In turn, consumers interested on that data type query that partic

In turn, consumers interested on that data type query that particular rendezvous node to retrieve the information.Ratnasamy et al. [9], introduced the Data Centric Storage term in 2002 as a novel mechanism that disseminates and stores data inside the WSN. This work is the cornerstone of the DCS research selleck kinase inhibitor field.Based Inhibitors,Modulators,Libraries on this research other authors have studied DCS aspects in order to improve and extend the original proposal. Some of these improvements and extensions are focused on: DCS routing, multi-replication DCS systems that employ several rendezvous nodes, combining DCS with other storage mechanisms Inhibitors,Modulators,Libraries depending on network conditions, finding the optimal location to place the rendezvous node, etc.This paper discusses the most relevant works studying DCS in the literature and presents their main contributions.

In addition, we introduce in this paper a Quadratic Adaptive Replication (QAR) scheme for multi-replication DCS that is demonstrated to perform better than previous works addressing the same problem. Finally, we discuss future research lines in the area of DCS for WSNs.Next, we introduce the scenarios where DCS can be applied as well as the terminology used Inhibitors,Modulators,Libraries in this paper.1.1. Scenarios for DCSThere are two kinds of sensor networks where DCS could be applied. On one hand, standard WSNs have a central node named sink, base station, gateway, etc, that connects the WSN with the external world. In this kind of networks, sensor nodes gather information from the environment and store it either locally, in a rendezvous node (if DCS is being applied) or push it to the sink that stores all data.

In such a network, usually the consumers of the WSN data are external users that query the network through the sink.On the Inhibitors,Modulators,Libraries other hand, nowadays not only sensors but actuators are becoming more relevant in the WSN community, and Wireless Sensor Networks are becoming Wireless Sensor and Actuator Networks (WSANs) [10, 11]. An actuator is a node that, based on the information retrieved from one or more sensors, performs some action. For instance a node controlling a water valve in a plantation Cilengitide opens it depending on the humidity and temperature conditions obtained from the sensors in the field. It must be noted that a node could perform both roles in the network, being a sensor and an actuator at the same time.

The use of WSANs opens up the possibility of autonomous WSANs that, in a distributed way, measure the environment, process the collected data and perform some actions to get a common goal without any external intervention. Therefore, in this case, DCS seems to be a suitable storage and information retrieval nearly system because there is no a clear central, powerful node as in the sink case.1.2. TerminologyThis subsection defines some common terminology to be used in the rest of the paper, since different works use different names to refer to the same thing.

To the best of our knowledge, this is the first time that a dispo

To the best of our knowledge, this is the first time that a disposable Ach amperometric biosensor NSC639966 has been used for the high sensitive and selective determination of arsenic. The enzyme was immobilized by covalent linkage on the surface of screen-printed carbon electrodes (SPCEs).2.?Results and DiscussionIn order to build the Ach/SPE biosensor, several experiments were done with the aim to find the optimum conditions for enzyme immobilization. The maximum inhibition response recorded was reached using the immobilization procedure described [18,19].The Ach/SPE biosensor produces an amperometric signal, which is sensitive to the concentration of acetylthiocholine iodide. The principle of the determinations is based on the inhibition effect of AsO33? on the activity of the enzyme Ach, immobilized on a SPCEs.

Acetylthiocholine iodide+H2O��Achthiocholine iodide+acetic acidIt has been proved that thiocholine, the product Inhibitors,Modulators,Libraries of this process, is electroactive. This specie suffers an anodic oxidation, providing a suitable signal for the arsenic determinationThe As(III), interacting with the Ach, inactivates this enzyme; the quantity of thiocholine generated diminishes and the value of the registered oxidation current also decreases as a function of AsO33? concentration, under similar conditions.As it is well known, arsenate(V) is not an Ach inhibitor, contrary to arsenite(III) [20].As(III) inhibition action was quantitatively evaluated by determining the difference between the steady-state current in the absence of arsenic (I0) and the steady-state current in the presence of arsenic (I) (Figure 1).

The parameter ��I (I0 ? I) depends on acetylthiocholine iodide concentration, applied potential (Eap) and pH of the buffer solution. Inhibitors,Modulators,Libraries Therefore, it is necessary to optimize all of these variables in order to Inhibitors,Modulators,Libraries ensure the quality of the results. Several experiments were carried out at different values of the experimental variables. From these experiments, the following optimum values can be set: concentration of the substrate 3.6 �� 10?4 M, Britton-Robinson buffer pH 7 and applied potential +0.6 V (versus Ag/AgCl), because under these conditions high analytical quality signals were obtained.Figure 1.Typical amperometric Inhibitors,Modulators,Libraries recording for an acetylthiocholine iodide concentration: (1) 3.64 �� 10?4 M and consecutive addition of aliquots of As(III) solution into the cell to give an overall concentration of: (2) 1.

90 �� 10?8 …2.1. Calibration and detection limitOnce the optimal experimental conditions were found for the analysis of arsenic by means of Ach/SPCE, a calibration was performed Batimastat using a least-median-squares regression (LMS) to detect the existence of anomalous points [21], which might have led to incorrect inhibitor Dasatinib adjustments altering the sensitivity and the detection limit. Several calibration curves were obtained in the concentration range 1 �� 10?8 to 1 �� 10?7 M for AcH/SPCE.

(b) Scanning electron microscope image of 9 0 �� 1 5 ��m2 SiON wa

(b) Scanning electron microscope image of 9.0 �� 1.5 ��m2 SiON waveguide after annealing the top BPSG cladding.2.2. Microfluidic Channel FabricationThe second lithographic step was performed after the anneal step and similar conditions were used as in the first lithographic step, albeit with a 3.3 ��m selleck inhibitor thick layer of resist. A thicker resist layer was necessary since an 8 ��m deep etch was required for the microfluidic/pillar regions in order to go through the waveguide core layer. Figure 3 illustrates Inhibitors,Modulators,Libraries a part of the pillar array (before removing the resist mask), where a clea
Fluorescence-based detection of low molecular weight compounds can be accomplished using indicators of widely varying selectivity. Antibody-based recognition, for example, may offer highly selective binding of a single Inhibitors,Modulators,Libraries analyte.

Other approaches may use a ��fingerprint�� response pattern across a number of indicators of lower selectivity. The effort described here employs a porphyrin for detection of targets. Porphyrins can be designed to offer semi-selective binding characteristics through altering the peripheral substituent groups of the compounds or through incorporation of a metal via coordination Inhibitors,Modulators,Libraries to the central nitrogen atoms. They have been employed for detection of a number of analytes from oxygen [1] to DNA [2]. Here, we do not seek to offer a review of these numerous efforts, instead we refer the reader to reviews of the topic [3,4]. The unique spectrophotometric characteristics of porphyrins result from their highly conjugated, macrocyclic structure (Figure 1).

This structure yields large extinction coefficients especially in the blue region of the absorbance spectrum. The prominent electronic transitions of porphyrins Inhibitors,Modulators,Libraries and their metal complexes are the �С���* transitions associated with the macrocycle. Several studies have shown that cyclic compounds bind cofacially to this macrocycle [5] even when the compound bears a AV-951 nitrogen [6] or the porphyrin bears a metal [7].Figure 1.Structures of the porphyrin parent compound and those porphyrins used in the presented studies; from left to right: porphine, meso-tetra(4-carboxyphenyl) porphine (C4TPP); 5-mono(4-carboxyphenyl)-10, 15, 20-triphenyl porphine (C1TPP); meso-tri(4-sulfonatophenyl)mono(4-carboxyphenyl) …The semi-selective nature of porphyrin-based detection can impede the applicability of detection protocols to real-world scenarios.

False-positive Seliciclib detection events can be reduced through careful selection of the porphyrin component so that detection relies on a shift (a peak/trough pair) in absorbance or fluorescence as opposed to a quench at a single wavelength. In order to further eliminate non-specific responses by the porphyrins, they can be used in conjunction with a scaffold that offers increased selectivity and protection of the indicators from irrelevant changes in the sensing environment. Porous silicate and organosilicate materials can be used to provide this protection.

The measurement area is increased by placing micro sensors in a r

The measurement area is increased by placing micro sensors in a reaction area intensively. Additionally, the size of a micro temperature sensor is decreased to measure the temperature variation of a small area and decrease the thickness of sensing membranes in order to increase the reaction selleck bio rate. Electrode length of the proposed micro temperature Inhibitors,Modulators,Libraries sensor is 260 ��m and the width is 300 ��m; both the line width and interval are 20 ��m as shown in Figure 1. Three micro temperature sensors are placed in one group, in which nine groups are set at the upstream, midstream and downstream of the micro flow plate, as well as one group at the inlet and one at the outlet. Figure 2 shows the micro temperature sensors distribution.Figure 1.Electrode dimensions of a micro temperature sensor (units: ��m).

Figure 2.Micro temperature sensor distribution.2.3. Fabrication of a Micro Flow PlateA micro flow plate is manufactured Inhibitors,Modulators,Libraries based on MEMS technology, in which SUS 304 is used as the substrate and the thickness of SUS 304 is 600 ��m. Figure 3 illustrates the process. An E-beam evaporator is used to Inhibitors,Modulators,Libraries deposit 500 ? of titanium on stainless steel to function as a sticking layer, followed by use of spin coater coating Hexamethyldisilazane (HMDS) as the sticking layer for the Inhibitors,Modulators,Libraries photoresist. Photoresist AZ-4620 is then coated on stainless steel to function as the etching mask. Next, an aligner and mask are used to make a transition graph for the photoresist, followed by use of MP2500 + H2O to define the pattern. Following development, titanium and aqua regia are etched using hydrofluoric acid to etch stainless steel, followed by removal of the titanium mask and photoresist.

Next, pattern is cut to the desired shape by wire cutting. Figure 4 illustrates the finished product.Figure 3.Process sketch of a micro flow plate.Figure 4.Planer GSK-3 micro flow plate: (a) surface; (b) sectional.2.4. Fabrication of a Flexible Micro Temperature SensorThe material of the micro temperature sensor is gold, and 40 ��m thick stainless steel foil was used as the substrate. Regardless of whether wet etching or metal lift-off is used, a layer of insulating material is coated, explaining our use of RF sputter to grow 1,000 ? of aluminum nitride (AlN).Next an E-beam evaporator is used to deposit 200 ? of chromium to be sticking layer. 2,000 ? of gold is then deposited.

Next, photolithography is performed to define the pattern of a micro temperature sensor, followed by use of etching liquid to etch gold. Finally, fabrication of the micro temperature sensor is completed after removing the photoresist layer. Figure 5 shows the micro temperature sensor, while Figure 6 shows the optical microscope diagram of the micro temperature sensor. Figure 7 schematically depicts the structure of the micro reformer combined with flexible micro temperature sensors.Figure 5.Process sketch of a micro temperature sensor.Figure 6.

That is to say,

That is to say, concerning HEED considers both energy and communication cost when selecting CHs. Unlike LEACH, the probability that two nodes within each other��s transmission range becoming CHs is small which means that the CHs are well distributed.The Energy-Efficient Unequal Clustering (EEUC) [6] partitions the network into clusters of unequal sizes where the clusters closer to the Sink have smaller sizes than those farther away from the Sink. Thus, CHs closer to the Sink, can save some energy for the data relaying. Unlike others protocols such as LEACH and EEUC uses an energy-aware multi-hop routing protocol for inter-cluster communication, however, the setup phase in EEUC has a lot of overhead and as a result it consumes more energy in the setup phase when compared to LEACH.
In stable state phase, EEUC saves energy by using an inter-cluster multi-hop data routing mechanism.Energy-Efficient Level-based and Time-based Clustering (EELTC) [7] is a hierarchical clustering algorithm with multi-hop communication that establishes unequal clusters with very low controlling overhead. In this protocol the network is divided into radial regions using a heuristic formula. The Sink calculates upper bound and lower bound of each level and it then broadcasts the results across the network via a ��hello�� message. All sensors determine their level by receiving this message from the Sink. Based on its level and energy each node sets a time to start advertising itself in the network to form clusters. The algorithm shows good energy efficiency and even load distribution across the whole network.
A modified version called EELTC-M is proposed in [8]. This modification builds upon the previously proposed algorithm EELTC; the lengths of levels are modeled as an optimization problem based on the energy saved for each cluster. This energy is the difference of energy used by cluster head when using single-hop versus Cilengitide multi-hop communication model. In this manner, the cluster in the next level expands its size to cover some extra nodes instead. A comparative simulation was performed and EELTC-M showed to have a longer network lifetime compared to both the previous version and the EEUC protocol.The remainder of the article is organized as follows. We discuss the original star-based communication architecture and sensor networks as well as the original energy model in Section 2.
After that we provide a detailed investigation of current requirements for a real WSN Deployment. In Section 4 we propose a new energy efficient, robust and scalable architecture for WSN that satisfies the real requirements studied in previous section. Section 5 explains the simulation setup and presents the simulation results. In Section selleck chemicals 6 we present the practical application and the experimental results of the proposed architecture. We then conclude our article in Section 7.2.

For this reason, we defined eight most common motion states durin

For this reason, we defined eight most common motion states during pedestrian navigation in this paper. In order to classify the motion states, twenty-seven features are investigated in this section.3.1. Motion DefinitionThe selleck inhibitor motion states, as defined in Table 1, are grouped into four series as follows:S-series motion states (Figure 1) refer to the stationary behavior during a navigation process. ST is a state where a user keeps a phone in hand without any movement. In contrast, SS is a category of the movement where user’s location does not change, but the phone is moving in a swinging.Figure 1.S-series (left and middle: ST, right: SS).W-series is relevant to walking. After observing the walking behaviour of the user when navigating, three types of walking motion states have been defined.
As shown in the left image of Figure 2, WH represents the motion state where the user is using the navigation application on the handset while walking. The user often keeps his or her eyes on the screen of a smartphone in this state. WS stands for the normal walking behaviour, when the user is not using the navigation application but is holding the smartphone in his or her hand. As the center image of Figure 2 indicates, a small arm swinging motion exists when the user is walking in normal speed, while the right image of Figure 2 shows the WF state, which represents a fast walking behaviour with significantly arm swinging.Figure 2.W-series (left: WH, middle: WS, and right: WF).T-series is related to turning motions. UT represents so-called U-turning, which is a spot turn without any horizontal displacement.
As shown in Figure 3, a UT motion results in a heading change of 180�� after turning.Figure 3.T-series (UT).V-series concerns motions in the vertical dimension. In Figure 4, US and DS are going up/down the stairs, respectively.Figure 4.V-series (left: US and right: DS).Table 1.Motion state definition.3.2. Feature DefinitionWhen using tri-axis accelerometer sensors, the sensor orientation determines the local coordinate system of each (x, y, z) reading. Most previous research work on motion recognition has used body-worn accelerometer sensors, i.e., sensors attached to the body in a constrained Entinostat orientation. When smartphones are used as portable sensors, the orientatio
With the development of mechatronics, automatic systems consisting of sensors for perception and actuators for action are more and more widely used in applications [1�C4].
Besides the proper choices of sensors and actuators and an elaborate fabrication of mechanical structures, the control law design also plays a crucial role in the implementation of automatic systems especially for those with complicated dynamics. For most mechanical sensor-actuator systems, it is possible to model them in Euler-lagrange equations [4,5].

Only the basic scheme was verified through static laboratory test

Only the basic scheme was verified through static laboratory tests, so the efficiency and feasibility for dynamic measurements are still under scrutiny. More recently, Jeon et al. [3] introduced in 2011 a paired structured light for displacement measurement of structures. This system could simultaneously obtain the displacement and rotation of the measurement point. However, the system inhibitor had a complicated setup and required a transformation matrix to obtain the final results.Some current commercial contact-type tilt-meter sensors can provide a resolution up to 0.002 degree, as shown in Table 1. Basically, a commercial rotation angle measurement system usually consists of a tilt sensor, data cable, data logger, and data processing software, so the total cost of the system is still expensive.
The total cost of a commercial rotation angle measurement system usually varies from several hundred to several thousand US dollars, depending on a specific application.Table 1.Commercial tilt sensors.Painter and Shkel [1] have briefly reviewed some typical contact-type direct angle measurement technologies and pointed out their disadvantages. Inclinometers and magnetometers are highly sensitive to linear acceleration and environmentally induced magnetic fields, respectively. In addition, inclinometers only measure rotations about axes perpendicular to gravity, while magnetometers can only capture angular changes about axes parallel to gravity. Gyroscopes can produce large errors in as short as an hour, due to integration bias and noise.
The objective of this study is to develop a single-point rotational angle measurement system for large-scale civil structures using a multi-point vision-based system with high resolutions. Compared to commercial rotation angle measurement systems, the proposed system can measure static and dynamic rotational angles with high resolutions. Moreover, the system can be easily expanded to multi-point rotation angle measurement using the partitioning approach [2]. To verify the performance of this system, several laboratory and field tests were carried out on a three-story steel frame model, and on a five-story modal testing tower with a hybrid mass damper (HMD).2.?A Vision-Based Rotation Angle Measurement SystemThe configuration of the proposed system AV-951 is provided in Figure 1.
The initial assumption is that two camcorders, Cam01 and Cam02, are placed at the fixed location base of the structure or beyond the structure where there is no displacement and rotation (��0 = 0, and ��0 = 0). Cam01 and Cam02 will track targets T1 and T2, respectively. Another camcorder (Cam12) will aim at target T2. Displacements ��1 and ��2 are directly measured by Cam01 and Cam02, and reference 4 the displacement ��12 is the relative displacement of target T2 with respect to the tangential line of Cam12.

Many researchers have studied in vivo combinatorial biotechnology

Many researchers have studied in vivo combinatorial biotechnology, e.g., either phage or cell-surface display techniques, and developed polypeptide sequences, which can specifically bind to metals [5�C7], oxides [8,9], and semiconductors [10�C12]. Among them, gold-binding polypeptide (GBP) is one of the genetically engineered proteins for a strong binding onto the gold surface [7,13,14]. Whereas many proteins and chemicals bind to the gold surface with thiol linkage, GBP does not contain a cysteine residue having thiol group. Although the definite mechanism is not clear yet, it is estimated that these polar groups in GBPs seem to coordinately interact with the gold surface within a monolayer [15,16]. In addition, the kinetics and thermodynamics of biomimetic interactions between the GBP, and the gold surface were investigated by surface plasmon resonance (SPR) [14,17,18].
Compared with other thiol-based systems, the GBP binds tightly to the gold surface due to the lower standard Gibbs free energy for the bond, and the binding process is fast under aqueous conditions compatible with biological environments [14,17,19]. These characteristics suggest its potential applications in nano- and bio-technologies as novel agents for surface functionalization [13].Moreover, immobilization with correct orientation of biological material is a problem of prime importance in biosensors. We employed GBP-fusion proteins in the construction of biosensor for the detection of hepatitis B viral surface antigen (HBsAg) as a model (Figure 1), which is a biomarker for diagnosing the hepatitis B virus (HBV).
The strong affinity between the GBP and the gold surface guarantees the stability of this sensor system and orients the sensing parts outward from the solid surface, exposing them directly to the target sample [13,16]. Furthermore, electrochemical detection has attracted considerable interest recently for miniaturized analytical systems [20,21], including remarkable sensitivity (approaching that of fluorescence), inherent miniaturization of both the detector and control instrumentation, independence of optical path length or sample turbidity, low cost, low-power requirements and high compatibility [22,23]. Besides, one of the most attractive points of this method is its potential for portable assays in a variety of point-of-care testing (POCT) environments.
We here developed a simple platform biosensor technology by mediating Cilengitide the recognition parts and the solid surface on the gold substrate. SPR analyses were used for optimization of sample concentrations and verification of target sensing. Electrical signal-based selleck Ponatinib detection methods for HBV such as electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV) were developed on the gold electrode surface, which has been a very versatile material in the field of biosensors.Figure 1.

importance for optimal growth of Toxoplasma Dictyostelium develo

importance for optimal growth of Toxoplasma. Dictyostelium development is ultrasensitive to O2 making it a good model for understanding the mechan ism of O2 sensing by other organisms that conserve the Skp1 thoroughly modification pathway. Development is induced by starvation, which signals the normally solitary phagocytic amoebae to form a multicellular fruiting body, which consists of a cellular stalk that aerially supports thou sands of spores for potential dispersal to other locations. Initially, the amoebae chemotax together to form a multicellular aggregate, which polarizes in response to environmental cues and elongates into a migratory slug consisting of prestalk cells mostly at its anterior end and prespore cells in the remainder.

The slug responds to environmental signals that direct its migration and regulate the slug to fruit switch the process of culmination leading to formation of the fruiting body. Signals include light, low NH3, low moisture, higher temperature, and high O2 which, in the native environment of the soil, draw the subterranean slug to above ground where culmination is most pro ductive. In the laboratory, the process takes place over the course of 24 h after deposition of amoebae on moist agar or filter surfaces wetted with low salt buffers. Whereas amoebae grow and form slugs at an air water interface in the presence of as little as 2. 5% O2, 10% is required for culmination, and slugs immersed in mineral oil require atmospheric hyperoxia to culminate. Overexpression of Skp1 or absence of pathway activity drives the O2 requirement up to 18 21%, whereas decreased Skp1 or overexpression of PhyA drives the O2 requirement down to 5% or less.

These genetic manipulations also revealed effects on timing of slug formation and on sporulation. Together with studies on a Skp1 mutant lacking the modifiable Pro143 residue, and double mutants between Skp1 and pathway enzyme genes, the findings suggested that the Skp1 modification pathway mediates at least some O2 responses. However, O2 con tingent modification AV-951 of the steady state pool of Skp1 has not been demonstrated. To address this issue, and to investigate the generality of O2 regulation of development, we turned to a previ ously described submerged development model in which terminal cell differentiation depends on high at mospheric O2.

The wider range of O2 concentra tions presented to cells in this setting may facilitate analysis of the dependence of Skp1 hydroxylation on O2, and absence of the morphogenetic movements of cul mination might reveal later developmental steps that are dependent on Skp1 and its modifications. In a static adaptation of the previous shaking cultures, we now observed that terminal cell differentiation occurs in a novel radi ally symmetrical fashion in multicellular cyst like struc tures. Under these conditions, we find that O2 is apparently rate limiting for Skp1 hydroxylation, and that cyst formation and terminal spore differentiation that re quire high O2 also

luble proteins into and out of the ER Both we and Trueman et al

luble proteins into and out of the ER. Both we and Trueman et al. observed much milder effects or none on the integration of transmembrane proteins into the ER in our respective mutants. Trueman et al. did not investigate the effects of their L7 and gating motif mutants on ERAD. We have shown here that a Sec61p mutant lacking ER lumenal loop 7 displays severe ERAD defects for soluble substrates. In contrast, ERAD of single spanning KWW was only moderately slower than in wildtype yeast. For soluble misfolded protein export to the cytosol through the Sec61 channel L7 is the only possible starting point, because it is the only large extramembrane domain of the channel in the ER lumen.

If L7 is missing, chaperone export substrate complexes have no contact point from which to open the lateral gate, and exit from the ER is compromised, transmembrane proteins, however, can still enter lat erally into the gate using their hydrophobic TMDs. Collectively, our data suggest that lateral gate opening of the Sec61 channel for entry or exit can proceed inde pendently of L7, whereas transverse gating for soluble protein transport in either direction requires the pres ence of L7. Methods Yeast strains growth conditions Two restriction sites surrounding L7 were introduced within the SEC61 ORF by site directed mutagenesis using the Strategene kit. After restriction with AatII and BstZ17I, self ligation of the ORF resulted in sec61L7pRS315. In sec61L7pRS315, amino acids 305 371 of wildtype Sec61p had been replaced by two amino acids only, arginine, glutamate.

Point mutants sec61Y345H and sec61 32 were established by site directed mutagenesis in bacterial pUC19 vector and cloned into yeast plasmid pRS315, resulting in sec61Y345HpRS315 and sec61 32pRS315. The plasmids were transformed into KRY461, selected on minimal medium without leucine, then on 5 FOA in minimal medium without leucine at 30 C for 4 d, and used for assays described below. Solid media, Yeast were grown in YPD to an OD600 of 0. 8 1. 5 and counted in a Neubauer Chamber. Cells were dropped on YPD plates without or with 0. 25 ug ml Tunicamycin, 0. 5 ug ml Tunicamycin or 5 mM EGTA. Plates were incubated for 3 d or 7 d and growth was examined. Liquid media, YPD was inoculated to an OD600 0. 005 or 5 x 104 cells ml and growth was moni tored at 2 h intervals by counting in a Neubauer chamber or by photometric measuring at 600 nm.

YTX69. UPR assay SEC61 Drug_discovery wildtype and mutant cells were transformed with pJC30 or pJC31, and beta galactosidase activity was assayed after growth overnight in SC without Trp to early log phase. Cells selleck catalog were harvested by cen trifugation and resuspended in 1 ml Z buffer and yeast were lysed with 100 ul chloroform, 50 ul 0. 1% SDS and vortexing for 10 sec. Sus pension was preincubated for 5 min at 28 C and 200 ul ONPG were added. After 30 min, the reaction was stopped by adding 700 ul Na2CO3, the absorbance at 420 nm was determined and Miller units were calculated. SEC61 yeast were treated