Bacterial accumulation in intestinal folds induced by physical and biological factors.

BACKGROUND: The gut microbiota, vital for host health, influences metabolism, immune function, and development. Understanding the dynamic processes of bacterial accumulation within the gut is crucial, as it is closely related to immune responses, antibiotic resistance, and colorectal cancer. We investigated Escherichia coli behavior and distribution in zebrafish larval intestines, focusing on the gut microenvironment. RESULTS: We discovered that E. coli spread was considerably suppressed within the intestinal folds, leading to a strong physical accumulation in the folds. Moreover, a higher concentration of E. coli on the dorsal side than on the ventral side was observed. Our in vitro microfluidic experiments and theoretical analysis revealed that the overall distribution of E. coli in the intestines was established by a combination of physical factor and bacterial taxis. CONCLUSIONS: Our findings provide valuable insight into how the intestinal microenvironment affects bacterial motility and accumulation, enhancing our understanding of the behavioral and ecological dynamics of the intestinal microbiota.

Reciprocating intestinal flows enhance glucose uptake in C. elegans.

Despite its physiological and pathological importance, the mechanical relationship between glucose uptake in the intestine and intestinal flows is unclear. In the intestine of the nematode Caenorhabditis elegans, the defecation motor program (DMP) causes reciprocating intestinal flows. Although the DMP is frequently activated in the intestines, its physiological function is unknown. We evaluated the mechanical signature of enhanced glucose uptake by the DMP in worms. Glucose uptake tended to increase with increasing flow velocity during the DMP because of mechanical mixing and transport. However, the increase in input energy required for the DMP was low compared with the calorie intake. The findings suggest that animals with gastrointestinal motility exploit the reciprocating intestinal flows caused by peristalsis to promote nutrient absorption by intestinal cells.

Vulnerability of the skin barrier to mechanical rubbing.

Skin barrier function is the battlefront for preventing permeation of harmful substances and infectious diseases. However, it can be destroyed by mechanical forces, as shown in many studies. Excess rubbing may increase the permeability of the skin to aqueous material. Although the skin barrier plays an important physiological role in humans, the vulnerability of skin to mechanical rubbing is poorly understood. Therefore, we investigated the effects of rubbing on the skin in vitro; skin damage was quantified by laser-induced fluorescence. Microscopic observation showed that keratinocytes in the stratum corneum sustained traumatic damage, which reduced the barrier function in that region. The permeability of the skin to an aqueous solution increased with rubbing frequency and load, and rubbing markedly reduced the barrier function of the stratum corneum. To understand the mechanisms underlying the skin damage, we developed a simple mathematical model assuming that the skin is a viscoelastic material. We hypothesized that the increased skin permeability was caused by the damage sustained by keratinocytes in the stratum corneum, and that the permeability was proportional to the time-averaged strain. Our theoretical results showed quantitative agreement with the experimental results and illustrated that rubbing and strain relaxation play key roles in rubbing-induced permeation.

How do Caenorhabditis elegans worms survive in highly viscous habitats?

The nematode Caenorhabditis elegans is a filter feeder that lives in various viscous habitats such as soil, the intestines of slugs, and rotting materials such as fruits and stems. Caenorhabditis elegans draws in suspensions of bacteria and separates bacteria from water using the pharyngeal pump. Although these worms often live in highly viscous habitats, it is still unclear how they survive in these environments by eating bacteria. In this study, we investigated the effects of suspension viscosity on the survival rate of malnourished worms by combining live imaging and scaling analyses. We found that survival rate decreased with increases in viscosity because the high viscosity suppressed the amount of food ingested. The same tendency was found in two feeding-defective mutants, eat-6(ad467) and eat-6(ad997). We also found that the high viscosity weakened pump function, but the velocities in the pharynx were not zero, even in the most viscous suspensions. Finally, we estimated the amount of ingested food using scaling analyses, which provided a master curve of the experimental survival rates. These results illustrate that the survival rate of C. elegans worms is strongly dependent on the ingested bacteria per unit time associated with physical environments, such as the viscosity of food suspensions and the cell density of bacteria. The pump function of the C. elegans pharynx is not completely lost even in fluids that have 105 times higher viscosity than water, which may contribute to their ability to survive around the world in highly viscous environments.

Mechanical roles of anterograde and retrograde intestinal peristalses after feeding in a larval fish (Danio rerio).

Transport in gut is important, not only for digestion, metabolism, and nutrient uptake, but also for microbiotic circumstance in the digestive tract; however, the effects of mixing and pumping in the intestine have not been fully clarified. Therefore, in this study, we quantitatively explored intestinal mixing and pumping, represented using a dispersion coefficient and pressure rise in zebrafish larvae, which is a model organism for vertebrate digestive studies, over time by measuring transport phenomena after feeding. Here we provide the first quantitative evidence of the roles of anterograde and retrograde intestinal peristalses in the larval fish of Danio rerio after feeding in terms of digestive pumping and mixing functions by an in vivo imaging of intestinal propagation waves in the larval intestine. Peristaltic velocities in the anterior and posterior intestines change considerably after feeding for 5 h, while the intervals and amplitudes remain almost constant. The intestinal transport is successively visualized after feeding to elimination. Moreover, the particle tracking velocimetry in the chyme leads our quantitative understanding of outstanding mixing and pumping functions in the anterior and posterior intestines by adopting physical parameters of diffusivity and pressure rise, respectively. From scaling analysis, we found that the anterior intestine maintains mixing for 5 h from feeding, whereas the posterior intestine activates gradually pumping up. These results suggest that time change of pumping and mixing functions of intestinal peristalsis could considerably influence the nutrient uptake and microbiotic circumstance in the larval fish intestine.NEW & NOTEWORTHY Transport in gut is important, not only for digestion, metabolism, and nutrient uptake, but also for microbiotic circumstance; however, hydrodynamic effects in the intestine have not been fully clarified. We provide the first quantitative evidence of the mechanical roles of anterograde and retrograde intestinal peristalses in the larval fish of Danio rerio by adopting physical parameters of diffusivity and pressure rise. The intestine transitionally regulates mixing and pumping functions by peristaltic propagations after feeding.

Pax6-dependent regulation of the rat Fabp7 promoter activity.

Fabp7 gene encodes a brain-specific fatty acid-binding protein that is widely used as a marker for neural stem cells. Here, we report that the activity of rat Fabp7 promoter was regulated directly by a transcription factor, Pax6. Deletion analyses identified an essential region (-837 to -64 from transcription start site) in the rat Fabp7 promoter. This region controls promoter activity in rat embryos and in the mouse cultured cell line MEB5. Over-expressing wild-type Pax6 or a dominant-negative Pax6 mutant enhanced and suppressed, respectively, the promoter activity. Pax6 can bind the region directly, although the region contains no clear binding motif for Pax6. The rat Fabp7 promoter also contains conserved binding sites for Pbx/POU (-384 to -377) and CBF1 (-270 to -262). However, specific deletion of the sites showed no significant reduction in the promoter activity, although a gel mobility shift assay confirmed that CBF1 binds the conserved sequence. Taken together, these results suggest that the rat Fabp7 promoter is mainly regulated by Pax6. The Pax6-dependent regulation of the rat Fabp7 expression might have an evolutionary aspect between rat and mouse; the former may need to efficiently use fatty acids to make the brain bigger than the latter.

Collective spreading of red blood cells flowing in a microchannel.

Due to recent advances in micro total analysis system technologies, microfluidics provides increased opportunities to manipulate, stimulate, and diagnose blood cells. Controlling the concentration of cells at a given position across the width of a channel is an important aspect in the design of microfluidic devices. Despite its biomedical importance, the collective spreading of red blood cells (RBCs) in a microchannel has not yet been fully clarified. In this study, we experimentally investigated the collective spreading of RBCs in a straight microchannel, and found that RBCs initially distributed in one side of the microchannel spread to the spanwise direction during downstream flow. Spreading increased considerably as the hematocrit increased, though the flow rate had a small effect. We proposed a scaling argument to show that this spreading phenomenon was diffusive and mainly induced by cell-cell interactions. The dispersion coefficient was approximately proportional to the flow rate and the hematocrit. These results are useful in understanding collective behaviors of RBCs in a microchannel and in microcirculation.

Effect of Fluid Viscosity on the Cilia-Generated Flow on a Mouse Tracheal Lumen.

Mucous flow in a tracheal lumen is generated by the beat motion of ciliated cells to provide a clearance function by discharging harmful dust particles and viruses. Due to its physiological importance, the cilia-generated flow and the rheological properties of mucus have been investigated intensively. The effects of viscosity on the cilia-generated flow, however, have not been fully clarified. In this study, we measured bulk background velocity of ciliary flow using a micro particle tracking velocimetry method under various viscosity conditions in mice. The results showed that the flow velocity decreased as the increase with viscosity of ambient fluid. Moreover, no previous study has clarified the pump power generated by cilia, which provides important information with regard to understanding the molecular motor properties of cilia. Measurements of both the ciliary flow and the ciliary motion were conducted to determine the cilia pump power. Our results indicated that the cilia pump during the effective stroke did not drive the ciliary flow efficiently under high viscosity conditions; these findings are necessary to resolve the clearance function.

Inhomogeneous distribution of Chlamydomonas in a cylindrical container with a bubble plume.

Swimming microalgae show various taxes, such as phototaxis and gravitaxis, which sometimes result in the formation of a cell-rich layer or a patch in a suspension. Despite intensive studies on the effects of shear flow and turbulence on the inhomogeneous distribution of microalgae, the effect of a bubble plume has remained unclear. In this study, we used Chlamydomonas as model microalgae, and investigated the spatial distribution of cells in a cylindrical container with a bubble plume. The results illustrate that cells become inhomogeneously distributed in the suspension due to their motility and photo-responses. A vortical ring distribution was observed below the free surface when the bubble flow rate was sufficiently small. We performed a scaling analysis on the length scale of the vortical ring, which captured the main features of the experimental results. These findings are important in understanding transport phenomena in a microalgae suspension with a bubble plume.

Separation of motile bacteria using drift velocity in a microchannel.

Separation of certain bacteria from liquids is important in the food, water quality management, bioengineering, and pharmaceutical industries. In this study, we developed a microfluidic device for the hydrodynamic separation of motile bacteria (Escherichia coli) using drift velocity. We first investigated drift tendencies of bacteria and found that cells tended to move in a spanwise direction with similar velocities regardless of the flow rate. When the drift distance was small compared to the wetted perimeter of the cross section, the cells were not separated efficiently. We then investigated the drift phenomenon in more detail using a numerical simulation. Interestingly, the drift phenomenon was observed even without a wall boundary, indicating that drift was caused mainly by the interaction of moving cells with the background shear flow. Finally, we developed a microfluidic device to separate motile bacteria from tracer particles or less motile cells. By decreasing the channel height, the device could successfully separate motile bacteria from other particles or cells with a separation efficiency of about 40%. Connecting microchannels in a series was also found to be effective, which achieved the separation efficiency of about 60%. The knowledge obtained in this study will facilitate the development of other microfluidics devices for use with bacteria.

Fluctuation of cilia-generated flow on the surface of the tracheal lumen.

Although we inhale air that contains many harmful substances, including, for example, dust and viruses, these small particles are trapped on the surface of the tracheal lumen and transported towards the larynx by cilia-generated flow. The transport phenomena are affected not only by the time- and space-average flow field but also by the fluctuation of the flow. Because flow fluctuation has received little attention, we investigated it experimentally in mice. To understand the origin of flow fluctuation, we first measured the distribution of ciliated cells in the trachea and individual ciliary motions. We then measured the detailed flow field using a confocal micro-PTV system. Strong flow fluctuations were observed, caused by the unsteadiness of the ciliary beat and the spatial inhomogeneity of ciliated cells. The spreading of particles relative to the bulk motion became diffusive if the time scale was sufficiently larger than the beat period. Finally, we quantified the effects of flow fluctuation on bulk flow by evaluating the Peclet number of the system, which indicated that the directional transport was an order of magnitude larger than the isotropic diffusion. These results are important in understanding transport phenomena in the airways on a cellular scale.

Entrapment of ciliates at the water-air interface.

The importance of water-air interfaces (WAI) on microorganism activities has been recognized by many researchers. In this paper, we report a novel phenomenon: the entrapment of ciliates Tetrahymena at the WAI. We first characterized the behavior of cells at the interface and showed that the cells' swimming velocity was considerably reduced at the WAI. To verify the possible causes of the entrapment, we investigated the effects of positive chemotaxis for oxygen, negative geotaxis and surface properties. Even though the taxes were still effective, the entrapment phenomenon was not dependent on the physiological conditions, but was instead affected by the physical properties at the interface. This knowledge is useful for a better understanding of the physiology of microorganisms at interfaces in nature and in industry.

Dmrta1 regulates proneural gene expression downstream of Pax6 in the mammalian telencephalon.

The transcription factor Pax6 balances cell proliferation and neuronal differentiation in the mammalian developing neocortex by regulating the expression of target genes. Using microarray analysis, we observed the down-regulation of Dmrta1 (doublesex and mab-3-related transcription factor-like family A1) in the telencephalon of Pax6 homozygous mutant rats (rSey(2) /rSey(2) ). Dmrta1 expression was restricted to the neural stem/progenitor cells of the dorsal telencephalon. Overexpression of Dmrta1 induced the expression of the proneural gene Neurogenin2 (Neurog2) and conversely repressed Ascl1 (Mash1), a proneural gene expressed in the ventral telencephalon. We found that another Dmrt family molecule, Dmrt3, induced Neurog2 expression in the dorsal telencephalon. Our novel findings suggest that dual regulation of proneural genes mediated by Pax6 and Dmrt family members is crucial for cortical neurogenesis.

Separation of cancer cells from a red blood cell suspension using inertial force.

The circulating tumor cell (CTC) test has recently become popular for evaluating prognosis and treatment efficacy in cancer patients. The accuracy of the test is strongly dependent on the precision of the cancer cell separation. In this study, we developed a multistage microfluidic device to separate cancer cells from a red blood cell (RBC) suspension using inertial migration forces. The device was able to effectively remove RBCs up to the 1% hematocrit (Hct) condition with a throughput of 565 µL min(-1). The collection efficiency of cancer cells from a RBC suspension was about 85%, and the enrichment of cancer cells was about 120-fold. Further improvements can be easily achieved by parallelizing the device. These results illustrate that the separation of cancer cells from RBCs is possible using only inertial migration forces, thus paving the way for the development of a novel microfluidic device for future CTC tests.

Inertial migration of cancer cells in blood flow in microchannels.

The circulating tumor cell test is used to evaluate the condition of breast cancer patients by counting the number of cancer cells in peripheral blood samples. Although microfluidic systems to detect or separate cells using the inertial migration effect may be applied to this test, the hydrodynamic forces acting on cancer cells in high hematocrit blood flow are incompletely understood. In the present study, we investigated the inertial migration of cancer cells in high hematocrit blood flow in microchannels. The maximum hematocrit used in this study was about 40%. By measuring the cell migration probability, we examined the effects of cell-cell interactions, cell deformability, and variations in cell size on the inertial migration of cancer cells in blood. The results clearly illustrate that cancer cells can migrate towards equilibrium positions up to a hematocrit level of 10%. We also performed simple scaling analysis to explain the differences in migration length between rigid particles and cancer cells as well as the effect of hematocrit on cancer cell migration. These results will be important for the design of microfluidic devices for separating cells from blood.

Distinctive effects of arachidonic acid and docosahexaenoic acid on neural stem /progenitor cells.

Arachidonic acid (ARA) and docosahexaenoic acid (DHA), which are the dominant polyunsaturated fatty acids in the brain, have crucial roles in brain development and function. Recent studies have shown that ARA and DHA promote postnatal neurogenesis. However, the direct effects of ARA on neural stem/progenitor cells (NSPCs) and the effects of ARA and DHA on NSPCs at the neurogenic and subsequent gliogenic stages are still unknown. Here, we analyzed the effects of ARA and DHA on neurogenesis, specifically maintenance and differentiation, using neurosphere assays. We confirmed that primary neurospheres are neurogenic NSPCs and that tertiary neurospheres are gliogenic NSPCs. Regarding the effects of ARA and DHA on neurogenic NSPCs, ARA and DHA increased the number of neurospheres, whereas neither ARA nor DHA had a detectable effect on NSPCs in the differentiation condition. In gliogenic NSPCs, DHA increased the number of neurospheres, whereas ARA had no such effect. In contrast, ARA increased the number of astrocytes, whereas DHA increased the number of neurons in the differentiation condition. These results suggest that ARA promotes the maintenance of neurogenic NSPCs and might induce the glial differentiation of gliogenic NSPCs and that DHA promotes the maintenance of both neurogenic and gliogenic NSPCs and might lead to the neuronal differentiation of gliogenic NSPCs.

Downstream genes of Pax6 revealed by comprehensive transcriptome profiling in the developing rat hindbrain.

BACKGROUND: The transcription factor Pax6 is essential for the development of the central nervous system and it exerts its multiple functions by regulating the expression of downstream target molecules. To screen for genes downstream of Pax6, we performed comprehensive transcriptome profiling analyses in the early hindbrain of Pax6 homozygous mutant and wild-type rats using microarrays. RESULTS: Comparison of quadruplicate microarray experiments using two computational methods allowed us to identify differentially expressed genes that have relatively small fold changes or low expression levels. Gene ontology analyses of the differentially expressed molecules demonstrated that Pax6 is involved in various signal transduction pathways where it regulates the expression of many receptors, signaling molecules, transporters and transcription factors. The up- or down-regulation of these genes was further confirmed by quantitative RT-PCR. In situ staining of Fabp7, Dbx1, Unc5h1 and Cyp26b1 mRNAs showed that expression of these transcripts not only overlapped with that of Pax6 in the hindbrain of wild-type and Pax6 heterozygous mutants, but also was clearly reduced in the hindbrain of the Pax6 homozygous mutant. In addition, the Pax6 homozygous mutant hindbrain showed that Cyp26b1 expression was lacked in the dorsal and ventrolateral regions of rhombomeres 5 and 6, and that the size of rhombomere 5 expanded rostrocaudally. CONCLUSIONS: These results indicate that Unc5h1 and Cyp26b1 are novel candidates for target genes transactivated by Pax6. Furthermore, our results suggest the interesting possibility that Pax6 regulates anterior-posterior patterning of the hindbrain via activation of Cyp26b1, an enzyme that metabolizes retinoic acid.

Transactivation activity of LBP-1 proteins and their dimerization in living cells.

LBP-1 proteins form dimers and act as transcription factors that activate a number of genes related to cell growth and differentiation. LBP-1a and LBP-1c are localized in the cytoplasm when transiently expressed in cultured cells, but translocated into the nucleus after forming heterodimers with LBP-1b, which is a splicing variant of LBP-1a with an intrinsic nuclear localization signal (NLS). Here, we report that LBP-1b showed potent transactivation activity, and that forcibly expressed LBP-1a and LBP-1c in the nucleus essentially exhibited very little or no transactivation activity. Mutations in the NLS that abolished the NLS activity of LBP-1b also abrogated the transactivation activity. We have found that LBP-1 proteins contain a putative sterile alpha motif domain indispensable for their dimerization capability in the C-terminal region. To demonstrate whether homo- and heterodimers composed of LBP-1a and/or LBP-1c are generated in the nucleus, we applied the FLIM-based fluorescence resonance energy transfer imaging technique to living cells. It revealed that dimers composed of LBP-1a and LBP-1c were re-formed probably by a partner-exchange of LBP-1b-containing heterodimers.

Concise review: Pax6 transcription factor contributes to both embryonic and adult neurogenesis as a multifunctional regulator.

Pax6 is a highly conserved transcription factor among vertebrates and is important in various developmental processes in the central nervous system (CNS), including patterning of the neural tube, migration of neurons, and formation of neural circuits. In this review, we focus on the role of Pax6 in embryonic and postnatal neurogenesis, namely, production of new neurons from neural stem/progenitor cells, because Pax6 is intensely expressed in these cells from the initial stage of CNS development and in neurogenic niches (the subgranular zone of the hippocampal dentate gyrus and the subventricular zone of the lateral ventricle) throughout life. Pax6 is a multifunctional player regulating proliferation and differentiation through the control of expression of different downstream molecules in a highly context-dependent manner.

Role of Fabp7, a downstream gene of Pax6, in the maintenance of neuroepithelial cells during early embryonic development of the rat cortex.

Pax6 is a transcription factor with key functional roles in the developing brain. Pax6 promotes neuronal differentiation via transcriptional regulation of the Neurogenin2 (Ngn2) gene, although Pax6 expression appears in proliferating neuroepithelial cells before the onset of neurogenesis. Here, we identified Fabp7 (BLBP/B-FABP), a member of the fatty acid-binding protein (FABP) family, as a downregulated gene in the embryonic brain of Pax6 mutant rat (rSey2/rSey2) by microarray analysis. Marked reduction of Fabp7 expression was confirmed by quantitative PCR. Spatiotemporal expression patterns of Fabp7 in the wild-type rat embryos from embryonic day 10.5 (E10.5) to E14.5 were similar to those of Pax6, and expression of Fabp7 was undetectable in the rSey2/rSey2 cortex. The expression pattern of Fabp7 in the wild-type mouse embryo at E10.5 (corresponding to E12.5 rat) was different from that in the rat embryo, and no change of expression was observed in the Sey/Sey mouse embryo. Overexpression of exogenous Pax6 mainly induced ectopic expression of Fabp7, rather than of Ngn2, in the early cortical primordium. Interestingly, knocking-down FABP7 function by electroporation of Fabp7 small interfering RNA severely curtailed cell proliferation but promoted neuronal differentiation. We conclude that Fabp7 is a downstream gene of Pax6 transcription factor in the developing rat cortex and essential for maintenance of neuroepithelial cells during early cortical development.