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Consolidated server racks are quickly becoming the backbone of IT infrastructure for science, engineering, and business, alike. These servers are still largely built and organized as when they were distributed, individual entities. Given that many fields increasingly rely on analytics of huge datasets, it makes sense to support flexible resource utilization across servers to improve cost-effectiveness and performance. We introduce Venice, a family of data-center server architectures that builds a strong communication substrate as a first-class resource for server chips. Venice provides a diverse set of resource-joining mechanisms that enables user programs to efficiently leverage non-local resources. To better understand the implications of design decisions about system support for resource sharing we have constructed a hardware prototype that allows us to more accurately measure end-to-end performance of at-scale applications and to explore tradeoffs among performance, power, and resource-sharing transparency. We present results from our initial studies analyzing these tradeoffs when sharing memory, accelerators, or NICs. We find that it is particularly important to reduce or hide latency, that data-sharing access patterns should match the features of the communication channels employed, and that inter-channel collaboration can be exploited for better performance.

Thread-level redundancy is an efficient approach for transient fault detection and recovery in Chip Multiprocessors (CMPs), in which two adjacent cores are statically coupled to form a functional Dual Modular Redundancy (DMR). Manufacturing process variations cause core-to-core (C2C) performance asymmetry across the chip, which can be further divided into the asymmetry among core-pairs and the asymmetry within a core-pair. We call them inter- and intra-pair asymmetries, respectively, both of which should be taken into considerations in application scheduling for CMPs with static core coupling. In this paper, we first formulate the above scheduling problem as a 0-1 programming problem to maximize the system Weighted Throughput. An efficient IVF&AppSen algorithm is then proposed, which we prove to be optimal when the number of applications equals to that of core-pairs. We also adapt the Simulated Annealing technique to tackle this problem when applications are less than core-pairs on chip. Simulations on a 64-core CMP shows that the proposed algorithms achieve 2.5-9.3% improvement in Weighted Throughput when compared to prior VarF&AppIPC algorithm.

The standard of treatment for botulism, equine antitoxin, is a foreign protein with associated safety issues and a short serum half-life which excludes its use as a prophylactic antitoxin and makes it a less-than-optimal therapeutic. Due to these limitations, a recombinant monoclonal antibody (mAb) product is preferable. It has been shown that combining three mAbs that bind non-overlapping epitopes leads to highly potent botulinum neurotoxin (BoNT) neutralization. Recently, a triple human antibody combination for BoNT/A has demonstrated potent toxin neutralization in mouse models with no serious adverse events when tested in a Phase I clinical trial. However, a triple antibody therapeutic poses unique development and manufacturing challenges. Thus, potentially to streamline development of BoNT antitoxins, we sought to achieve the potency of multiple mAb combinations in a single IgG-based molecule that has a long serum half-life. The design, production, and testing of a single tri-epitopic IgG1-based mAb (TeAb) containing the binding sites of each of the three parental BoNT/A mAbs yielded an antibody of nearly equal potency to the combination. The approach taken here could be applied to the design and creation of other multivalent antibodies that could be used for a variety of applications, including toxin elimination.

Existing antibodies (Abs) used to treat botulism cannot enter the cytosol of neurons and bind to botulinum neurotoxin (BoNT) at its site of action, and thus cannot reverse paralysis. However, Abs targeting the proteolytic domain of the toxin could inhibit the proteolytic activity of the toxin intracellularly and potentially reverse intoxication, if they could be delivered intracellularly. As such, antibodies that neutralize toxin activity could serve as potent inhibitory cargos for therapeutic antitoxins against botulism. BoNT serotype B (BoNT/B) contains a zinc endopeptidase light chain (LC) domain that cleaves synaoptobrevin-2, a SNARE protein responsible for vesicle fusion and acetylcholine vesicle release. To generate monoclonal Abs (mAbs) that could reverse paralysis, we targeted the protease domain for Ab generation. Single-chain variable fragment (scFv) libraries from immunized mice or humans were displayed on yeast, and 19 unique BoNT/B LC-specific mAbs isolated by fluorescence-activated cell sorting (FACS). The equilibrium dissociation constants (KD) of these mAbs for BoNT/B LC ranged from 0.24 nM to 14.3 nM (mean KD 3.27 nM). Eleven mAbs inhibited BoNT/B LC proteolytic activity. The fine epitopes of selected mAbs were identified by alanine-scanning mutagenesis, revealing that inhibitory mAbs bound near the active site, substrate-binding site or the extended substrate-binding site. The results provide mAbs that could prove useful for intracellular reversal of paralysis and identify epitopes that could be targeted by small molecules inhibitors.

The paralytic disease botulism is caused by botulinum neurotoxins (BoNT), multi-domain proteins containing a zinc endopeptidase that cleaves the cognate SNARE protein, thereby blocking acetylcholine neurotransmitter release. Antitoxins currently used to treat botulism neutralize circulating BoNT but cannot enter, bind to or neutralize BoNT that has already entered the neuron. The light chain endopeptidase domain (LC) of BoNT serotype A (BoNT/A) was targeted for generation of monoclonal antibodies (mAbs) that could reverse paralysis resulting from intoxication by BoNT/A. Single-chain variable fragment (scFv) libraries from immunized humans and mice were displayed on the surface of yeast, and 19 BoNT/A LC-specific mAbs were isolated by using fluorescence-activated cell sorting (FACS). Affinities of the mAbs for BoNT/A LC ranged from a KD value of 9.0×10−11 M to 3.53×10−8 M (mean KD 5.38×10−9 M and median KD 1.53×10−9 M), as determined by flow cytometry analysis. Eleven mAbs inhibited BoNT/A LC catalytic activity with IC50 values ranging from 8.3 ~73×10−9 M. The fine epitopes of selected mAbs were also mapped by alanine-scanning mutagenesis, revealing that the inhibitory mAbs bound the α-exosite region remote from the BoNT/A LC catalytic center. The results provide mAbs that could prove useful for intracellular reversal of paralysis post-intoxication and further define epitopes that could be targeted by small molecule inhibitors.

Rab7 is a key regulatory protein governing early to late endocytic membrane transport. In this study the proteasome alpha-subunit XAPC7 (also known as PSMA7, RC6-1, and HSPC in mammals) was identified to interact specifically with Rab7 and was recruited to multivesicular late endosomes through this interaction. The protein interaction domains were localized to the C terminus of XAPC7 and the N terminus of Rab7. XAPC7 was not found on early or recycling endosomes, but could be recruited to recycling endosomes by expression of a Rab7-(1-174)Rab11-(160-202) chimera, establishing a central role for Rab7 in the membrane recruitment of XAPC7. Although XAPC7 could be shown to associate with membranes bearing ubiquitinated cargo, overexpression had no impact on steady-state ubiquitinated protein levels. Most notably, overexpression of XAPC7 was found to impair late endocytic transport of two different membrane proteins, including EGFR known to be highly dependent on ubiquitination and proteasome activity for proper endocytic sorting and lysosomal transport. Decreased late endocytic transport caused by XAPC7 overexpression was partially rescued by coexpression of wild-type Rab7, suggesting a negative regulatory role for XAPC7. Nevertheless, Rab7 itself was not subject to XAPC7-dependent proteasomal degradation. Together the data establish the first direct molecular link between the endocytic trafficking and cytosolic degradative machineries.

The exploding growth of digitalized information has led to the rapid growth of data centers, both in numbers and in size. Cluster has been the dominating system architecture used in most data centers. However, the increasingly diversified data center applications have requirements beyond what the cluster architecture can deliver. For instance, clouding computing requires flexible sharing of all data center resources. Big data applications often need large memory capacity. A few applications can use GPGPU effectively. Existing system might be extended to a certain degree to meet those needs. Those extensions however would often be prohibitively expensive. The paper presents our attempt to design a system using commodity products that can meet the varying needs of many emerging data center applications in a cost-effective way. Our attempt is to create a system by connecting multiple nodes through a PCIe switch and then extend the software stack to support resource sharing among these nodes. In particular, a node can directly use the memory, NIC, and GPGPU of other nodes through the PCIe switch with no or little involvement from other nodes. We build a prototype as our evaluation platform. Our evaluation results indicate that those resources can be shared effectively in many cases. For using remote memory as block device, our prototype system has 5 times bandwidth, 11 times IOPS and 1/12 latency compared with the system connected by 10GigE in average for Orion benchmark; Using remote GPGPU via PCIe switch achieves average 60 times speedup than the case without GPGPU, and the performance loss is also acceptable (its average execution time is 1/3 of local GPGPU) for micro-benchmarks from GPU computing SDK; And using remote NIC via PCIe switch achieves average 95% bandwidth and 1.4 times latency of local NIC in httperf testing. While our prototype system offers multiple benefits, it is not perfect and has a lot room for further optimization and extension. We hope the outcome presented in this paper will encourage more researchers to join us in designing highly efficient and cost-effective servers.

The limited write endurance of phase change random access memory (PRAM) is one of the major obstacles for PRAM-based main memory. Wear leveling techniques were proposed to extend its lifetime by balancing writes traffic. Another important concern that need to be considered is endurance variation in PRAM chips. When different PRAM cells have distinct endurance, balanced writes will result in lifetime degradation due to the weakest cells. Instead of balancing writes traffic, in this paper we propose wear rate leveling (WRL), a variant of wear leveling, to balance wear rates (i.e., writes traffic/edudrance) of cells across the PRAM chip. After investigating writing behavior of applications and endurance variation, we propose an architecture-level WRL mechanism. Moreover, there is an important tradeoff between endurance improvement and swapping data volume. To co-optimize endurance and swapping, a novel algorithm, Max Hyper-weight Rematching, is proposed to maximize PRAM lifetime and minimize performance degradation. Experimental results show 19x endurance improvement to prior Wear Leveling.

Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1more » inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.« less

Thread-Level Redundancy in Chip Multiprocessors(TLR-CMP) is efficient for soft error tolerance. Process variation causes core-to-core (C2C) performance asymmetry across a chip, which should be taken into consideration for application scheduling. In this paper, two types of variations beyond C2C are introduced, i.e., inter-pair and intra-pair variation in TLR-CMP. Intra-pair performance asymmetry can affect the performance of applications differently. Based on the above observation, we firstly formalize the variation aware scheduling in TLR-CMP as a 0-1 programming problem,to maximize the system weighted throughput. An efficient scheduling algorithm, named IntraVarF&AppSen, is then proposed to tackle this problem, which can be proved to be optimal when the number of applications to be scheduled is equal to the number of core pairs. Simulation on a 64-core CMP shows 2.8%-4% improvement in weighted throughput when compared to prior VarF&AppIPC algorithm.

Proteasomes have long been known to mediate the degradation of polyubiquitinated proteins in the cytoplasm and the nucleus. Additionally, proteasomes have been identified as participating in cellular degradative pathways involving the endomembrane system. In conjunction with the endoplasmic reticulum, proteasomes serve as a quality control mechanism for disposing of malfolded newly synthesized proteins, while on the endocytic pathway they serve to facilitate the degradation of key signaling and nutrient receptors as well as the destruction of phagocytosed pathogens. Our laboratory has identified a direct interaction between the late endocytic Rab7 GTPase and the alpha-proteasome subunit, XAPC7, thus providing the first molecular link between the endocytic trafficking and cytosolic degradative machineries. In this chapter reagents and methods for studying the regulation and interactions between XAPC7, the 20S proteasome, and Rab7 are described.

MthK is a Ca2+-gated K+ channel from Methanobacterium autotrophicum. The crystal structure of the MthK channel in a Ca2+-bound open state was previously determined at 3.3 A and revealed an octameric gating ring composed of eight intracellular ligand-binding RCK (regulate the conductance of K+) domains. It was suggested that Ca2+ binding regulates the gating ring conformation, which in turn leads to the opening and closing of the channel. However, at 3.3 AA resolution, the molecular details of the structure are not well defined, and many of the conclusions drawn from that structure were hypothetical. Here we have presented high resolution structures of the MthK RCK domain with and without Ca2+ bound from three different crystals. These structures revealed a dimeric architecture of the RCK domain and allowed us to visualize the Ca2+ binding and protein-protein contacts at atomic detail. The dimerization of RCK domains is also conserved in other RCK-regulated K+ channels and transporters, suggesting that the RCK dimer serves as a basic unit in the gating ring assembly. A comparison of these dimer structures confirmed that the dimer interface is indeed flexible as suggested previously. However, the conformational change at the flexible interface is of an extent smaller than the previously hypothesized gating ring movement, and a reconstruction of these dimers into octamers by applying protein-protein contacts at the fixed interface did not generate enclosed gating rings. This indicated that there is a high probability that the previously defined fixed interface may not be fixed during channel gating. In addition to the structural studies, we have also carried out biochemical analyses and have shown that near physiological pH, isolated RCK domains form a stable octamer in solution, supporting the notion that the formation of octameric gating ring is a functionally relevant event in MthK gating. Additionally, our stability studies indicated that Ca2+ binding stabilizes the RCK domains in this octameric state.

Proteasomes have long been known to mediate the degradation of polyubiquitinated proteins in the cytoplasm and the nucleus. Additionally, proteasomes have been identified as participating in cellular degradative pathways involving the endomembrane system. In conjunction with the endoplasmic reticulum, proteasomes serve as a quality control mechanism for disposing of malfolded newly synthesized proteins, while on the endocytic pathway they serve to facilitate the degradation of key signaling and nutrient receptors as well as the destruction of phagocytosed pathogens. Our laboratory has identified a direct interaction between the late endocytic Rab7 GTPase and the alpha-proteasome subunit, XAPC7, thus providing the first molecular link between the endocytic trafficking and cytosolic degradative machineries. In this chapter reagents and methods for studying the regulation and interactions between XAPC7, the 20S proteasome, and Rab7 are described.

Rab GTPases serve as master regulators of vesicular membrane transport on both the exo- and endocytic pathways. In their active forms, rab proteins serve in cargo selection and as scaffolds for the sequential assembly of effectors requisite for vesicle budding, cytoskeletal transport, and target membrane fusion. Rab protein function is in turn tightly regulated at the level of protein expression, localization, membrane association, and activation. Alterations in the rab GTPases and associated regulatory proteins or effectors have increasingly been implicated in causing human disease. Some diseases such as those resulting in bleeding and pigmentation disorders (Griscelli syndrome), mental retardation, neuropathy (Charcot-Marie-Tooth), kidney disease (tuberous sclerosis), and blindness (choroideremia) arise from direct loss of function mutations of rab GTPases or associated regulatory molecules. In contrast, in a number of cancers (prostate, liver, breast) as well as vascular, lung, and thyroid diseases, the overexpression of select rab GTPases have been tightly correlated with disease pathogenesis. Unique therapeutic opportunities lie ahead in developing strategies that target rab proteins and modulate the endocytic pathway.