Flagellated bacterial motility in polymer solutions

It is widely believed that the swimming speed, v, of many flagellated bacteria is a nonmonotonic function of the concentration, c, of high-molecular-weight linear polymers in aqueous solution, showing peaked v(c) curves. Pores in the polymer solution were suggested as the explanation. Quantifying this picture led to a theory that predicted peaked v(c) curves. Using high-throughput methods for characterizing motility, we measured v and the angular frequency of cell body rotation, Ω, of motile Escherichia coli as a function of polymer concentration in polyvinylpyrrolidone (PVP) and Ficoll solutions of different molecular weights. We find that nonmonotonic v(c) curves are typically due to low-molecular-weight impurities. After purification by dialysis, the measured v(c) and Ω(c) relations for all but the highest-molecular-weight PVP can be described in detail by Newtonian hydrodynamics. There is clear evidence for non-Newtonian effects in the highest-molecular-weight PVP solution. Calculations suggest that this is due to the fast-rotating flagella seeing a lower viscosity than the cell body, so that flagella can be seen as nano-rheometers for probing the non-Newtonian behavior of high polymer solutions on a molecular scale.The motility of microorganisms in polymer solutions is a topic of vital biomedical interest. For example, mucus covers the respiratory (1), gastrointestinal (2), and reproductive (3) tracks of all metazoans. Penetration of this solution of biomacromolecules by motile bacterial pathogens is implicated in a range of diseases, e.g., stomach ulcers caused by Helicobacter pylori (4). Oviduct mucus in hens provides a barrier against Salmonella infection of eggs (5). Penetration of the exopolysaccharide matrix of biofilms by swimming bacteria (6) can stabilize or destabilize them in vivo (e.g., the bladder) and in vitro (e.g., catheters). In reproductive medicine (human and veterinary), the motion of sperms in seminal plasma and vaginal mucus, both non-Newtonian polymer solutions, is a strong determinant of fertility (3), and polymeric media are often used to deliver spermicidal and other vaginal drugs (7).Microorganismic propulsion in non-Newtonian media such as high-polymer solutions is also a hot topic in biophysics, soft matter physics, and fluid dynamics (8). Building on knowledge of propulsion modes at low Reynolds number in Newtonian fluids (8), current work seeks to understand how these are modified to enable efficient non-Newtonian swimming. In particular, there is significant interest in a flapping sheet (9, 10) or an undulating filament (11) (modeling the sperm tail) and in a rotating rigid helix (modeling the flagella of, e.g., Escherichia coli) (12, 13) in non-Newtonian fluids.An influential set of experiments in this field was performed 40 years ago by Schneider and Doetsch (SD) (14), who measured the average speed, v¯, of seven flagellated bacterial species (including E. coli) in solutions of polyvinylpyrrolidone (PVP, molecular weight given as M = 360?kDa) and in methyl cellulose (MC, M unspecified) at various concentrations, c. SD claimed that v¯(c) was always nonmonotonic and peaked.A qualitative explanation was suggested by Berg and Turner (BT) (15), who argued that entangled linear polymers formed “a loose quasi-rigid network easily penetrated by particles of microscopic size.” BT measured the angular speed, Ω, of the rotating bodies of tethered E. coli cells in MC solutions. They found that adding MC hardly decreased Ω. However, in solutions of Ficoll, a branched polymer, Ω is proportional to η^?1, where η is the solution’s viscosity, which was taken as evidence for Newtonian behavior. In MC solutions, however, BT suggested that there were E. coli-sized pores, so that cells rotated locally in nearly pure solvent. Magariyama and Kudo (MK) (16) formulated a theory based on this picture and predicted a peak in v(c) by assuming that a slender body in a linear-polymer solution experienced different viscosities for tangential and normal motions in BT’s “easily penetrated” pores.This standard model is widely accepted in the biomedical literature on flagellated bacteria in polymeric media. It also motivates much current physics research in non-Newtonian low-Reynolds-number propulsion. Nevertheless, there are several reasons for a fundamental reexamination of the topic.First, polymer physics (17) casts a priori doubt on the presence of E. coli-sized pores in an entangled solution. Entanglement occurs above the overlap concentration, c^?, where coils begin to touch. The mesh size at c^?, comparable to a coil’s radius of gyration, r_g, gives the maximum possible pore size in the entangled network. For 360-kDa PVP in water, r_g ? 60?nm (see below), which is well under the cross section of E. coli (0.8 μm). Thus, the physical picture suggested by BT (15) and used by MK (16) has doubtful validity.Second, SD’s data were statistically problematic. They took movies, from which cells with “the 10 greatest velocities were used to calculate the average velocity” (14). Thus, their peaks in v(c) could be no more than fluctuations in measurements that were in any case systematically biased.Finally, although MK’s theory indeed predicts a peak in v(c), we find that their formulas also predict a monotonic increase in Ω(c) in the same range of c (Fig. S1), which is inconsistent with the data of BT, who observed a monotonic decrease.We therefore performed a fresh experimental study of E. coli motility using the same polymer (PVP) as SD, but varying the molecular weight, M, systematically. High-throughput methods for determining v and Ω enabled us to average over  ～ 10⁴ cells at each data point. Using polymers as purchased, we indeed found peaked v(c) curves at all M studied. However, purifying the polymers removed the peak in all but a single case. Newtonian hydrodynamics can account in detail for the majority of our results, collapsing data onto master curves. We show that the ratio v(c)/Ω(c) is a sensitive indicator of non-Newtonian effects, which we uncover for 360-kDa PVP. We argue that these are due to shear-induced changes in the polymer around the flagella.Below, we first give the necessary theoretical and experimental background before reporting our results.     

The effects of pneumatic tube transport on fresh and stored platelets in additive solution

Background

Limited scientific work has been conducted on potential in vitro effects of transport on pneumatic tube systems on blood components, in particular platelets.

Materials and methods

To evaluate the possible effects of the Swisslog TranspoNet system on the cellular, metabolic, phenotypic and secreting properties of fresh and stored platelets, we set up a four-arm paired study comparing transported and non-transported platelets. Platelets were aliquoted, prepared with the OrbiSac system and suspended in 70% SSP+ (n=8). All in vitro parameters were monitored over a 7-day storage period.

Results

Throughout storage, no differences were observed in glucose consumption, lactate production, pH, pCO₂, ATP, hypotonic shock response reactivity, CD62P, PAC-1, platelet endothelial cell adhesion molecule-1 or CD42b. The release of sCD40L increased (p<0.01) in all units but without any significant differences between groups.

Conclusion

The storage stability of all platelets conveyed by the Swisslog TranspoNet system was not impaired throughout 7 days of storage. The Swisslog TranspoNet system does not, therefore, seem to be a risk for increased metabolic activity, activation or release reactions from the platelets. This lack of effect of the pneumatic tube transport system did not seem to be affected by the age of the platelets or repeated transport.     

Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency

The capacity to produce therapeutically relevant quantities of multipotent mesenchymal stromal cells (MSCs) via in vitro culture is a common prerequisite for stem cell-based therapies. Although culture expanded MSCs are widely studied and considered for therapeutic applications, it has remained challenging to identify a unique set of characteristics that enables robust identification and isolation of the multipotent stem cells. New means to describe and separate this rare cell type and its downstream progenitor cells within heterogeneous cell populations will contribute significantly to basic biological understanding and can potentially improve efficacy of stem and progenitor cell-based therapies. Here, we use multivariate biophysical analysis of culture-expanded, bone marrow-derived MSCs, correlating these quantitative measures with biomolecular markers and in vitro and in vivo functionality. We find that, although no single biophysical property robustly predicts stem cell multipotency, there exists a unique and minimal set of three biophysical markers that together are predictive of multipotent subpopulations, in vitro and in vivo. Subpopulations of culture-expanded stromal cells from both adult and fetal bone marrow that exhibit sufficiently small cell diameter, low cell stiffness, and high nuclear membrane fluctuations are highly clonogenic and also exhibit gene, protein, and functional signatures of multipotency. Further, we show that high-throughput inertial microfluidics enables efficient sorting of committed osteoprogenitor cells, as distinct from these mesenchymal stem cells, in adult bone marrow. Together, these results demonstrate novel methods and markers of stemness that facilitate physical isolation, study, and therapeutic use of culture-expanded, stromal cell subpopulations.The biophysical state of a cell is potentially a rich source of information indicative of cell identity and physiology. When the underlying biochemical activity that occurs as cells replicate, senesce, differentiate, become malignant, or undergo apoptosis is manifest as measurable changes in biophysical characteristics, then parameters such as cell size or mechanical stiffness (1–4) may serve as predictive markers of cellular fate. For example, the metastatic competence of cancer cell lines has been correlated with average mechanical creep compliance (5, 6), and the stiffness of malaria-infected (7) and sickle red blood cells (8) has been related to disease stage and severity.The successful application of mechanobiology to the analysis of human disease has prompted development of biophysical cytometry methods to study the functional multipotency of stem cells (9, 10). However, such efforts to predict multipotency or “stemness” are challenging due to potential coupling or plurality of biophysical changes in response to distinct cues. For example, changes in cell size are not only related to cell cycle events (11) and cell proliferation rates (12), but also to the reported differentiation capacity of progenitor cells derived from corneal epithelium (13), adipose tissue (14), or adult bone marrow (15, 16). It is thus evident that a more comprehensive physical profile of stem cells is required to consider whether biophysical markers are robust indicators of inducible function in vitro and in vivo. Herein, we demonstrate that multivariate biophysical analysis of cells can readily identify subpopulations of multipotent, as well as osteochondral progenitor, cells within in vitro culture-expanded mesenchymal stromal cells (MSCs).Although often referred to and treated as a uniform stem cell population, culture-expanded MSCs derived from adult bone marrow (aMSCs) are actually a heterogeneous cell mixture (16, 17). These cell populations exhibit reduced multilineage potential during in vitro culture expansion (18, 19). This decreased multipotency has been attributed to environmental cues (20–25) during in vitro culture and results in MSC subpopulations that render it difficult to study and engineer “stem cell behavior.” Such desirable behavior includes self-renewal and multilineage differentiation in vitro or production of uniform, robust therapeutic responses in vivo. However, the long-term and large-scale expansion of aMSCs is necessary to obtain a clinically relevant number of cells for many envisioned tissue regeneration therapies. Conventional high-throughput sorting of multipotent MSCs from this heterogeneous, putative MSC population via flow cytometry has proven insufficient, due to the lack of biomolecular surface markers that select specifically for multipotency (15, 26, 27); such molecular labeling approaches also restrict viability and use of such cells for therapeutic applications (28). Thus, it is common to verify the multipotency of MSC subpopulations or clones via in vitro experiments that directly quantify MSC capacity to form colonies and differentiate along multiple tissue lineages. These Schrodinger’s cat-like assessments of viable stem cell function are both retrospective and confer obvious limitations for robust studies of stem cell biology and for clinical applications of culture-expanded MSCs. Such considerations illustrate the need for alternative, multivariate, and functional cytometry platforms and methods that can identify marrow stromal cell subpopulations of predictable potency or progenitor status, without labeling or differentiating those cells.Here, we quantify several biophysical characteristics of MSCs subpopulations derived from human adult and fetal bone marrow. These potential multivariate biomarkers of MSC potency are as follows: (i) suspended cell diameter; (ii) adherent cell spread area; (iii) cell stiffness; (iv) nuclear to cytoplasmic ratio; and (v) relative nuclear membrane fluctuations. We correlated each property with molecular surface markers, in vitro multilineage differentiation potential, and in vivo regenerative potential (see SI Appendix for discussion of previous studies that noted one or more of these properties to be potential indicators of differentiation capacity or commitment). Of particular interest is whether any of these physical signatures, or combinations thereof, could prospectively identify and sort multipotent MSC subpopulations from precommitted progenitor cells. We find that cell size is a necessary but insufficient predictor of MSC multipotency: not all subpopulations of small diameter are multipotent, as might be inferred from previous in vitro studies that compared smaller and larger MSCs (16). Among the several other biophysical markers considered, we find that only cell stiffness and nuclear fluctuations correlated strongly with in vitro differentiation potential and in vivo bone and muscle regeneration capacity. Specifically, adult and fetal MSC subpopulations of sufficiently low mean diameter (D < 20 μm), low mechanical stiffness (E < 375 Pa), and high nuclear fluctuations (NF > 1.2%) consistently exhibited multipotency in vitro and in vivo. All other MSC subpopulations exhibited commitment toward the osteogenic lineage. Together these findings suggest a minimal set of biophysical markers exist for the identification of MSC and progenitor subpopulations toward clinical applications.     

Good longterm survival after primary living donor liver transplantation for solitary hepatocellular carcinomas up to 8 cm in diameter

Objectives

There is controversy over whether hepatocellular carcinoma (HCC) should be primarily treated with living donor liver transplantation (LDLT) if liver resection (LR) can be effective. This retrospective study was conducted to compare survival outcomes in patients treated with either modality for solitary HCC measuring ≤8 cm in diameter.

Methods

Outcomes in patients with solitary HCC primarily treated by LDLT were analysed. Patients with solitary HCC of similar sizes with or without microvascular invasion primarily treated with LR were selected at a ratio of 6 : 1 for comparison.

Results

In-hospital mortality amounted to 0% and 1.3% in the LDLT (n = 50) and LR (n = 300) groups, respectively (P = 0.918). Complication rates were 34% and 20% in the LDLT and LR groups, respectively (P = 0.027). Rates of 1-, 3-, 5- and 10-year overall survival were 98%, 94%, 89% and 83%, respectively, in the LDLT group and 95%, 85%, 76% and 56%, respectively, in the LR group (P = 0.013). Rates of 1-, 3-, 5- and 10-year disease-free survival were 96%, 90%, 87% and 81%, respectively, in the LDLT group and 81%, 64%, 57% and 40%, respectively, in the LR group (P < 0.0001).

Conclusions

Living donor liver transplantation surpassed LR in survival outcomes, achieving a 10-year overall survival rate 1.5 times as high and a 10-year disease-free survival rate twice as high as those facilitated by LR. However, it entailed more complications, in addition to the inevitable risks to the donor.