DNA Capture by Nanopore Sensors under Flow.

Integrating nanopore sensors within microfluidic architectures is key to providing advanced sample processing capabilities upstream of the biosensor. When confined in a microchannel, the nanopore capture and translocation characteristics are altered when subjected to cross-flow, affecting sensor performance. Here, we study the capture rate and translocation of 1-5 kbp double-stranded DNA molecules through solid-state nanopores in the presence of tangential fluid flow over the nanopore aperture. Experiments reveal a trend of increased capture rate with cross-flow, reaching a 5-fold enhancement (dependent on DNA length) at moderate flow rates, before decreasing at higher flow rates. By modeling DNA dynamics in microchannels under the combined effect of laminar flow, Brownian motion and electrophoretic drift, it is shown that the observed trend is the result of two competing mechanisms: enhanced DNA transport by convection and reduction in the nanopore's capture volume with increased flow velocity. Moreover, it is shown that the viscous drag force exerted by flow on a translocating DNA can be exploited to tune the kinetics of DNA translocation.

A vacuum-actuated microtissue stretcher for long-term exposure to oscillatory strain within a 3D matrix.

Although our understanding of cellular behavior in response to extracellular biological and mechanical stimuli has greatly advanced using conventional 2D cell culture methods, these techniques lack physiological relevance. To a cell, the extracellular environment of a 2D plastic petri dish is artificially flat, extremely rigid, static and void of matrix protein. In contrast, we developed the microtissue vacuum-actuated stretcher (MVAS) to probe cellular behavior within a 3D multicellular environment composed of innate matrix protein, and in response to continuous uniaxial stretch. An array format, compatibility with live imaging and high-throughput fabrication techniques make the MVAS highly suited for biomedical research and pharmaceutical discovery. We validated our approach by characterizing the bulk microtissue strain, the microtissue strain field and single cell strain, and by assessing F-actin expression in response to chronic cyclic strain of 10%. The MVAS was shown to be capable of delivering reproducible dynamic bulk strain amplitudes up to 13%. The strain at the single cell level was found to be 10.4% less than the microtissue axial strain due to cellular rotation. Chronic cyclic strain produced a 35% increase in F-actin expression consistent with cytoskeletal reinforcement previously observed in 2D cell culture. The MVAS may further our understanding of the reciprocity shared between cells and their environment, which is critical to meaningful biomedical research and successful therapeutic approaches.

Manipulating Electrical and Fluidic Access in Integrated Nanopore-Microfluidic Arrays Using Microvalves.

On-chip microvalves regulate electrical and fluidic access to an array of nanopores integrated within microfluidic networks. This configuration allows for on-chip sequestration of biomolecular samples in various flow channels and analysis by independent nanopores.

Polycystin deficiency induces dopamine-reversible alterations in flow-mediated dilatation and vascular nitric oxide release in humans.

Autosomal dominant polycystic kidney disease (ADPKD) is a renal hereditary disorder associated with increased cardiovascular mortality, due to mutations in polycystin-1 and polycystin-2 genes. Endothelial polycystin-deficient cells have an altered mechanosensitivity to fluid shear stress and subsequent deficit in calcium-induced nitric oxide release, prevented by dopamine receptor stimulation. However, the impact of polycystin deficiency on endothelial function in ADPKD patients is still largely unknown. Here we assessed endothelium-dependent flow-mediated dilatation in 21 normotensive ADPKD patients and 21 healthy control subjects, during sustained (hand skin heating) and transient (postischemic hyperemia) flow stimulation. Flow-mediated dilatation was less marked in ADPKD patients than in controls during heating, but it was similar during postischemic hyperemia. There was no difference in endothelium-independent dilatation in response to glyceryl trinitrate. Local plasma nitrite, an indicator of nitric oxide availability, increased during heating in controls but not in patients. Brachial infusion of dopamine in a subset of ADPKD patients stimulated plasma nitrite increase during heating and improved flow-mediated dilatation. Thus, ADPKD patients display a loss of nitric oxide release and an associated reduction in endothelium-dependent dilatation of conduit arteries during sustained blood flow increase. The correction of these anomalies by dopamine suggests future therapeutic strategies that could reduce the occurrence of cardiovascular events in ADPKD.

Light chain deposition disease and proximal tubulopathy in two successive kidney allografts.

Light chain proximal tubulopathy (LCPT) is a rare kidney disease associated with plasma cell dyscrasias, characterized by light chain deposits in the proximal tubular cells, with or without crystal formation. We describe an exceptional case of LCPT without crystal formation in a kidney allograft, in a patient who underwent two renal transplants for a light chain deposition disease (LCDD) complicating smoldering myeloma. This is the first description of this association in successive kidney allografts. We concisely describe pathology of LCDD and LCPT and discuss potential pathophysiological mechanisms relating these two conditions.

High-efficiency on-line haemodiafiltration improves conduit artery endothelial function compared with high-flux haemodialysis in end-stage renal disease patients.

BACKGROUND: Middle molecular weight uraemic toxins are considered to play an important role in vascular dysfunction and cardiovascular outcomes in end-stage renal disease (ESRD) patients. Recent dialysis techniques based on convection, specifically high-efficiency on-line haemodiafiltration (HDF), enhance the removal of middle molecular weight toxins and reduce all-cause mortality in haemodialysis (HD) patients. However, the mechanisms of these improved outcomes remain to be established. METHODS: This prospective study randomly assigned 42 ESRD patients to switch from high-flux HD to high-efficiency on-line HDF (n=22) or to continue HD (n=20). Brachial artery endothelium-dependent flow-mediated dilatation, central pulse pressure, carotid artery intima-media thickness (IMT), internal diastolic diameter and distensibility and circulating markers of uraemia, inflammation and oxidative stress were blindly assessed before and after a 4-month follow-up. RESULTS: Brachial flow-mediated dilatation and carotid artery distensibility increased significantly in the HDF group compared with HD, while carotid IMT and diameter remained similar. HDF decreased predialysis levels of the uraemic toxins ß2-microglobulin, phosphate and blood TNFα mRNA expression. Oxidative stress markers were not different between the HD and HDF groups. Blood mRNA expression of protein kinase C ß2, an endothelial NO-synthase (eNOS) inhibitor, decreased significantly with HDF. CONCLUSIONS: High-efficiency on-line HDF prevents the endothelial dysfunction and stiffening of the conduit arteries in ESRD patients compared with high-flux HD. HDF decreases uraemic toxins, vascular inflammation, and is associated with subsequent improvement in eNOS functionality. These results suggest that reduced endothelial dysfunction may be an intermediate mechanism explaining the beneficial outcomes associated with HDF.

A microscale anisotropic biaxial cell stretching device for applications in mechanobiology.

A multi-layered polydimethylsiloxane microfluidic device with an integrated suspended membrane has been fabricated that allows dynamic and multi-axial mechanical deformation and simultaneous live-cell microscopy imaging. The transparent membrane's strain field can be controlled independently along two orthogonal directions. Human foreskin fibroblasts were immobilized on the membrane's surface and stretched along two orthogonal directions sequentially while performing live-cell imaging. Cyclic deformation of the cells induced a reversible reorientation perpendicular to the direction of the applied strain. Cells remained viable in the microdevice for several days. As opposed to existing microfluidic or macroscale stretching devices, this device can impose changing, anisotropic and time-varying strain fields in order to more closely mimic the complexities of strains occurring in vivo.

Field-flow fractionation and hydrodynamic chromatography on a microfluidic chip.

We present gravitational field-flow fractionation and hydrodynamic chromatography of colloids eluting through 18 µm microchannels. Using video microscopy and mesoscopic simulations, we investigate the average retention ratio of colloids with both a large specific weight and neutral buoyancy. We consider the entire range of colloid sizes, including particles that barely fit in the microchannel and nanoscopic particles. Ideal theory predicts four operational modes, from hydrodynamic chromatography to Faxén-mode field-flow fractionation. We experimentally demonstrate, for the first time, the existence of the Faxén-mode field-flow fractionation and the transition from hydrodynamic chromatography to normal-mode field-flow fractionation. Furthermore, video microscopy and simulations show that the retention ratios are largely reduced above the steric-inversion point, causing the variation of the retention ratio in the steric- and Faxén-mode regimes to be suppressed due to increased drag. We demonstrate that theory can accurately predict retention ratios if hydrodynamic interactions with the microchannel walls (wall drag) are added to the ideal theory. Rather than limiting the applicability, these effects allow the microfluidic channel size to be tuned to ensure high selectivity. Our findings indicate that particle velocimetry methods must account for the wall-induced lag when determining flow rates in highly confining systems.

Using buoyant mass to measure the growth of single cells.

We used a suspended microchannel resonator (SMR) combined with picoliter-scale microfluidic control to measure buoyant mass and determine the 'instantaneous' growth rates of individual cells. The SMR measures mass with femtogram precision, allowing rapid determination of the growth rate in a fraction of a complete cell cycle. We found that for individual cells of Bacillus subtilis, Escherichia coli, Saccharomyces cerevisiae and mouse lymphoblasts, heavier cells grew faster than lighter cells.

Hollow core photonic crystal fiber as a reusable Raman biosensor.

We report that a single hollow core photonic crystal fiber (HC-PCF) can be used for repetitive characterization of multiple samples by Raman spectroscopy. This was achieved by integrating the HC-PCF to a differential pressure system that allowed effective filling, draining and re-filling of samples into a HC-PCF under identical optical conditions. Consequently, high-quality and reliable spectral data could be obtained which were suitable for multivariate analysis (partial least squares). With the present scheme, we were able to accurately predict different concentrations of heparin and adenosine in serum. Thus the detection scheme as presented here paves a path for the inclusion of HC-PCFs in point-of-care technologies and environmental monitoring where rapid sample characterization is of utmost importance.

Kidney graft dysfunction in simultaneous pancreas-kidney recipients after pancreas failure: analysis of early and late protocol biopsies.

BACKGROUND: Kidney graft survival in simultaneous pancreas-kidney (SPK) recipients is known to decrease after pancreas graft failure. METHODS: Sixty-three consecutive SPK recipients were retrospectively reviewed. Kidney graft function and proteinuria were evaluated at three months after the transplantation and at last follow-up. Histopathologic findings of protocol biopsies performed three months and one yr after transplantation were analyzed. RESULTS: Twelve patients lost the pancreas graft. Donors' characteristics were similar in patients with or without pancreas failure. After a median follow-up of 36 months, mean eGFR with a functional pancreas was 69.5 mL/min/1.73 m² vs. 56.3 mL/min/1.73 m² (p = 0.01) after pancreas loss. Patients who lost pancreas had a median proteinuria of 0.28 g vs. 0.13 g per 24 h (p = 0.02). Analysis of three-month protocol biopsies revealed more frequent isolated glomerulitis after pancreas failure (p = 0.0001), without peritubular capillaritis or C4d deposition. No donor-specific anti-HLA antibodies were detectable in these patients. Chronic tubulointerstitial changes were more frequent in patients with pancreas loss. There was no evidence of diabetic nephropathy recurrence. CONCLUSION: SPK recipients develop an early kidney graft dysfunction after pancreas failure. Histopathologic findings revealed frequent glomerulitis without antibody-mediated rejection and early chronic changes.

Weighing of biomolecules, single cells and single nanoparticles in fluid.

Nanomechanical resonators enable the measurement of mass with extraordinary sensitivity. Previously, samples as light as 7 zeptograms (1 zg = 10(-21) g) have been weighed in vacuum, and proton-level resolution seems to be within reach. Resolving small mass changes requires the resonator to be light and to ring at a very pure tone-that is, with a high quality factor. In solution, viscosity severely degrades both of these characteristics, thus preventing many applications in nanotechnology and the life sciences where fluid is required. Although the resonant structure can be designed to minimize viscous loss, resolution is still substantially degraded when compared to measurements made in air or vacuum. An entirely different approach eliminates viscous damping by placing the solution inside a hollow resonator that is surrounded by vacuum. Here we demonstrate that suspended microchannel resonators can weigh single nanoparticles, single bacterial cells and sub-monolayers of adsorbed proteins in water with sub-femtogram resolution (1 Hz bandwidth). Central to these results is our observation that viscous loss due to the fluid is negligible compared to the intrinsic damping of our silicon crystal resonator. The combination of the low resonator mass (100 ng) and high quality factor (15,000) enables an improvement in mass resolution of six orders of magnitude over a high-end commercial quartz crystal microbalance. This gives access to intriguing applications, such as mass-based flow cytometry, the direct detection of pathogens, or the non-optical sizing and mass density measurement of colloidal particles.

Monitoring of hemodialysis quality-of-care indicators: why is it important?

BACKGROUND: Meeting specific guideline targets is associated with improved survival rates and reduced hospitalizations in the dialysis population. This prospective work evaluated the adequacy of hemodialysis quality indicators in an in-center hemodialysis population with severe comorbidities, and assessed whether clinical practice could impact intermediate outcomes. METHODS: All the chronic hemodialysis patients treated in Rouen University Hospital hemodialysis Unit between January 2009 and April 2010 were included in this observational study. Every quarter, mean levels and prevalence of conformity were collected for the following indicators: anemia, dialysis dose, serum calcium and phosphorus, PTH, 25OH-vitamin D, albumin, serum bicarbonate, LDL-cholesterol, serum ß2-microglobulin, systolic and diastolic blood pressure, intradialytic hypotension and vascular access. Conformity of quality-of-care indicators was determined according to targets defined by international guidelines, whenever available. RESULTS: Altogether, 124 patients were included in the study. Thirty-three patients were evaluated during the entire follow-up period. An improvement in the percentage of conformity was observed for hemoglobin, dialysis dose, phosphates, PTH, serum bicarbonate and ß2-microglobulin in the global population. Failure to improve conformity rates for several indicators, including serum albumin, was found, possibly depending on patients' comorbidities rather than on quality of care. CONCLUSION: Overall, this study shows that following quality-of-care indicators can improve clinical practice by identifying center-specific weaknesses, prompting the establishment of corrective measures. Finally, we suggest that the definition and targets of some indicators, especially hypertension and LDL-cholesterol, be reviewed, since evidence of their association with mortality is not demonstrated.

Recurrent membranous nephropathy in an allograft caused by IgG3κ targeting the PLA2 receptor.

Up to 80% of patients with idiopathic membranous nephropathy have non-complement-fixing IgG4 autoantibodies to the phospholipase A2 receptor (PLA2R). Membranous nephropathy recurs in approximately 40% of patients after kidney transplantation, but the mechanism is unknown. Here, we describe a patient with recurrent membranous nephropathy 13 days after kidney transplantation whose graft biopsy specimen showed granular staining for C3, C5b-9, C1q, and IgG3κ; electron microscopy revealed subepithelial nonorganized deposits. A search for hematologic disorders was negative. Retrospective evaluation of a biopsy sample from the native kidney revealed a similar pattern: monotypic IgG3κ deposits together with C3, C1q, and C5b-9. Glomerular deposits contained PLA2R in both the graft and the native kidney, suggesting that the recurrence was the result of circulating anti-PLA2R antibodies binding to PLA2R antigen expressed on donor podocytes. Confocal analysis of anti-PLA2R and antihuman IgG3 showed co-localization, and the patient had IgG3κ-restricted circulating anti-PLA2R antibodies. Treatment with rituximab stabilized both proteinuria and serum creatinine, and circulating anti-PLA2R became undetectable. In summary, this case of recurrent membranous nephropathy in a graft suggests that circulating monoclonal anti-PLA2R IgG3κ caused the disease and activated complement by the classic pathway.

Encapsulating therapeutic cells in RGD-modified agarose microcapsules.

Current cell-based strategies for repairing damaged tissue often show limited efficacy due to low cell retention at the site of injury. Encapsulation of cells within hydrogel microcapsules demonstrably increases cell retention but benefits can be limited due to premature cell escape from the hydrogel microcapsules and subsequent clearance from the targeted tissue. We propose a method of encapsulating cells in agarose microcapsules that have been modified to increase cell retention by providing cell attachment domains within the agarose hydrogel allowing cells to adhere to the microcapsules. We covalently modified agarose with the addition of the cell adhesion peptide, RGD (arginine, glycine, aspartic acid). We then used a microfluidic platform to encapsulate single cells within 50 µm agarose microcapsules. We tracked encapsulated cells for cell viability, egress from microcapsules and attachment to microcapsules at 2 h, 24 h, and 48 h after encapsulation. Many encapsulated cells eventually egress their microcapsule. Those that were encapsulated using RGD-modified agarose adhered to the outer surface of the microcapsule following egress. NIH 3T3 cells showed nearly 45% of egressed cells attached to the outside of RGD modified agarose microcapsules, while minimal cellular adhesion was observed when using unmodified agarose. Similarly, human umbilical vein endothelial cells had up to 33% of egressed cells attached and explant-derived cardiac cells showed up to 20% attachment with the presence of RGD binding domains within the agarose microcapsules.

Targeting extracellular vesicle delivery to the lungs by microgel encapsulation.

Extracellular vesicles (EVs) secreted by stem and progenitor cells have significant potential as cell-free 'cellular' therapeutics. Yet, small EVs (<200 nm) are rapidly cleared after systemic administration, mainly by the liver, presenting challenges targeting EVs to a specific organ or tissue. Microencapsulation using natural nano-porous hydrogels (microgels) has been shown to enhance engraftment and increase the survival of transplanted cells. We sought to encapsulate EVs within microgels to target their delivery to the lung by virtue of their size-based retention within the pulmonary microcirculation. Mesenchymal stromal cell (MSC) derived EVs were labelled with the lipophilic dye (DiR) and encapsulated within agarose-gelatin microgels. Endothelial cells and bone marrow derived macrophages were able to take up EVs encapsulated in microgels in vitro, but less efficiently than the uptake of free EVs. Following intrajugular administration, microgel encapsulated EVs were selectively retained within the lungs for 72h, while free EVs were rapidly cleared by the liver. Furthermore, microgel-loaded EVs demonstrated greater uptake by lung cells, in particular CD45+ immune cells, as assessed by flow cytometry compared to free EVs. Microencapsulation of EVs may be a novel tool for enhancing the targeted delivery of EVs for future therapeutic applications.

Transcutaneous oxymetry as predictive test of peripheral vascular revascularization in haemodialysis population.

BACKGROUND: Peripheral arterial disease (PAD) occurs frequently among haemodialysis patients but it is underestimated. Vascular treatment and amputations are more frequent in end stage renal disease (ESRD) population compared to the general population possibly because of a diagnosis of PAD delayed. Transcutaneous oxymetry (TcPO2) is commonly used in vascular medicine to reflect local arterial blood flow and skin oxygenation.The aim of this study was to assess the accuracy of the TcPO2 measurements to screen PAD and to predict vascular outcomes in haemodialysis population. METHODS: In a 1-year prospective study, the value of TcPO2 was assessed in a cohort of 48 patients when starting haemodialysis. RESULTS: Twenty one patients had at least one vascular stenosis (42%) on Doppler examination and were considered as affected by PAD. At inclusion a pathologic resting TcPO2 (<40mmHg) was found in 13 patients (29%). A severe ischemia (TcPO2 <30mmHg) was noted in 8 patients (16.7%) and a critical limb ischemia (TcPO2 <10mmHg) in 3 patients.(6%). Eleven (25.5%) and 6 patients (15%) had a TcPO2 <40mmHg at 6 and 12 months respectively. During the follow-up, death was seven times more frequent in patients with abnormal TcPO2 at T0 compared to patients with normal TcPO2 (38% vs 5.7%; p = 0.04). Revascularization (n = 6) or amputation (n = 5) were required for 5 patients. TcPO2 was pathologic in all patients and legs requiring a vascular treatment. The sensitivity, specificity, positive predictive value and negative predictive value were 100%, 85.2%, 38% and 100% respectively. CONCLUSIONS: This study confirms the underestimated PAD diagnosis and the severity of PAD in haemodialysis population. A TcPO2 less than 40mmHg at the onset of the haemodialysis could identify patients at high risk of death and patients requiring vascular treatment. Moreover, since haemodialysis seems to be an accelerating factor of atherosclerosis, TcPO2 might be perform as a complement to traditional vascular explorations to assess the distal vascular conditions of limbs of haemodialysis patients.

Spectrum of mutations in Gitelman syndrome.

Gitelman's syndrome (GS) is a rare, autosomal recessive, salt-losing tubulopathy caused by mutations in the SLC12A3 gene, which encodes the thiazide-sensitive NaCl cotransporter (NCC). Because 18 to 40% of suspected GS patients carry only one SLC12A3 mutant allele, large genomic rearrangements may account for unidentified mutations. Here, we directly sequenced genomic DNA from a large cohort of 448 unrelated patients suspected of having GS. We found 172 distinct mutations, of which 100 were unreported previously. In 315 patients (70%), we identified two mutations; in 81 patients (18%), we identified one; and in 52 patients (12%), we did not detect a mutation. In 88 patients, we performed a search for large rearrangements by multiplex ligation-dependent probe amplification (MLPA) and found nine deletions and two duplications in 24 of the 51 heterozygous patients. A second technique confirmed each rearrangement. Based on the breakpoints of seven deletions, nonallelic homologous recombination by Alu sequences and nonhomologous end-joining probably favor these intragenic deletions. In summary, missense mutations account for approximately 59% of the mutations in Gitelman's syndrome, and there is a predisposition to large rearrangements (6% of our cases) caused by the presence of repeated sequences within the SLC12A3 gene.

Association of PKD2 (polycystin 2) mutations with left-right laterality defects.

Mutations in the PKD1 (polycystin 1) and PKD2 (polycystin 2) genes cause autosomal dominant polycystic kidney disease (ADPKD). Most Pkd2-null mouse embryos present with left-right laterality defects. For the first time, we report the association of ADPKD resulting from a mutation in PKD2 and left-right asymmetry defects. PKD1 and PKD2 were screened for mutations or large genomic rearrangements in 3 unrelated patients with ADPKD presenting with laterality defects: dextrocardia in one and situs inversus totalis in 2 others. A large gene deletion, a single-exon duplication, and an in-frame duplication respectively, were found in the 3 patients. These polymorphisms were found in all tested relatives with ADPKD, but were absent in unaffected related individuals. No left-right anomalies were found in other members of the 3 families. A possible association between heterotaxia and a PKD2 mutation in our 3 patients is suggested by: (1) the existence of laterality defects in Pkd2-null mouse and zebrafish models and (2) detection of a pathogenic PKD2 mutation in the 3 probands, although PKD2 mutations account for only 15% of ADPKD families. The presence of left-right laterality defects should be systematically screened in larger cohorts of patients with ADPKD harboring PKD2 mutations.

Acute renal failure and Fanconi syndrome due to deferasirox.

Deferasirox is the first oral iron chelator and, as such, is widely used for the treatment of chronic iron overload. However, recent data from large studies confirmed the renal toxicity of deferasirox. We report a case of Fanconi syndrome associated with acute renal failure in a patient receiving deferasirox. In particular, new insights regarding the pathophysiology of the renal disease due to this treatment are discussed. This case highlights the importance of a careful monitoring of kidney function, markers of proximal tubulopathy and ferritinaemia in patients receiving deferasirox.