Cytomorphic Electronics With Memristors for Modeling Fundamental Genetic Circuits.

Cytomorphic engineering attempts to study the cellular behavior of biological systems using electronics. As such, it can be considered analogous to the study of neurobiological concepts for neuromorphic engineering applications. To date, digital and analog translinear electronics have commonly been used in the design of cytomorphic circuits; Such circuits could greatly benefit from lowering the area of the digital memory via memristive circuits. In this article, we propose a novel approach that utilizes the Boltzmann-exponential stochastic transport of ionic species through insulators to naturally model the nonlinear and stochastic behavior of biochemical reactions. We first show that two-terminal memristive devices can capture the non-linear and stochastic behavior of biochemical reactions. Then, we present the design of several building blocks based on analog memristive circuits that inherently model the biophysical mechanisms of gene expression. The circuits model induction by small molecules, activation and repression by transcription factors, biological promoters, cooperative binding, and transcriptional and translational regulation of gene expression. Finally, we utilize the building blocks to form complex mixed-signal networks that can simulate the delay-induced oscillator and the p53-mdm2 interaction in the cancer signaling pathway. Our approach can provide a fast and simple emulative framework for studying genetic circuits and arbitrary large-scale biological networks in systems and synthetic biology. Some challenges may be that memristive devices with frequent learning and programming do not have the same longevity as traditional transistor-based electron-transport devices, and operate with significantly slower time constants, which can limit emulation speed.

Ligation of the intersphincteric fistula tract procedure and its modifications / Procedimento de ligadura de trato de fístula interesfincteriana e suas modificações

ABSTRACT Purpose: Treatment of anal fistulae is regarded as a challenge due to the diverse nature of this disease and its countless complications. Ligation of the intersphincteric fistula tract procedure and its modifications have been popularized among many surgeons worldwide due to their simplicity and promising outcomes. The main purpose of this article was to conduct a comprehensives review of the published literature on ligation of the intersphincteric fistula tract procedure and its modifications. Method: PubMed, the Cochrane database and Ovid were searched from January 2007 to June 2017. Fully published peer-reviewed studies which applied ligation of the intersphincteric fistula tract procedure and its modifications for the treatment of anal fistulae of cryptogenic origin with follow-up of median 12 months were eligible. Uncompleted studies, case reports, reviews, abstracts, letters, short communication, comments, and studies which did not fulfill inclusion criteria were excluded. The primary outcome was to measure primary healing, overall healing, failure, and recurrence of ligation of the intersphincteric fistula tract procedure and its modifications. Results: Twenty-two studies were identified with only ten studies meeting criteria of inclusion. Original ligation of the intersphincteric fistula tract was performed in five studies with a population of 199 patients while the remaining five studies showed four different modifications of the ligation of the intersphincteric fistula tract with a total number of 147 patients. Both original LIFT and its modifications have promising as well as potentially similar outcomes; primary healing in the original ligation of the intersphincteric fistula tract (73.95%) (95% CI 60.3-85.6) performed less than the modifications (82.3%) (95% CI 64.8-94.7). Overall healing in the original ligation of the intersphincteric fistula tract (78.9%) (95% CI 58.5-93.7) performed relatively less than in the modifications (93.6%) (95% CI 81.4-99.6). Failure in the original ligation of the intersphincteric fistula tract (17.9%) (95% CI 4.9-36.5) performed almost the same as the modifications (17.7%) (95% CI 5.3-35.2). Recurrence in the original ligation of the intersphincteric fistula tract was 9.7% (95% CI 1.7-23.2). However, there was no recurrence in the modifications. Conclusion: Ligation of the intersphincteric fistula tract and its modifications are effective and simple procedures in treating simple anal fistulae, especially high transsphincteric ones. However, more trials should be performed to evaluate its effectiveness regarding complex fistulae.

RESUMO Objetivo: O tratamento de fístulas anais é considerado um desafio devido à natureza diversa dessa doença e suas incontáveis complicações. O procedimento de ligadura do trato da fístula interesfincteriana e suas modificações foi popularizado entre cirurgiões em todo o mundo devido a sua simplicidade e desfechos promissores. O principal objetivo deste artigo foi conduzir uma revisão abrangente da literatura publicada sobre o procedimento de ligadura do trato da fístula interesfincteriana e suas modificações. Método: as bases de dados PubMed, Cochrane e Ovid foram pesquisadas de janeiro de 2007 a junho de 2017. Estudos publicados com revisão por pares que aplicaram o procedimento de ligadura do trato da fístula interesfincteriana e suas modificações para o tratamento de fístulas anais de origem criptogênica com acompanhamento de mediana de 12 meses foram elegíveis. Estudos incompletos, relatos de casos, revisões, resumos, cartas, comunicação breve, comentários e estudos que não preenchiam os critérios de inclusão foram excluídos. O desfecho primário foi medir a cicatrização primária, a cicatrização geral, falhas e recorrência do procedimento de ligadura do trato da fístula interesfincteriana e suas modificações. Resultados: Vinte e dois estudos foram identificados com apenas dez estudos atendendo aos critérios de inclusão. A ligadura original do trato da fístula interesfincteriana foi realizada em cinco estudos com uma população de 199 pacientes, enquanto os cinco estudos restantes apresentaram quatro modificações diferentes da ligadura do trato da fístula interesfincteriana com um total de 147 pacientes. Tanto o LIFT original quanto suas modificações têm resultados promissores e desfechos potencialmente semelhantes; cicatrização primária na ligadura original do trato da fístula interesfincteriana de 73,95% (IC 95% 60,3-85,6) menos realizada que as modificações de 82,3% (IC 95% 64,8-94,7). Cicatrização geral na ligadura original do trato da fístula interesfincteriana de 78,9% (IC 95% 58,5-93,7) realizada relativamente menos do que as modificações (93,6%, IC 95% 81,4-99,6). A falha na ligadura original do trato da fístula interesfincteriana (17,9%; IC 95% 4,9-36,5) realizada quase tanto quanto as modificações (17,7%; IC 95% 5,3-35,2). Recidiva na ligadura original do trato da fístula interesfincteriana em 9,7% (IC 95% 1,7-23,2). No entanto, não houve recorrência nas modificações. Conclusão: A ligadura do trato da fístula interesfincteriana e suas modificações são procedimentos eficazes e simples no tratamento de fístulas anais simples, especialmente as transesfincterianas altas. No entanto, mais estudos devem ser realizados para avaliar sua eficácia em relação às fístulas complexas.

Altered arterial baroreflex-muscle metaboreflex interaction in heart failure.

Two powerful reflexes controlling cardiovascular function during exercise are the muscle metaboreflex and arterial baroreflex. In heart failure (HF), the strength and mechanisms of these reflexes are altered. Muscle metaboreflex activation (MMA) in normal subjects increases mean arterial pressure (MAP) primarily via increases in cardiac output (CO), whereas in HF the mechanism shifts to peripheral vasoconstriction. Baroreceptor unloading increases MAP via peripheral vasoconstriction, and this pressor response is blunted in HF. Baroreceptor unloading during MMA in normal animals elicits an enormous pressor response via combined increases in CO and peripheral vasoconstriction. The mode of interaction between these reflexes is intimately dependent on the parameter (e.g., MAP and CO) being investigated. The interaction between the two reflexes when activated simultaneously during dynamic exercise in HF is unknown. We activated the muscle metaboreflex in chronically instrumented dogs during mild exercise (via graded reductions in hindlimb blood flow) followed by baroreceptor unloading [via bilateral carotid occlusion (BCO)] before and after induction of HF. We hypothesized that BCO during MMA in HF would cause a smaller increase in MAP and a larger vasoconstriction of ischemic hindlimb vasculature, which would attenuate the restoration of blood flow to ischemic muscle observed in normal dogs. We observed that BCO during MMA in HF increases MAP by substantial vasoconstriction of all vascular beds, including ischemic active muscle, and that all cardiovascular responses, except ventricular function, exhibit occlusive interaction. We conclude that vasoconstriction of ischemic active skeletal muscle in response to baroreceptor unloading during MMA attenuates restoration of hindlimb blood flow. NEW & NOTEWORTHY We found that baroreceptor unloading during the muscle metaboreflex in heart failure results in occlusive interaction (except for ventricular function) with significant vasoconstriction of all vascular beds. In addition, restoration of blood flow to ischemic active muscle, via preferentially larger vasoconstriction of nonischemic beds, is significantly attenuated in heart failure.

In vitro osteoblastic differentiation of mesenchymal stem cells generates cell layers with distinct properties.

BACKGROUND: Differentiation of mesenchymal stem cells to osteoblasts is widely performed in research laboratories. Classical tests to prove this differentiation employ procedures such as cell fixation, cell lysis or cell scraping. Very few studies report gentle dissociation of mesenchymal stem cells undergoing an osteodifferentiation process. Here we used this technique to reveal the presence of several cell layers during osteogenesis and to study their different properties. METHODS: Through the sequential enzymatic detachment of the cells, we confirm the presence of several layers of differentiated cells and we compare them in terms of enzymatic sensitivity for dissociation, expression of cluster of differentiation, cytosolic calcium oscillations and osteogenic potential. Adipogenic and neurogenic differentiations were also performed in order to compare the cell layers. RESULTS: The cells undergoing differentiation formed one layer in the neurogenic differentiation, two layers in the adipogenic differentiation and at least four layers in the osteogenic differentiation. In the latter, the upper layers, maintained by a collagen I extracellular matrix, can be dissociated using collagenase I, while the remaining lowest layer, attached to the bottom of the dish, is sensitive only to trypsin-versene. The action of collagenase I is more efficient before the mineralization of the extracellular matrix. The collagenase-sensitive and trypsin-sensitive layers differ in their cluster of differentiation expression. The dissociation of the cells on day 15 reveals that cells could resume their growth (increase in cell number) and rapidly differentiate again in osteoblasts, in 2 weeks (instead of 4 weeks). Cells from the upper layers displayed a higher mineralization. CONCLUSIONS: MSCs undergoing osteogenic differentiation form several layers with distinct osteogenic properties. This could allow the investigators to use upper layers to rapidly produce differentiated osteoblasts and the lowest layer to continue growth and differentiation until an ulterior dissociation.

Muscle metaboreflex-induced vasoconstriction in the ischemic active muscle is exaggerated in heart failure.

When oxygen delivery to active muscle is insufficient to meet the metabolic demand during exercise, metabolites accumulate and stimulate skeletal muscle afferents, inducing a reflex increase in blood pressure, termed the muscle metaboreflex. In healthy individuals, muscle metaboreflex activation (MMA) during submaximal exercise increases arterial pressure primarily via an increase in cardiac output (CO), as little peripheral vasoconstriction occurs. This increase in CO partially restores blood flow to ischemic muscle. However, we recently demonstrated that MMA induces sympathetic vasoconstriction in ischemic active muscle, limiting the ability of the metaboreflex to restore blood flow. In heart failure (HF), increases in CO are limited, and metaboreflex-induced pressor responses occur predominantly via peripheral vasoconstriction. In the present study, we tested the hypothesis that vasoconstriction of ischemic active muscle is exaggerated in HF. Changes in hindlimb vascular resistance [femoral arterial pressure ÷ hindlimb blood flow (HLBF)] were observed during MMA (via graded reductions in HLBF) during mild exercise with and without α1-adrenergic blockade (prazosin, 50 µg/kg) before and after induction of HF. In normal animals, initial HLBF reductions caused metabolic vasodilation, while reductions below the metaboreflex threshold elicited reflex vasoconstriction, in ischemic active skeletal muscle, which was abolished after α1-adrenergic blockade. Metaboreflex-induced vasoconstriction of ischemic active muscle was exaggerated after induction of HF. This heightened vasoconstriction impairs the ability of the metaboreflex to restore blood flow to ischemic muscle in HF and may contribute to the exercise intolerance observed in these patients. We conclude that sympathetically mediated vasoconstriction of ischemic active muscle during MMA is exaggerated in HF. NEW & NOTEWORTHY We found that muscle metaboreflex-induced vasoconstriction of the ischemic active skeletal muscle from which the reflex originates is exaggerated in heart failure. This results in heightened metaboreflex activation, which further amplifies the reflex-induced vasoconstriction of the ischemic active skeletal muscle and contributes to exercise intolerance in patients.

Electropermeabilization of Inner and Outer Cell Membranes with Microsecond Pulsed Electric Fields: Quantitative Study with Calcium Ions.

Microsecond pulsed electric fields (µsPEF) permeabilize the plasma membrane (PM) and are widely used in research, medicine and biotechnology. For internal membranes permeabilization, nanosecond pulsed electric fields (nsPEF) are applied but this technology is complex to use. Here we report that the endoplasmic reticulum (ER) membrane can also be electropermeabilized by one 100 µs pulse without affecting the cell viability. Indeed, using Ca2+ as a permeabilization marker, we observed cytosolic Ca2+ peaks in two different cell types after one 100 µs pulse in a medium without Ca2+. Thapsigargin abolished these Ca2+ peaks demonstrating that the calcium is released from the ER. Moreover, IP3R and RyR inhibitors did not modify these peaks showing that they are due to the electropermeabilization of the ER membrane and not to ER Ca2+ channels activation. Finally, the comparison of the two cell types suggests that the PM and the ER permeabilization thresholds are affected by the sizes of the cell and the ER. In conclusion, this study demonstrates that µsPEF, which are easier to control than nsPEF, can permeabilize internal membranes. Besides, µsPEF interaction with either the PM or ER, can be an efficient tool to modulate the cytosolic calcium concentration and study Ca2+ roles in cell physiology.

Role of endothelial nitric oxide in control of peripheral vascular conductance during muscle metaboreflex activation.

The muscle metaboreflex is a powerful pressor reflex induced by the activation of chemically sensitive muscle afferents as a result of metabolite accumulation. During submaximal dynamic exercise, the rise in arterial pressure is primarily due to increases in cardiac output, since there is little systemic vasoconstriction. Indeed, in normal animals, we have often shown a small, but significant, peripheral vasodilation during metaboreflex activation, which is mediated, at least in part, by release of epinephrine and activation of vascular ß2-receptors. We tested whether this vasodilation is in part due to increased release of nitric oxide caused by the rise in cardiac output eliciting endothelium-dependent flow-mediated vasodilation. The muscle metaboreflex was activated via graded reductions in hindlimb blood flow during mild exercise with and without nitric oxide synthesis blockade [NG-nitro-l-arginine methyl ester (l-NAME); 5 mg/kg]. We assessed the role of increased cardiac output in mediating peripheral vasodilation via the slope of the relationship between the rise in nonischemic vascular conductance (conductance of all vascular beds excluding hindlimbs) vs. the rise in cardiac output. l-NAME increased mean arterial pressure at rest and during exercise. The metaboreflex-induced increases in mean arterial pressure were unaltered by l-NAME, whereas the increases in cardiac output and nonischemic vascular conductance were attenuated. However, the slope of the relationship between nonischemic vascular conductance and cardiac output was not affected by l-NAME, indicating that the rise in cardiac output did not elicit vasodilation via increased release of nitric oxide. Thus, although nitric oxide is intrinsic to the vascular tonus, endothelial-dependent flow-mediated vasodilation plays little role in the small peripheral vasodilation observed during muscle metaboreflex activation.

Electrical control of calcium oscillations in mesenchymal stem cells using microsecond pulsed electric fields.

BACKGROUND: Human mesenchymal stem cells are promising tools for regenerative medicine due to their ability to differentiate into many cellular types such as osteocytes, chondrocytes and adipocytes amongst many other cell types. These cells present spontaneous calcium oscillations implicating calcium channels and pumps of the plasma membrane and the endoplasmic reticulum. These oscillations regulate many basic functions in the cell such as proliferation and differentiation. Therefore, the possibility to mimic or regulate these oscillations might be useful to regulate mesenchymal stem cells biological functions. METHODS: One or several electric pulses of 100 µs were used to induce Ca2+ spikes caused by the penetration of Ca2+ from the extracellular medium, through the transiently electropermeabilized plasma membrane, in human adipose mesenchymal stem cells from several donors. Attached cells were preloaded with Fluo-4 AM and exposed to the electric pulse(s) under the fluorescence microscope. Viability was also checked. RESULTS: According to the pulse(s) electric field amplitude, it is possible to generate a supplementary calcium spike with properties close to those of calcium spontaneous oscillations, or, on the contrary, to inhibit the spontaneous calcium oscillations for a very long time compared to the pulse duration. Through that inhibition of the oscillations, Ca2+ oscillations of desired amplitude and frequency could then be imposed on the cells using subsequent electric pulses. None of the pulses used here, even those with the highest amplitude, caused a loss of cell viability. CONCLUSIONS: An easy way to control Ca2+ oscillations in mesenchymal stem cells, through their cancellation or the addition of supplementary Ca2+ spikes, is reported here. Indeed, the direct link between the microsecond electric pulse(s) delivery and the occurrence/cancellation of cytosolic Ca2+ spikes allowed us to mimic and regulate the Ca2+ oscillations in these cells. Since microsecond electric pulse delivery constitutes a simple technology available in many laboratories, this new tool might be useful to further investigate the role of Ca2+ in human mesenchymal stem cells biological processes such as proliferation and differentiation.

Interaction between the muscle metaboreflex and the arterial baroreflex in control of arterial pressure and skeletal muscle blood flow.

The muscle metaboreflex and arterial baroreflex regulate arterial pressure through distinct mechanisms. During submaximal exercise muscle metaboreflex activation (MMA) elicits a pressor response virtually solely by increasing cardiac output (CO) while baroreceptor unloading increases mean arterial pressure (MAP) primarily through peripheral vasoconstriction. The interaction between the two reflexes when activated simultaneously has not been well established. We activated the muscle metaboreflex in chronically instrumented canines during dynamic exercise (via graded reductions in hindlimb blood flow; HLBF) followed by simultaneous baroreceptor unloading (via bilateral carotid occlusion; BCO). We hypothesized that simultaneous activation of both reflexes would result in an exacerbated pressor response owing to both an increase in CO and vasoconstriction. We observed that coactivation of muscle metaboreflex and arterial baroreflex resulted in additive interaction although the mechanisms for the pressor response were different. MMA increased MAP via increases in CO, heart rate (HR), and ventricular contractility whereas baroreflex unloading during MMA caused further increases in MAP via a large decrease in nonischemic vascular conductance (NIVC; conductance of all vascular beds except the hindlimb vasculature), indicating substantial peripheral vasoconstriction. Moreover, there was significant vasoconstriction within the ischemic muscle itself during coactivation of the two reflexes but the remaining vasculature vasoconstricted to a greater extent, thereby redirecting blood flow to the ischemic muscle. We conclude that baroreceptor unloading during MMA induces preferential peripheral vasoconstriction to improve blood flow to the ischemic active skeletal muscle.

Muscle metaboreflex activation during dynamic exercise vasoconstricts ischemic active skeletal muscle.

Metabolite accumulation due to ischemia of active skeletal muscle stimulates group III/IV chemosensitive afferents eliciting reflex increases in arterial blood pressure and sympathetic activity, termed the muscle metaboreflex. We and others have previously demonstrated sympathetically mediated vasoconstriction of coronary, renal, and forelimb vasculatures with muscle metaboreflex activation (MMA). Whether MMA elicits vasoconstriction of the ischemic muscle from which it originates is unknown. We hypothesized that the vasodilation in active skeletal muscle with imposed ischemia becomes progressively restrained by the increasing sympathetic vasoconstriction during MMA. We activated the metaboreflex during mild dynamic exercise in chronically instrumented canines via graded reductions in hindlimb blood flow (HLBF) before and after α1-adrenergic blockade [prazosin (50 µg/kg)], ß-adrenergic blockade [propranolol (2 mg/kg)], and α1 + ß-blockade. Hindlimb resistance was calculated as femoral arterial pressure/HLBF. During mild exercise, HLBF must be reduced below a threshold level before the reflex is activated. With initial reductions in HLBF, vasodilation occurred with the imposed ischemia. Once the muscle metaboreflex was elicited, hindlimb resistance increased. This increase in hindlimb resistance was abolished by α1-adrenergic blockade and exacerbated after ß-adrenergic blockade. We conclude that metaboreflex activation during submaximal dynamic exercise causes sympathetically mediated α-adrenergic vasoconstriction in ischemic skeletal muscle. This limits the ability of the reflex to improve blood flow to the muscle.