Biomolecular motor-driven microtubule translocation in the presence of shear flow: analysis of redirection behaviours

We suggest a concept for powering microfluidic devices with biomolecular motors and microtubules to meet the demands for highly efficient microfluidic devices. However, to successfully implement such devices, we require methods for active control over the direction of microtubule translocation. While most previous work has employed largely microfabricated passive mechanical patterns designed to guide the direction of microtubules, in this paper we demonstrate that hydrodynamic shear flow can be used to align microtubules translocating on a kinesin-coated surface in a direction parallel to the fluid flow. Our evidence supports the hypothesis that the mechanism of microtubule redirection is simply that drag force induced by viscous shear bends the leading end of a microtubule, which may be cantilevered beyond its kinesin supports. This cantilevered end deflects towards the flow direction, until it is subsequently bound to additional kinesins; as translocation continues, the process repeats until the microtubule is largely aligned with the flow, to a limit determined by random fluctuations created by thermal energy. We present statistics on the rate of microtubule alignment versus various strengths of shear flow as well as concentrations of kinesin, and also investigate the effects of shear flow on the motility.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/58134/2/nano7_2_025101.pd

Biomolecular motor-driven molecular sorter

We have developed a novel, microfabricated, stand-alone microfluidic device that can efficiently sort and concentrate (bio-)analyte molecules by using kinesin motors and microtubules as a chemo-mechanical transduction machine. The device removes hundreds of targeted molecules per second from an analyte stream by translocating functionalized microtubules with kinesin across the stream and concentrating them at a horseshoe-shaped collector. Target biomolecule concentrations increase up to three orders of magnitude within one hour of operation.close191

Large Fragment Pre-S Deletion and High Viral Load Independently Predict Hepatitis B Relapse after Liver Transplantation

Hepatitis B virus (HBV) associated end-stage liver diseases are the leading causes of liver transplantation (LT) in Taiwan. Relapse of hepatitis B occurs after LT, raising the risk of graft failure and reducing patient survival. Although several oral antiviral agents have been approved for anti-HBV treatment, lamivudine (LAM) remained to be the most widely used preventive regimen in Taiwan. While several clinical predictors have been identified for hepatitis B relapse, the predictive roles of the histopathological characteristics in liver explants as well as the genotypic features of the viruses in pre-LT serum samples have not been assessed. Between September 2002 and August 2009, 150 consecutive hepatitis B surface antigen (HBsAg) positive patients undergoing LT were included for outcome analysis following assessment of the clinicopathological and virological factors prior to LT. Kaplan-Meier analyses discovered that pre-operative LAM treatment ≤3 months; membranous distribution and higher expression of tissue HBsAg in liver explants; preoperative viral load ≧106 copies/ml; and presence of large fragment (>100 base pairs) pre-S deletion (LFpreSDel) correlated significantly with hepatitis B relapse. Multivariate Cox regression analysis showed that the presence of LFpreSDel (P = 0.001) and viral load ≧106 copies/mL (P = 0.023) were independent predictors for hepatitis B relapse. In conclusion, besides high viral load, LFpreSDel mutation is an important independent predictor for hepatitis B relapse after LT. More aggressive preventive strategies should be applied for patients carrying these risk factors

Kinesin: Directional Properties, Strain Coordination and Nanotechnology Applications.

Kinesin motors are biological motor proteins that evolved for a range of biological transport functions in cells. Kinesin’s small size and robustness of movement in vitro provide tremendous advantages for using kinesin in engineering application. Moreover, kinesin’s ability to utilize chemical energy from their ambient environment simplifies microdevice design and eliminates the requirement of large external power supplies. Here, I present three devices into which kinesin motors are integrated. Two of the devices efficiently rectify the mechanical power produced by kinesins into designated directions. The third device leverages the techniques of rectifying kinesin’s power to achieve highly sensitive bio-molecule sorting. These devices demonstrate that kinesin-powered devices are practical and have significant potential for future applications. To enhance future technological application, it is important to understand the molecular mechanisms of kinesin. Kinesin has been intensively studied for decades; however, one major gap relates to the mechanism(s) that control the direction of kinesin motors. Here, I used mutagenesis to investigate which structural domains determine the directionality of conventional kinesin and Ncd, the most frequently used models for directionality studies. The result suggests that structural components that control kinesins’ directionality are also directly involved generating the motor’s motility. Therefore, it is challenging to alter kinesins’ directionality and simuteneously keeping their motility intact. My data show that both kinesin and Ncd use components close to their head domains for controlling their directionality: they are neck-linker and C-terminal neck domain for kinesin and Ncd, respectively. An important physiological property of conventional kinesin is ability of taking a large number steps along the microtubule. This processive motion is believed to be based on a strain-coordinated, alternate catalysis. Little work has directly investigated the intra-molecular strain coordination of kinesin’s movement. To test this intra-molecular strain hypothesis I inserted a set of flexible residues at the junction between kinesin’s neck domain and neck-linker. The single motor gliding assays show that the wild-type and mutated kinesins move in vitro with the same velocity. These biophysical properties of these kinesin mutants suggest that kinesins may use different mechanism(s) other than the mechanical strain to coordinate their movement.Ph.D.Biomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/63629/1/mtkao_1.pd

Active alignment of microtubules with electric fields

The direction of translocation of microtubules on a surface coated with kinesin is usually random. Here we demonstrate and quantify the rate at which externally applied electric fields can direct moving microtubules parallel to the field by deflecting their leading end toward the anode. Effects of electric field strength, kinesin surface density, and microtubule translocation speed on the rate of redirection of microtubules were analyzed statistically. Furthermore, we demonstrated that microtubules can be steered in any desired direction via manipulation of externally applied E-fields.close394

Low Galectin-3 Expression Level in Primary Tumors Is Associated with Metastasis in T1 Lung Adenocarcinoma

Background and objective: Although nodal and distant metastasis is rare in T1 lung adenocarcinoma, it is related to poor clinical prognosis. Association between galectin-3 (Gal-3) expression level, and clinical outcome of T1 lung adenocarcinoma has not been clarified. Methods: From January 2009 to December 2014, 74 patients with surgically resected T1 lung adenocarcinoma were enrolled in this retrospective cohort study. Patient outcomes were followed up until December 2019. Gal-3 expression level in primary tumors was assessed immunohistochemically and evaluated based on the staining intensity and percentage. Patient characteristics and correlation between Gal-3 expression level and clinical outcomes were reviewed. Results: Low Gal-3 expression was associated with increased metastatic events (p = 0.03), especially distant metastasis (p = 0.007), and mortality rate (p = 0.04). Kaplan–Meier analysis revealed that high Gal-3 expression level was associated with favorable recurrence-free survival in T1 lung adenocarcinoma (log-rank p = 0.048) and T1a (≤ 2 cm, American Joint Committee on Cancer (AJCC) 7th edition) lung adenocarcinoma (log-rank p = 0.043). Gal-3 expression along with tumor size showed a larger area under curve (AUC) than tumor size alone for predicting metastatic events (AUC = 0.747 vs. 0.681) and recurrence (AUC = 0.813 vs. 0.766) in T1a lung adenocarcinoma in the receiver-operating characteristic curve. Conclusion: Low Gal-3 expression level in primary tumors was remarkably associated with increased metastatic events and reduced recurrence-free survival in T1 lung adenocarcinoma. We suggest that Gal-3 expression level in addition to tumor size may potentially be stronger than tumor size alone in predicting metastasis in T1a lung adenocarcinoma patients

MicroRNA-133a Suppresses Multiple Oncogenic Membrane Receptors and Cell Invasion in Non-Small Cell Lung Carcinoma

<div><p>Non-small cell lung cancers (NSCLCs) cause high mortality worldwide, and the cancer progression can be activated by several genetic events causing receptor dysregulation, including mutation or amplification. MicroRNAs are a group of small non-coding RNA molecules that function in gene silencing and have emerged as the fine-tuning regulators during cancer progression. MiR-133a is known as a key regulator in skeletal and cardiac myogenesis, and it acts as a tumor suppressor in various cancers. This study demonstrates that miR-133a expression negatively correlates with cell invasiveness in both transformed normal bronchial epithelial cells and lung cancer cell lines. The oncogenic receptors in lung cancer cells, including insulin-like growth factor 1 receptor (IGF-1R), TGF-beta receptor type-1 (TGFBR1), and epidermal growth factor receptor (EGFR), are direct targets of miR-133a. MiR-133a can inhibit cell invasiveness and cell growth through suppressing the expressions of IGF-1R, TGFBR1 and EGFR, which then influences the downstream signaling in lung cancer cell lines. The cell invasive ability is suppressed in IGF-1R- and TGFBR1-repressed cells and this phenomenon is mediated through AKT signaling in highly invasive cell lines. In addition, by using the in <i>vivo</i> animal model, we find that ectopically-expressing miR-133a dramatically suppresses the metastatic ability of lung cancer cells. Accordingly, patients with NSCLCs who have higher expression levels of miR-133a have longer survival rates compared with those who have lower miR-133a expression levels. In summary, we identified the tumor suppressor role of miR-133a in lung cancer outcome prognosis, and we demonstrated that it targets several membrane receptors, which generally produce an activating signaling network during the progression of lung cancer.</p></div