designed in our microfluidic device is useful in selecting bacteria with plethora morphological aberrancies other than the branched type. Interestingly, disparate morphological changes, rather than those being directly induced under a stressful environment, can be generated in the stress-released bacteria experiencing morphological reversion. Further, the discovery of novel transitions between the Min patterns in most reverting bacteria implicates its regulatory effect of cell filamentation. However, by exploiting the micro-nanofluidic approach, further investigations of the mechanism underlying the development of morphological plasticity in bacteria adapting to physical constraints are expected in future studies to gain more insights into the molecular basis of shape generation.  相似文献   

Construction and Validation of a Chinese Value of Learning Scale (CVLS)     

Angela Leung  Iris Chi  Nelson Chow  Kin Sun Chan  Kee Lee Chou 《Educational gerontology》2013,39(10):907-920

The Chinese Value of Learning Scale (CVLS) is a 5-item instrument for assessing the value of learning in Chinese culture. The psychometric properties of CVLS were investigated in a convenient sample of 583 Chinese soon-to-be-aged adults in Hong Kong. CVLS possesses high internal consistency and exhibited satisfactory short-term test-retest reliability. CVLS also exhibited high face validity and mild convergent validity with other 48-item openness-to-experience scale (OTES) from Revised NEO Personality Inventory, and is significantly related to the past and actual learning experience. In view of its brevity and easy administration, CVLS offers a useful instrument to further investigate the value of learning in Chinese culture and its possible relationship with adults' learning behaviour.  相似文献   

Powerful moments in sports: the most significant sporting events in American history     

Wen-hao Winston Chou 《Sport in History》2019,39(1):99-101

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Endoscopic vein harvest in elective off-pump coronary artery bypass grafting     

Nai-kuan Chou  Meng-lin Lee  Shoei-shen Wang 《Journal of Zhejiang University. Science. B》2009,10(10):748-752

While traditional open vein harvest was related to postoperative wound complications, endoscopic vein harvest was developed to minimize the morbidity in the greater saphenous vein harvest procedure. In this study, these two procedures were compared for postoperative wound healing and long-term graft patency. We reviewed all consecutive patients undergoing elective off-pump coronary artery bypass grafting from January 2004 to December 2005 and collected data regarding wound complications and coronary events. Wound complications included dehiscence, excessive discharge, edema, altered sensation, cellulitis, hema-toma, pain scale, and superlicial and deep wound infection. Coronary events were defined as diagnosis of myocardial infarction during the first year's follow-up. A total of 392 patients were included in our series, among whom 44 were excluded from the study due to emergent operation, preoperative intra-aortic balloon pump support, or the greater saphenous vein varicose characteristic, 78 belonged to open vein harvest group, and 270 to endoscopic vein harvest group. Wound complications were significantly less in the endoscopic group (5.2%) compared to the open group (19.2%) (P=0.0002). There was no significant difference on preopera-tive risk factors, total operative time, or hospitalization days. During one-year follow-up, both the early and late graft patency rates were similar between the two groups. Endoscopic vein harvest is safe and effective, which carries less risk for wound complica-tions and is associated with better satisfaction and cosmetic result than the traditional greater saphenous vein harvest procedure. The endoscopic vein harvest also demonstrates a great long-term patency.  相似文献   

The role of IT in achieving operational agility: A case study of Haier,China     

Pei-Ying Huang  Tao Hua Ouyang  Shan L. Pan  Tzu-Chuan Chou 《International Journal of Information Management》2012

Operational agility has increasingly become essential for survival and prosperity for contemporary organizations. Given its significant role in a turbulent business environment, this concept has garnered considerable research attention over the past ten years. However, due to the complex nature of operational agility, how to achieve this important organizaitonal capability is still not clearly understood. Thus the present study attempts to open this black box through examing a case study of Haier, the largest household appliances producer in China. The findings of this case highlight the significant role played by IT leveraging competence in achieving operational agility. It shows that IT leveraging competence helps enhance firm's ability to process information in a turbulent business environment, which results in information processing efficiency and information processing effectiveness. With the enhancement in the ability to process information, the focal firm is capable to sense and respond to market changes more readily.  相似文献   

Child physical abuse and the related PTSD in Taiwan: The role of Chinese cultural background and victims' subjective reactions     

Chou CY  Su YJ  Wu HM  Chen SH 《Child abuse & neglect》2011,35(1):58-68

Objective

This study aimed to investigate child physical abuse (CPA) while taking into account the more rigorous definitions of CPA in the Chinese societies. The prevalence of CPA and CPA-related PTSD were estimated, together with the examination of peri-traumatic subjective reactions and their impacts on PTSD.

Methods

In a Taiwanese sample of 1966 4th to 8th graders, the Chinese version of UCLA PTSD Reaction Index for DSM-IV (Steinberg, Brymer, Decker, & Pynoos, 2004) was used to investigate the lifetime exposure to CPA. A sub-sample of 236 traumatized CPA victims was examined with respect to related PTSD symptoms.

Results

Thirty-four percent of the children had been exposed to CPA. The estimated current prevalence of full and partial PTSD was 13.6% and 16.9%, respectively.

Conclusions

The current CPA prevalence was found to be higher than the Western countries, but lower than the previous findings in other East Asian societies. The full PTSD prevalence was close to the findings in the Western countries, whereas sub-clinical PTSD was less observed in Taiwan. Peri-traumatic subjective reactions, that is, Criterion A2 and perceived threat, were shown to be major predictors of PTSD symptom severity. The role of attitudes of child discipline in the Chinese societies in the prevalence of CPA and CPA-related PTSD is discussed.

Practice implications

By providing explicit epidemiological information of CPA and CPA-related PTSD in Taiwan, the current study extends our understanding of CPA and CPA-related PTSD more broadly from Western countries to the Eastern societies. By separately investigating CPA relating to different perpetrators, cross-study comparison is enhanced. In the current study, the significance of considering cultural background in defining CPA and examining CPA-related PTSD was pointed out. Meanwhile, the role of victims’ subjective reactions in the psychopathology of PTSD is highlighted. The findings and discussions could contribute for generating a more sophisticated clinical practice, especially with Asian or Chinese cases.  相似文献   

Facial micro-expression states as an indicator for conceptual change in students' understanding of air pressure and boiling points     

Mei-Hung Chiu  Hongming Leonard Liaw  Yuh-Ru Yu  Chin-Cheng Chou 《British journal of educational technology : journal of the Council for Educational Technology》2019,50(1):469-480

Utilizing facial recognition technology, the current study has attempted to predict the likelihood of student conceptual change with decision tree models based on the facial micro-expression states (FMES) students exhibited when they experience conceptual conflict. While conceptual change through conceptual conflicts in science education is a well-studied field, there is little research done on conceptual change through conceptual conflict in terms of students' facial expressions. As facial expressions are one of the most direct and immediate responses one can get during instruction and that facial expressions are often representations student's emotions, a link between students' FMES and learning was explored. Facial data was collected from 90 tenth graders. Only data from the 72 students who made incorrect predictions were analyzed in this study. The concept taught was the relationship between boiling point and air pressure. Through facial recognition software analysis and decision tree models, the current study found Surprised, Sad and Disgusted to be key FMES that could be used to predict student conceptual change in a conceptual conflict-based scenario.  相似文献   

Course Management Systems and Blended Learning: An Innovative Learning Approach     

Amy Y. Chou  David C. Chou 《Decision Sciences Journal of Innovative Education》2011,9(3):463-484

This article utilizes Rogers ’ innovation‐decision process model (2003) and Beckman and Berry's innovation process model (2007) to create an innovative learning map that illustrates three learning methods (i.e., face‐to‐face learning, online learning, and blended learning) in two types of innovation (i.e., incremental innovation and radical innovation) that exhibits the behavior and dynamics on a two‐dimensional area of learning changes (i.e., change in learning model and change in technological infrastructure). The behavior and dynamics of face‐to‐face, online, and blended learning are depicted in the innovative learning map. Blended learning is identified as a radical innovative learning method in the innovative learning map. A course management system (CMS) is an information and communication technology (ICT) tool that can be used to facilitate and balance communication channel within a blended learning environment. Two propositions are proposed in this article: (1) CMS facilitates communication channel, enhances learning practice for learners and instructors, and is an enabler for blended learning; and (2) adopting CMS is suitable for blended learning practice. A case study to a college System Analysis and Design (SA&D) course reflects that the adoption of CMS in a blended learning environment is acceptable.  相似文献   

From a Disciplinary to an Interdisciplinary Design Research: Developing an Integrative Approach for Design          下载免费PDF全文

Wen Huei Chou  Ju‐Joan Wong 《The International Journal of Art & Design Education》2015,34(2):206-223

As the new generation of designers face more complex design issues, the forms of design research start to shift towards a user‐centred approach to problem‐solving. The cooperation and communication among various fields and specialisations are becoming more complex; in many practical design cases, in particular, technology developers face challenges in deciphering the creative ideas of designers and the needs and restraints of users, society, law and science. The emergence of integrative design based on a wide range of disciplines has prompted discussion and exploration among various participants. The direction of contemporary design research has already transformed from one based on production of artefact to one focused on the integration of varied knowledge and fields at different stages. We examine such design research based on a problem‐driven approach in two case studies characterised by interdisciplinary collaboration for solving complex problems. We focus on the transformation and implementation of integrative design research methods and suggest a ‘reflection‐in‐action’ problem‐solving process for strengthening the capabilities of multidisciplinary design research.  相似文献   

The influence of learning style and training method on self-efficacy and learning performance in WWW homepage design training     

Huey-Wen Chou  Tsung-Bin Wang 《International Journal of Information Management》2000,20(6)

The present study compares the relative effects of two training methods on learner's computer self-efficacy and learning performance in WWW homepage design. A field experiment was conducted by employing two classes of 10th graders. Results indicated that the behavior modeling training method yielded consistently superior performance and higher computer self-efficacy as compared with the instruction-based approach. Subjects with various learning styles performed substantially different in some learning tasks. The significant two-way interaction indicates the critical roles that gender and learning style played in interacting with training method. For learning performance, male students benefited more from the instruction-based and female students learned better in the behavior modeling condition. Concerning computer self-efficacy, female students gained more from the instruction and male students benefited more from behavior modeling approaches. For different learning style students, there exists a best-fit training approach. In addition, the best-fit training approach is task dependent. These results suggest that each individual training method has its unique merit to meet designated training objectives for learners with specific traits. Future research directions conclude the paper.  相似文献