Determining the Young's Modulus of the Bacterial Cell Envelope.

Bacteria experience substantial physical forces in their natural environment, including forces caused by osmotic pressure, growth in constrained spaces, and fluid shear. The cell envelope is the primary load-carrying structure of bacteria, but the mechanical properties of the cell envelope are poorly understood; reports of Young's modulus of the cell envelope of Escherichia coli range from 2 to 18 MPa. We developed a microfluidic system to apply mechanical loads to hundreds of bacteria at once and demonstrated the utility of the approach for evaluating whole-cell stiffness. Here, we extend this technique to determine Young's modulus of the cell envelope of E. coli and of the pathogens Vibrio cholerae and Staphylococcus aureus. An optimization-based inverse finite element analysis was used to determine the cell envelope Young's modulus from observed deformations. The Young's modulus values of the cell envelope were 2.06 ± 0.04 MPa for E. coli, 0.84 ± 0.02 MPa for E. coli treated with a chemical (A22) known to reduce cell stiffness, 0.12 ± 0.03 MPa for V. cholerae, and 1.52 ± 0.06 MPa for S. aureus (mean ± SD). The microfluidic approach allows examination of hundreds of cells at once and is readily applied to Gram-negative and Gram-positive organisms as well as rod-shaped and cocci cells, allowing further examination of the structural causes behind differences in cell envelope Young's modulus among bacterial species and strains.

Mechanical stimuli activate gene expression via a cell envelope stress sensing pathway.

Mechanosensitive mechanisms are often used to sense damage to tissue structure, stimulating matrix synthesis and repair. While this kind of mechanoregulatory process is well recognized in eukaryotic systems, it is not known whether such a process occurs in bacteria. In Vibrio cholerae, antibiotic-induced damage to the load-bearing cell wall promotes increased signaling by the two-component system VxrAB, which stimulates cell wall synthesis. Here we show that changes in mechanical stress within the cell envelope are sufficient to stimulate VxrAB signaling in the absence of antibiotics. We applied mechanical forces to individual bacteria using three distinct loading modalities: extrusion loading within a microfluidic device, direct compression and hydrostatic pressure. In all cases, VxrAB signaling, as indicated by a fluorescent protein reporter, was increased in cells submitted to greater magnitudes of mechanical loading, hence diverse forms of mechanical stimuli activate VxrAB signaling. Reduction in cell envelope stiffness following removal of the endopeptidase ShyA led to large increases in cell envelope deformation and substantially increased VxrAB response, further supporting the responsiveness of VxrAB. Our findings demonstrate a mechanosensitive gene regulatory system in bacteria and suggest that mechanical signals may contribute to the regulation of cell wall homeostasis.

Targeted Fusogenic Liposomes for Effective Tumor Delivery and Penetration of Lipophilic Cargoes.

Nanoparticle-based drug delivery has been widely used for effective anticancer treatment. However, a key challenge restricting the efficacy of nanotherapeutics is limited tissue penetration within solid tumors. Here, we report a targeted fusogenic liposome (TFL) that can selectively deliver lipophilic cargo to the plasma membranes of tumor cells. TFL is prepared by directly attaching tumor-targeting peptides to the surface of FL instead of the cationic moieties. The lipophilic cargo loaded in the membrane of TFL is transferred to the plasma membranes of tumor cells and subsequently packaged in the extracellular vesicles (EVs) released by the cells. Systemically administered TFL accumulates in the perivascular region of tumors, where the lipophilic cargo is unloaded to the tumor cell membranes and distributed autonomously throughout the tumor tissue via extracellular vesicle-mediated intercellular transfer. When loaded with a lipophilic pro-apoptotic drug, thapsigargin (Tg), TFL significantly inhibits tumor growth in a mouse colorectal cancer model. Furthermore, the combination treatment with TFL (Tg) potentiates the antitumor efficacy of FDA-approved liposomal doxorubicin, whose therapeutic effect is limited to perivascular regions without significant toxicity.

Cardiac Kinetic Energy and Viscous Dissipation Rate From Radial Flow Data.

Recent studies have correlated kinetic energy (KE) and viscous dissipation rate (VDR) in the left ventricle (LV) with heart health. These studies have relied on 4D-flow imaging or computational fluid dynamics modeling, which are able to measure, or compute, all 3 components (3C) of the blood flow velocity in 3 dimensional (3D) space. This richness of data is difficult to acquire clinically. Alternatively, color Doppler echocardiography (CDE) is more widespread clinically, but only measures a single radial component of velocity and typically only over a planar section. Because of this limitation, prior CDE-based studies have first reconstructed a second component of velocity in the measurement plane prior to evaluating VDR or KE. Herein, we propose 1C-based surrogates of KE and VDR that can be derived directly from the radial component of the flow velocity in the LV. Our results demonstrate that the proposed 1C-based surrogates of KE and VDR are generally as well-correlated with the true KE and VDR values as surrogates that use reconstructed 2C flow data. Moreover, the correlation of these 1C-based surrogates with the true values indicate that CDE (3D in particular) may be useful in evaluating these metrics in practice.

Evaluation of the equation for predicting dry matter intake of lactating dairy cows in the Korean feeding standards for dairy cattle.

OBJECTIVE: This study aimed to validate and evaluate the dry matter (DM) intake prediction model of the Korean feeding standards for dairy cattle (KFSD). METHODS: The KFSD DM intake (DMI) model was developed using a database containing the data from the Journal of Dairy Science from 2006 to 2011 (1,065 observations 287 studies). The development (458 observations from 103 studies) and evaluation databases (168 observations from 74 studies) were constructed from the database. The body weight (kg; BW), metabolic BW (BW0.75, MBW), 4% fat-corrected milk (FCM), forage as a percentage of dietary DM, and the dietary content of nutrients (% DM) were chosen as possible explanatory variables. A random coefficient model with the study as a random variable and a linear model without the random effect was used to select model variables and estimate parameters, respectively, during the model development. The best-fit equation was compared to published equations, and sensitivity analysis of the prediction equation was conducted. The KFSD model was also evaluated using in vivo feeding trial data. RESULTS: The KFSD DMI equation is 4.103 (±2.994)+0.112 (±0.022)×MBW+0.284 (±0.020) ×FCM-0.119 (±0.028)×neutral detergent fiber (NDF), explaining 47% of the variation in the evaluation dataset with no mean nor slope bias (p>0.05). The root mean square prediction error was 2.70 kg/d, best among the tested equations. The sensitivity analysis showed that the model is the most sensitive to FCM, followed by MBW and NDF. With the in vivo data, the KFSD equation showed slightly higher precision (R2 = 0.39) than the NRC equation (R2 = 0.37), with a mean bias of 1.19 kg and no slope bias (p>0.05). CONCLUSION: The KFSD DMI model is suitable for predicting the DMI of lactating dairy cows in practical situations in Korea.

Simple transscleral fixation of IOL technique without tying up haptics.

AIM: To introduce a novel technique for transscleral fixation of the posterior chamber intraocular lens (PC-IOL) that requires no sutures on the IOL haptics. METHODS: Instead of suturing polypropylene onto the IOL haptics, the method simply winds the thread on the haptics. Fifteen eyes of 15 patients underwent this technique and were followed up for more than 18mo. Surgical outcomes and post-operative complications were evaluated and compared with those of the conventional transscleral fixation method. RESULTS: Postoperative cylinder was significantly lower in the thread winding group than in the conventional transscleral fixation method group (-1.02±0.46 diopters vs -1.57±0.77 diopters; P=0.01). Further, no postoperative complications, such as optic capture, IOL dislocation, and hyphema, were detected in the thread winding group. CONCLUSION: We believe that our thread winding technique is better than previously reported methods because it is simple, mechanically stable, and free from suture-related complications.

Effective Delivery of Exogenous Compounds to the Optic Nerve by Intravitreal Injection of Liposome.

PURPOSE: To improve the treatment efficiency of optic nerve diseases by delivering therapeutic materials to the optic nerve directly. METHODS: We tried to optimize liposomal composition to deliver a payload to the optic nerve efficiently when it is injected intravitreally. After loading dexamethasone into this liposome, we tested the therapeutic effect of liposomes in this treatment using a murine model of ischemic optic neuropathy. RESULTS: Our optimized liposome can deliver its payload to the optic nerve more efficiently than other tested compositions. Moreover, dexamethasone-loaded liposomes had a significant therapeutic effect in a murine model of ischemic optic neuropathy. CONCLUSIONS: Here, we demonstrate the optimal composition of liposomes that could efficiently deliver intravitreally injected exogenous compounds to the optic nerve. We expect that the intravitreal injection of liposomes with the suggested composition would improve the delivery efficacy of therapeutic compounds to the optic nerve.

Cooperative tumour cell membrane targeted phototherapy.

The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

Effective Retinal Penetration of Lipophilic and Lipid-Conjugated Hydrophilic Agents Delivered by Engineered Liposomes.

Efficient delivery of drugs to the retina is critical but difficult to achieve with current methods. There have been a number of attempts to use intravitreal injection of liposomes, artificial vesicles composed of a phospholipid bilayer, to overcome the limitations of conventional intravitreal injection (short retention time, toxicity, poor penetration, etc.). Here, we report an optimal liposomal formulation that can diffuse through the vitreous humor, deliver the incorporated agents to all retinal layers effectively, and maintain them for a relatively long time. We first delivered lipophilic compounds and phospholipid-conjugated hydrophilic agents to the inner limiting membrane using engineered liposomes. Subsequently, the agents penetrated the retina deeply, presumably via extracellular vesicles, nanoscale vesicles secreted from retinal-associated cells. These results suggest that this engineered liposomal formulation can leverage the biological transport system for effective retinal penetration of lipophilic and lipid-conjugated agents.

A novel NFIA-NFκB feed-forward loop contributes to glioblastoma cell survival.

Background: The nuclear factor I-A (NFIA) transcription factor promotes glioma growth and inhibits apoptosis in glioblastoma (GBM) cells. Here we report that the NFIA pro-survival effect in GBM is mediated in part via a novel NFIA-nuclear factor-kappaB (NFκB) p65 feed-forward loop. Methods: We examined effects of gain- and loss-of-function manipulations of NFIA and NFκB p65 on each other's transcription, cell growth, apoptosis and sensitivity to chemotherapy in patient-derived GBM cells and established GBM cell lines. Results: NFIA enhanced apoptosis evasion by activating NFκB p65 and its downstream anti-apoptotic factors tumor necrosis factor receptor-associated factor 1 (TRAF1) and cellular inhibitor of apoptosis proteins (cIAPs). Induction of NFκB by NFIA was required to protect cells from apoptosis, and inhibition of NFκB effectively reversed the NFIA anti-apoptotic effect. Conversely, NFIA knockdown decreased expression of NFκB and anti-apoptotic genes TRAF1 and cIAPs, and increased baseline apoptosis. NFIA positively regulated NFκB transcription and NFκB protein level. Interestingly, NFκB also activated the NFIA promoter and increased NFIA level, and knockdown of NFIA was sufficient to attenuate the NFκB pro-survival effect, suggesting a reciprocal regulation between NFIA and NFκB in governing GBM cell survival. Supporting this, NFIA and NFκB expression levels were highly correlated in human GBM and patient-derived GBM cells. Conclusions: These data define a previously unknown NFIA-NFκB feed-forward regulation that may contribute to GBM cell survival.

Cellular Engineering with Membrane Fusogenic Liposomes to Produce Functionalized Extracellular Vesicles.

Engineering of extracellular vesicles (EVs) without affecting biological functions remains a challenge, limiting the broad applications of EVs in biomedicine. Here, we report a method to equip EVs with various functional agents, including fluorophores, drugs, lipids, and bio-orthogonal chemicals, in an efficient and controlled manner by engineering parental cells with membrane fusogenic liposomes, while keeping the EVs intact. As a demonstration of how this method can be applied, we prepared EVs containing azide-lipids, and conjugated them with targeting peptides using copper-free click chemistry to enhance targeting efficacy to cancer cells. We believe that this liposome-based cellular engineering method will find utility in studying the biological roles of EVs and delivering therapeutic agents through their innate pathway.

One-Wave Optical Phase Conjugation Mirror by Actively Coupling Arbitrary Light Fields into a Single-Mode Reflector.

Rewinding the arrow of time via phase conjugation is an intriguing phenomenon made possible by the wave property of light. Here, we demonstrate the realization of a one-wave optical phase conjugation mirror using a spatial light modulator. An adaptable single-mode filter is created, and a phase-conjugate beam is then prepared by reverse propagation through this filter. Our method is simple, alignment free, and fast while allowing high power throughput in the time-reversed wave, which has not been simultaneously demonstrated before. Using our method, we demonstrate high throughput full-field light delivery through highly scattering biological tissue and multimode fibers, even for quantum dot fluorescence.

Enhanced Performance in Bulk Heterojunction Polymer Solar Cell Using Water Soluble Conjugated Polymer.

We have synthesized water-soluble polymer, poly[(9,9-bis((6'-(N,N,N-trimethylammonium)hexyl)-2,7-fluorene))-alt-bisphenylfumaronitrile]dibromide (AHF-alt-PFN), the polymer typically obtained by the Suzuki type of polymerization reaction and shows good solubility in methanol. Bulk heterojunction polymer solar cells (BHJ-PSCs) fabricated by using water soluble conjugated polymer and positive (Cs+) and negative (F-, CO2-(3)) charge ions doping as an interfacial layer for poly(3-hexylthiophene):phenyl-C61 butyric acid methyl ester (P3HT:PCBM). We have achieved an enhancement of the short circuit density and power conversion efficiency in solar cell by introducing poly(AHF-alt-PFN) layer between the active layer and the cathode metal. The device with poly(AHF-alt-PRN) layer containing F-, CO2-(3) showed a short circuit current density more 1.3, 2.3 times higher than those of the device without poly(AHF-alt-PFN) + ion layer. We explain the better performance in solar cell with poly(AHF-alt-PFN) + ion layer was due not only to the increase of electron mobility in poly(AHF-alt-PFN) layer but also to the decrease of the electron barrier near cathode by the addition of the negative ions.

Liposome-based engineering of cells to package hydrophobic compounds in membrane vesicles for tumor penetration.

Natural membrane vesicles (MVs) derived from various types of cells play an essential role in transporting biological materials between cells. Here, we show that exogenous compounds are packaged in the MVs by engineering the parental cells via liposomes, and the MVs mediate autonomous intercellular migration of the compounds through multiple cancer cell layers. Hydrophobic compounds delivered selectively to the plasma membrane of cancer cells using synthetic membrane fusogenic liposomes were efficiently incorporated into the membrane of MVs secreted from the cells and then transferred to neighboring cells via the MVs. This liposome-mediated MV engineering strategy allowed hydrophobic photosensitizers to significantly penetrate both spheroids and in vivo tumors, thereby enhancing the therapeutic efficacy. These results suggest that innate biological transport systems can be in situ engineered via synthetic liposomes to guide the penetration of chemotherapeutics across challenging tissue barriers in solid tumors.

Risk factors for fluoroquinolone resistance in ocular cultures.

PURPOSE: To identify the risk factors associated with fluoroquinolone resistance in patients undergoing cataract surgery. METHODS: A total of 1,125 patients (1,125 eyes) who underwent cataract surgery at Veterans Health Service Medical Center from May 2011 to July 2012 were enrolled in this study. Conjunctival cultures were obtained from the patients on the day of surgery before instillation of any ophthalmic solutions. The medical records of patients with positive coagulase negative staphylococcus (CNS) and Staphylococcus aureus (S. aureus) cultures were reviewed to determine factors associated with fluoroquinolone resistance. RESULTS: Of 734 CNS and S. aureus cultures, 175 (23.8%) were resistant to ciprofloxacin, levofloxacin, gatifloxacin, or moxifloxacin. Use of fluoroquinolone within 3 months and within 1 year before surgery, topical antibiotic use other than fluoroquinolone, systemic antibiotic use, recent hospitalization, ocular surgery, intravitreal injection and use of eyedrops containing benzalkonium chloride were significantly more frequent in resistant isolates than in susceptible isolates. In multivariable logistic regression analysis, ocular surgery (odds ratio [OR], 8.457), recent hospitalization (OR, 6.646) and use of fluoroquinolone within 3 months before surgery (OR, 4.918) were significant predictors of fluoroquinolone resistance, along with intravitreal injection (OR, 2.976), systemic antibiotic use (OR, 2.665), use of eyedrops containing benzalkonium chloride (OR, 2.323), use of fluoroquinolone within 1 year before surgery (OR, 1.943) and topical antibiotic use other than fluoroquinolone (OR, 1.673). CONCLUSIONS: Recent topical fluoroquinolone use, hospitalization and ocular surgery were significantly associated with fluoroquinolone resistance in CNS and S. aureus isolates from ocular culture.

Meta-analysis of factors affecting milk component yields in dairy cattle.

The objectives of this study were thus to identify most significant factors that determine milk component yield (MCY) using a meta-analysis and, if possible, to develop equations to predict MCY using variables that can be easily measured in the field. A literature database was constructed based on the research articles published in the Journal of Dairy Science from Oct., 2007 till May, 2010. The database consisted of a total of 442 observed means for MCY from 118 studies. The candidate factors that determine MCY were those which can be routinely measured in the field (e.g. DMI, BW, dietary forage content, chemical composition of diets). Using a simple linear regression, the best equations for predicting milk fat yield(MFY) and milk protein yield (MPY) were MFY = 0.351 (±0.068) + 0.038 (±0.003) DMI (R(2) = 0.27), and MPY = 0.552 (±0.071) + 0.031 (±0.002) DMI - 0.004 (±0.001) FpDM (%, forage as a percentage of dietary DM) (R(2) = 0.38), respectively. The best equation for predicting milk fat content (%) explained only 12% of variations in milk fat content, and none of a single variable can explain more than 5% of variations in milk protein content. We concluded that among the tested variables, DMI was the only significant factor that affects MFY and both DMI and FpDM significantly affect MPY. However, predictability of linear equations was relatively low. Further studies are needed to identify other variables that can predict milk component yield more accurately.

Ocular biometric parameters associated with intraocular pressure reduction after cataract surgery in normal eyes.

PURPOSE: To evaluate the ocular biometric parameters associated with intraocular pressure (IOP) reduction after phacoemulsification. DESIGN: Prospective, observational case series. METHODS: The study included 999 patients who had undergone uncomplicated phacoemulsification. IOP and ocular biometric parameters were checked preoperatively and 3 months postoperatively using anterior segment optical coherence tomography, optical biometry, and ultrasonic biomicroscopy. The relationship between IOP change and the parameters, including preoperative IOP, anterior chamber depth, axial length, angle opening distance at 500 µm, anterior chamber area, corneal thickness, lens thickness, and iris thickness at 750 µm, was evaluated. RESULTS: The mean patient age was 67.1 ± 4.3 years. The average change in IOP was -1.6 mm Hg (-11.8%). In univariate analysis, axial length, corneal thickness, and iris thickness were not significantly associated with IOP reduction. However, preoperative IOP, anterior chamber depth, angle opening distance, anterior chamber area, and lens thickness were significantly associated with IOP change (P < .05). Furthermore, changes in anterior chamber depth (standardized coefficient beta [B] = -0.082), angle opening distance (B = -0.095), and anterior chamber area (B = -0.380) were more strongly correlated with IOP change than were preoperative factors (B = -0.078, B = -0.071, and B = -0.067, respectively). In multivariate analysis, preoperative IOP, lens thickness, angle opening distance change, and anterior chamber area change were significantly associated with IOP change (P < .005). CONCLUSION: In addition to preoperative IOP and lens thickness, parameters such as changes in anterior chamber area and angle opening distance were significantly associated positively with reduced IOP after phacoemulsification.

Cytotoxic sesquiterpene lactones from Carpesium abrotanoides.

Eight sesquiterpene lactones, 4 alpha,5 alpha-epoxy-10 alpha,14-dihydro-inuviscolide (1), 2,3-dihydroaromomaticin (2), carpesiolin (3), carabrone (4), carabrol (5), telekin (6), ivalin (7) and 11,13-didehydroivaxillin (8), were isolated from the aerial parts of Carpesium abrotanoides Linne (Compositae). In vitro cytotoxicity testing was carried out against L1210, A549, SK-OV-3, SK-MEL-2, XF-498 and HCT-15 tumor cell lines. Sesquiterpene lactone compounds 1 - 8 showed significant cytotoxic activity (ED50 values, < 20 microM) against all tumor call lines tested. Among these compounds, 4 alpha,5 alpha-epoxy-10 alpha,14-dihydro-inuviscolide (1), 2,3-dihydroaromomaticin (2), telekin (6) and ivalin (7) showed cytotoxic activity (ED50, < 10 microM) comparable to that of cisplatin.