An In-Switch Architecture for Low-Latency Microservices

In recent time, there is has been a movement away from standard monolithic architecture in cloud and web services towards what is known as a microservice architecture. Microservice architecture decomposes the previous monolithic architecture into multiple independent services called "microservices". Examples of applications that use a microservice architecture include Netflix and Amazon. These applications typically send large numbers of microservice requests, which go through the OSI network layers to establish a client server connection. This trend towards microservices has developed interest by other researchers to make improvements in this field, due to the growing reliance importance on such architectures by consumers. There have been studies regarding the security of these microservices, performance analysis of various applications, and the use of these microservice applications in cloud technology. Any improvements in the speed, security, or organization of such network architecture would be very beneficial of these popular API's, and their user base. This project's objective is to investigate the potential of moving some of the processing that is done for these microservices within a network switch, and as a result the performance at the application level, by alleviating network communication. We formulate a high-level design for an in-switch architecture for low-latency microservice leveraging existing programmable-switches support. We investigate the implementation of NetCache as a microservice in our model and predict a significant latency reduction and subsequent performance increase

Health Insurance and Disclosure of Same-Sex Sexual Behaviors Among Gay and Bisexual Men in Same-Sex Relationships

Purpose: Gay and bisexual men (GBM) have poorer health outcomes than the general population. Improved health outcomes will require that GBM have access to healthcare and that healthcare providers are aware of their sexual behaviors. This study sought to examine factors associated with having health insurance and disclosure of same-sex sexual behaviors to primary care providers (PCPs) among GBM in primary same-sex relationships. Methods: We conducted an online survey of a national sample of 722 men in same-sex couples living in the United States. Logistic regression and multinomial regression models were conducted to assess whether characteristic differences existed between men who did and did not have health insurance, and between men who did and did not report that their PCP knew about their same-sex sexual activity. Results: Our national sample of same-sex partnered men identified themselves predominantly as gay and white, and most reported having an income and health insurance. Having health insurance and disclosing sexual behavior to PCPs was associated with increasing age, higher education, and higher income levels. Insurance was less prevalent among nonwhite participants and those living in the south and midwest United States. Disclosure of sexual behavior was more common in urban respondents and in the western United States. In 25% of couples, one partner was insured, while the other was not. Conclusions: Having health insurance and disclosing one's sexual behavior to PCPs was suboptimal overall and occurred in patterns likely to exacerbate health disparities among those GBM already more heavily burdened with poorer health outcomes. These factors need to be considered by PCPs and health policymakers to improve the health of GBM. Patient- and provider-targeted interventions could also improve the health outcomes of GBM.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140156/1/lgbt.2013.0050.pd

Novel biocatalytic modules for the cell-free conversion of cellodextrins to glucaric acid

Cell-free biocatalysis offers a versatile platform for the biomanufacturing of bulk or specialty chemicals due to the flexibility in assembling enzymes from different organisms in synthetic reaction pathways. Current challenges of this approach include costly enzyme preparation, low enzyme stability and efficient enzyme recycling. To overcome these challenges, we present a molecular toolbox that facilitates the simple construction of enzymes as low-cost and recyclable biocatalytic modules. The toolbox is composed of three interchangeable components: (i) inorganic matrices; (ii) matrix-specific solid-binding peptides (SBPs); and (iii) thermostable enzymes. SBPs are short amino acid sequences that can be fused genetically to proteins and direct the orientated immobilization of the resulting protein fusion onto solid matrices (1, 2). The biocatalytic module design relies on the affinity of the SBP for inorganic matrices. Single enzyme biocatalytic modules can be prepared easily consisting of one type of enzyme immobilized per matrix while a multiple enzyme biocatalytic module consists of multiple enzymes immobilized simultaneously onto the matrix. The modules can be combined rationally to generate product-specific reaction pathways and their subsequent removal from the reaction medium allows for a ‘pick, mix, and reuse’ approach, which can be optimized easily for low-cost cell-free biomanufacturing. Recently, we have shown that it is possible to assemble single and multiple enzyme biocatalytic modules using thermostable polysaccharide-degrading enzymes and that the enzymes retain their specific hydrolytic activities upon several rounds of recycling at high temperatures (2). Here, we applied the biocatalytic modules concept for the cell-free conversion of cellodextrins to glucaric acid, via a more complex seven enzyme synthetic pathway. Glucaric acid is one of the 12 top candidates for bio-based building blocks and is a precursor for polymers, including nylons and hyperbranched polyesters (3). Its bioproduction from cellodextrins, which can be derived from organic waste, provides a sustainable alternative to the fossil-derived production of polymers. Initially, single enzyme biocatalytic modules were prepared with a silica-specific SBP fused to two enzymes of the synthetic pathway allowing for their selective immobilization onto an inexpensive silica-based matrix. The SBP mediated the binding of each enzyme onto the matrix with over 85% immobilization efficiency. When comparing the enzyme activities of the biocatalytic modules against the free enzymes, 85 and 93% of their initial activities were retained upon immobilization, respectively. Furthermore, co-immobilization of these two enzymes as a multiple enzyme module resulted in similar immobilization yields. Performance of both enzymes in the multiple enzyme module in a successive reaction revealed that they retained 70% of their activity when compared to the free enzymes. Currently, the silica-specific SBP has been incorporated into other 5 enzymes of the pathway and we are proceeding with the construction of the single and multiple enzyme biocatalytic modules and pathway assembly. (1) Care A, Bergquist PL, Sunna A (2015) Trends in Biotechnology, 33: 259-268. (2) Care A, Petroll K, Gibson ESY, Bergquist PL, Sunna A (2017) Biotechnology for Biofuels 10:29. (3) Werpy T and G Petersen (2004). Results of Screening for Potential Candidates from Sugars and Synthesis Gas. National Renewable Energy Lab

Funktioneller und struktureller Nachweis einer ipsilateralen Verbindung zwischen dem dorsalen prämotorischen und dem primär motorischen Kortex

Der primär motorische Kortex (M1) steuert die Bewegungen der kontralateralen Körperseite. Der dorsale prämotorische Kortex (PMd) spielt eine wichtige Rolle bei der Abstimmung von Bewegungen auf äußere, vor allem somatosensorische Einflüsse sowie deren Integration in Bewegungsabläufe. Aus Studien an Primaten ist bekannt, dass zwischen dem ipsilateralen PMd und der Handregion des M1 direkte Verbindungen bestehen. Wie genau der PMd Einfluss auf den M1 im menschlichen Gehirn ausübt, wurde im Rahmen dieser Arbeit mit Hilfe der transkraniellen Magnetstimulation (TMS) nicht-invasiv untersucht. Hierfür kamen speziell entwickelte Präzisionsspulen (MAG & More) zur Anwendung, welche die Stimulation der dicht benachbarten Regionen erst möglich machten. Ein neuartiges Doppelpulsparadigma mit kurzen Latenzzeiten wurde entwickelt, um eine Interaktion mit den deszendierenden Wellen zu erreichen und so Auskunft über die zeitliche Diskrimination der Verbindung zu erhalten. Es wurden verschiedene Faktoren zur Änderung der kortikalen Erregbarkeit sowie ihr Einfluss auf die Verbindung der Areale untersucht: die Vorinnervation der Zielmuskeln in Experiment 2 sowie die Bearbeitung einer Wahl-Reaktionszeit-Aufgabe in Experiment 3. Durch die Diffusions-Tensor-Bildgebung (DTI) wurde die Mikrostruktur der subkortikalen weißen Substanz im Bereich des PMd dargestellt. Als Maß dafür diente die fraktionelle Anisotropie (FA). Im Rahmen der Doppelpulsstimulation wurde zunächst ein überschwelliger Puls über dem M1 appliziert, gefolgt von einer unterschwelligen Stimulation des PMd. In Experiment 1 erfolgte die Stimulation zunächst unter Ruhebedingungen. Die Pulse wurden in Interstimulusintervallen von 0,8 ms bis 2,0 ms in Schritten von 0,4 ms appliziert. Des Weiteren wurde in Experiment 2 unter sonst identischen Bedingungen der Einfluss einer willkürlichen Vorinnervation des Musculus interosseus dorsalis I untersucht. Im letzten Teilexperiment wurde 75, 100 und 125 ms vor der TMS- Applikation eine Wahl-Reaktionszeit-Aufgabe gestellt. Die Probanden wurden durch eine visuelle Anleitung dazu aufgefordert eine Reaktion mit der linken oder rechten Hand auszuführen. Die ISI betrugen 0,8, 1,2 und 1,6 ms. Die durch DTI erhobenen FA-Werte wurden schließlich mit den TMS-Daten korreliert. Durch die Doppelpulsstimulation unter Ruhebedingungen konnte eine signifikante Bahnung bei einem ISI von 1,2 ms festgestellt werden. Eine Vorspannung des Zielmuskels führte dazu, dass keine MEP mehr abzuleiten waren und bewirkte somit eine Modulation der Verbindung. Durch die Wahl-Reaktionszeit-Aufgabe konnte eine aufgabenspezifische Aktivierung bei einem ISI von 1,2 ms, einem SOA von 125 ms und einer Reaktion mit der rechten Hand erzielt werden. Mittels DTI gelang es das strukturelle Korrelat der Verbindung in der weißen Substanz unterhalb des PMd darzustellen (MNI-Koordinaten x, y, z = -15, -10, 55). Die Analyse ergab einen positiven linearen Zusammenhang zwischen der funktionellen Ausprägung der Verbindung und der Struktur der subkortikalen weißen Substanz. Die identifizierte Bahnung in Ruhe tritt nach einer kurzen Latenzzeit auf. Die Periodik dieser Bahnung entspricht dem Auftreten von I-Wellen und spricht dafür, dass die Signale vom PMd die Aktivität des M1 über intrakortikale Regelkreise der I-Wellen beeinflussen können. Diese Studie kann so eine direkte, ipsilaterale Verbindung bestätigen und liefert Hinweise dafür, dass diese mit der Entstehung von I-Wellen assoziiert ist. Die kurze Latenzzeit der Bahnung weist auf das Vorliegen einer schnell leitenden, monosynaptischen Verbindung hin. Weiterhin kann festgestellt werden, dass die I-Wellen im Ruhezustand einen größeren Einfluss auf den M1 haben als unter Vorinnervation. Die Bahnung durch die Wahl-Reaktionszeit-Aufgabe kann durch eine Veränderung der kortikalen Erregbarkeit erklärt werden und korreliert mit der Ausprägung der subkortikalen Bahnen im Bereich des PMd. Die Kontrollexperimente belegen, dass die Ergebnisse für den stimulierten Bereich spezifisch sind, und schließen eine reine Erregungsausbreitung oder Aufsummierung der Pulse aus. Mit dem verwendeten Doppelpulsparadigma, den Präzisionsspulen und DTI können in Zukunft weitere intrakortikale Bahnen funktionell und strukturell untersucht werden. Insbesondere kann deren Funktionszustand und Veränderung im Rahmen der Reorganisation nach einem Schlaganfall beurteilt und beeinflusst werden

Synthetic biocatalytic modules for enhanced transformation of biological waste products

Many insoluble materials can be used as carriers for the immobilisation of enzymes. Solid-binding peptides (SBPs) are short amino acid sequences that can act as molecular linkers to direct the orientated immobilisation of proteins onto solid materials without impeding their biological activity [1]. Silica-based materials like silica and zeolite have been found to be suitable matrices for enzyme immobilisation in industrial processes. They are inexpensive, offer high mechanical strength and stability, are chemically inert and can be deployed over a wide range of operating conditions. We have constructed biocatalytic modules that are based on the incorporation of a silica-binding SBP (‘linker’) sequence into several genes for thermostable enzymes to facilitate the immobilisation of the proteins onto silica-based matrices, enabling the hydrolysis of both simple and complex polysaccharides. We have shown also that the procedure is suitable for the construction of complex enzymological pathways. In proof of concept experiments, the linker (L) sequence was attached to the N- or C-terminus of three thermostable hemicellulases isolated from thermophilic bacteria using genetic engineering techniques [2]. The resulting L-enzymes remained active after fusion and displayed the same pH and temperature optima but differing thermostabilities in comparison to their corresponding enzymes without linker. The linker facilitated the rapid and simple immobilisation of each L-enzyme onto zeolite, resulting in the construction of ‘single enzyme biocatalytic modules’. All three L-enzymes co-immobilised onto the same zeolite matrix resulted in the formation of ‘multiple enzyme biocatalytic modules’, which were shown to degrade various hemicellulosic substrates effectively in a ‘one-pot’ reaction. Cell-free synthetic biology circumvents many of the limitations encountered by in vivo synthetic biology by operating without the constraints of a cell. It offers higher substrate and enzyme loading and the facile optimisation of enzyme ratios. Some of the challenges of this approach include costly enzyme preparation, biocatalyst stability, and the need for constant supplementation with co-factors. To overcome these challenges, we have developed a molecular toolbox that facilitates the construction of biocatalytic modules with predefined functions and catalytic properties. It consists of three interchangeable building blocks: (a) low-cost inorganic matrices (e.g., silica, zeolite), (b) matrix-specific SBPs and (c) thermostable enzymes. The rational combination of these building blocks allows for flexibility and a ‘pick, mix’ and re-use’ approach with multiple biocatalytic modules available for the assembly of natural and non-natural pathways. Individual immobilised enzymes can be combined rationally to assemble recyclable and product-specific reactions. We present preliminary results relating to the construction of two synthetic pathways for the conversion of organic wastes such as coffee and plant biomass. The pathway assembly process allows for rapid evaluation for proof of concept and for assessing the parameters for a synthetic pathway, which are very labour- and time-intensive by the in vivo approach. [1] Care, A, Bergquist, PL, Sunna, A. (2015) Trends Biotech. 33: 259-268 [2] Care, A, Petroll, K, Gibson, ESY, Bergquist, PL, Sunna, A. (2017) Biotech. Biofuels. 10: 2

Solid-binding peptides for immobilisation of thermostable enzymes to hydrolyse biomass polysaccharides.

BACKGROUND: Solid-binding peptides (SBPs) bind strongly to a diverse range of solid materials without the need for any chemical reactions. They have been used mainly for the functionalisation of nanomaterials but little is known about their use for the immobilisation of thermostable enzymes and their feasibility in industrial-scale biocatalysis. RESULTS: A silica-binding SBP sequence was fused genetically to three thermostable hemicellulases. The resulting enzymes were active after fusion and exhibited identical pH and temperature optima but differing thermostabilities when compared to their corresponding unmodified enzymes. The silica-binding peptide mediated the efficient immobilisation of each enzyme onto zeolite, demonstrating the construction of single enzyme biocatalytic modules. Cross-linked enzyme aggregates (CLEAs) of enzyme preparations either with or without zeolite immobilisation displayed greater activity retention during enzyme recycling than those of free enzymes (without silica-binding peptide) or zeolite-bound enzymes without any crosslinking. CLEA preparations comprising all three enzymes simultaneously immobilised onto zeolite enabled the formation of multiple enzyme biocatalytic modules which were shown to degrade several hemicellulosic substrates. CONCLUSIONS: The current work introduced the construction of functional biocatalytic modules for the hydrolysis of simple and complex polysaccharides. This technology exploited a silica-binding SBP to mediate effectively the rapid and simple immobilisation of thermostable enzymes onto readily-available and inexpensive silica-based matrices. A conceptual application of biocatalytic modules consisting of single or multiple enzymes was validated by hydrolysing various hemicellulosic polysaccharides

Modulation of Corneal Fibroblast Contractility within Fibrillar Collagen Matrices

PURPOSE. To investigate the migratory and contractile behavior of isolated human corneal fibroblasts in fibrillar collagen matrices. METHODS. A telomerase-infected, extended-lifespan human corneal fibroblast cell line (HTK) was transfected by using a vector for enhanced green fluorescent protein (GFP)-α-actinin. Cells were plated at low density on top of or within 100-μm-thick fibrillar collagen lattices. After 18 hours to 7 days, time-lapse imaging was performed. At each 1- to 3-minute interval, GFP and Nomarski differential interference contrast (DIC) images were acquired in rapid succession. Serum-containing (S+) medium was used initially for perfusion. After 2 hours, perfusion was switched to either serum-free (S-) or S+ medium containing the Rho-kinase inhibitor Y-27632 for 1 to 2 hours. Finally, perfusion was changed back to S+ medium for 1 hour. RESULTS. Two to 4 days after plating, many cells underwent spontaneous contraction and/or relaxation in S+ medium. A decrease in the distance between consecutive α-actinin-dense bodies along stress fibers was measured during contraction, and focal adhesion and matrix displacements correlated significantly. Removal of serum or inhibition of Rho-kinase induced cell body elongation and relaxation of matrix stress, as confirmed using finite element modeling. Rapid formation and extension of pseudopodia and filopodia were also observed, and transient tractional forces were generated by these extending processes. CONCLUSIONS. Cultured human corneal fibroblasts can undergo rapid changes in the subcellular pattern of force generation that are mediated, in part, by Rho-kinase. Sarcomeric shortening of stress fibers in contracting corneal fibroblasts is also demonstrated for the first time