skip to main content

Explore Research Products in the NSF-PAR

Title: Sidecar: in-network performance enhancements in the age of paranoid transport protocols
In response to ossification and privacy concerns, post-TCP transport protocols such as QUIC are designed to be “paranoid”—opaque to meddling middleboxes by encrypting and authenticating the header and payload—making it impossible for Performance-Enhancing Proxies (PEPs) to provide the same assistance as before. We propose a research agenda towards an alternate approach to PEPs, creating a sidecar protocol that is loosely-coupled to the unchanged and opaque, underlying transport protocol. The key technical challenge to sidecar protocols is how to usefully refer to the packets of the underlying connection without ossification. We have made progress on this problem by creating a tool we call a quACK (quick ACK), a concise representation of a multiset of numbers that can be used to efficiently decode the randomly-encrypted packet contents a sidecar has received. We implement the quACK and discuss how to achieve several applications with this approach: alternate congestion control, ACK reduction, and PEP-to-PEP retransmission across a lossy subpath.  more » « less
Award ID(s):
2045714 2039070 2028733 1909212 1763256
NSF-PAR ID:
10424939
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
The Twenty-first ACM Workshop on Hot Topics in Networks (HotNets 2022)
Page Range / eLocation ID:
221 to 227
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ( , Current Protocols)
    Abstract

    Conditional control of gene expression allows an experimenter to investigate many aspects of a gene's function. In the model organismSaccharomyces cerevisiae, a number of methods to control gene expression are widely practiced, including induction by metabolites, small molecules, and even light. However, all current methods suffer from at least one of a set of drawbacks, including need for specialized growth conditions, leaky expression, or requirement of specialized equipment. Here we describe protocols using two transformations to construct strains that carry a new controller in which all these drawbacks are overcome. In these strains, the expression of a controlled gene of interest is repressed by the bacterial repressor TetR and induced by anhydrotetracycline. TetR also regulates its own expression, creating an autorepression loop. This autorepression allows tight control of gene expression and protein dosage with low cell‐to‐cell variation in expression. A second repressor, TetR‐Tup1, prevents any leaky expression. We also present a protocol showing a particular workhorse application of such strains to generate synchronized cell populations. We turn off expression of the cell cycle regulatorCDC20completely, arresting the cell population, and then we turn it back on so that the synchronized cells resume cell cycle progression. This control system can be applied to any endogenous or exogenous gene for precise expression. © 2023 Wiley Periodicals LLC.

    Basic Protocol 1: Generating a parent WTC846strain

    Basic Protocol 2: Generating a WTC846strain with controlled expression of the targeted gene

    Alternate Protocol: CRISPR‐mediated promoter replacement

    Basic Protocol 3: Cell cycle synchronization/arrest and release using the WTC846‐K3::CDC20strain

     
    more » « less
  2. ; ; ; ; ; ; ( , Proceedings of the Annual Conference of the ACM Special Interest Group on Data Communication on the Applications, Technologies, Architectures, and Protocols for Computer Communication (SIGCOMM 2020))
    The shared nature of the wireless medium induces contention between data transport and backward signaling, such as acknowledgement. The current way of TCP acknowledgment induces control overhead which is counter-productive for TCP performance especially in wireless local area network (WLAN) scenarios.In this paper, we present a new acknowledgement called TACK ("Tame ACK"), as well as its TCP implementation TCP-TACK. TCP-TACK works on top of commodity WLAN, delivering high wireless transport goodput with minimal control overhead in the form of ACKs, without any hardware modification. To minimize ACK frequency, TACK abandons the legacy received-packet-driven ACK. Instead, it balances byte-counting ACK and periodic ACK so as to achieve a controlled ACK frequency. Evaluation results show that TCP-TACK achieves significant advantages over legacy TCP in WLAN scenarios due to less contention between data packets and ACKs. Specifically, TCP-TACK reduces over 90% of ACKs and also obtains an improvement of ~ 28% on good-put. We further find it performs equally well as high-speed TCP variants in wide area network (WAN) scenarios, this is attributed to the advancements of the TACK-based protocol design in loss recovery, round-trip timing, and send rate control. 
    more » « less
  3. ; ; ; ; ( , Current Protocols)
    Abstract

    Visualization of gene products inCaenorhabditis eleganshas provided insights into the molecular and biological functions of many novel genes in their native contexts. Single‐molecule fluorescencein situhybridization (smFISH) and immunofluorescence (IF) enable the visualization of the abundance and localization of mRNAs and proteins, respectively, allowing researchers to ultimately elucidate the localization, dynamics, and functions of the corresponding genes. Whereas both smFISH and immunofluorescence have been foundational techniques in molecular biology, each protocol poses challenges for use in theC. elegansembryo. smFISH protocols suffer from high initial costs and can photobleach rapidly, and immunofluorescence requires technically challenging permeabilization steps and slide preparation. Most importantly, published smFISH and IF protocols have predominantly been mutually exclusive, preventing the exploration of relationships between an mRNA and a relevant protein in the same sample. Here, we describe protocols to perform immunofluorescence and smFISH inC. elegansembryos either in sequence or simultaneously. We also outline the steps to perform smFISH or immunofluorescence alone, including several improvements and optimizations to existing approaches. These protocols feature improved fixation and permeabilization steps to preserve cellular morphology while maintaining probe and antibody accessibility in the embryo, a streamlined, in‐tube approach for antibody staining that negates freeze‐cracking, a validated method to perform the cost‐reducing single molecule inexpensive FISH (smiFISH) adaptation, slide preparation using empirically determined optimal antifade products, and straightforward quantification and data analysis methods. Finally, we discuss tricks and tips to help the reader optimize and troubleshoot individual steps in each protocol. Together, these protocols simplify existing workflows for single‐molecule RNA and protein detection. Moreover, simultaneous, high‐resolution imaging of proteins and RNAs of interest will permit analysis, quantification, and comparison of protein and RNA distributions, furthering our understanding of the relationship between RNAs and their protein products or cellular markers in early development. © 2021 Wiley Periodicals LLC.

    Basic Protocol 1: Sequential immunofluorescence and single‐molecule fluorescencein situhybridization

    Alternate Protocol: Abbreviated protocol for simultaneous immunofluorescence and single‐molecule fluorescencein situhybridization

    Basic Protocol 2: Simplified immunofluorescence inC. elegansembryos

    Basic Protocol 3: Single‐molecule fluorescencein situhybridization or single‐molecule inexpensive fluorescencein situhybridization

     
    more » « less
  4. ; ; ; ; ; ( , Current Protocols)
    Abstract

    Fluorescence fluctuation spectroscopy (FFS) encompasses a bevy of techniques that involve analyzing fluorescence intensity fluctuations occurring due to fluorescently labeled molecules diffusing in and out of a microscope's focal region. Statistical analysis of these fluctuations may reveal the oligomerization (i.e., association) state of said molecules. We have recently developed a new FFS‐based method, termed Two‐Dimensional Fluorescence Intensity Fluctuation (2D FIF) spectrometry, which provides quantitative information on the size and stability of protein oligomers as a function of receptor concentration. This article describes protocols for employing FIF spectrometry to quantify the oligomerization of a membrane protein of interest, with specific instructions regarding cell preparation, image acquisition, and analysis of images given in detail. Application of the FIF Spectrometry Suite, a software package designed for applying FIF analysis on fluorescence images, is emphasized in the protocol. Also discussed in detail is the identification, removal, and/or analysis of inhomogeneous regions of the membrane that appear as bright spots. The 2D FIF approach is particularly suited to assess the effects of agonists and antagonists on the oligomeric size of membrane receptors of interest. © 2022 Wiley Periodicals LLC.

    Basic Protocol 1: Preparation of live cells expressing protein constructs

    Basic Protocol 2: Image acquisition and noise correction

    Basic Protocol 3: Drawing and segmenting regions of interest

    Basic Protocol 4: Calculating the molecular brightness and concentration of individual image segments

    Basic Protocol 5: Combining data subsets using a manual procedure (Optional)

    Alternate Protocol 1: Combining data subsets using the advanced FIF spectrometry suite (Optional; alternative to Basic Protocol 5)

    Basic Protocol 6: Performing meta‐analysis of brightness spectrograms

    Alternate Protocol 2: Performing meta‐analysis of brightness spectrograms (alternative to Basic Protocol 6)

    Basic Protocol 7: Spot extraction and analysis using a manual procedure or by writing a program (Optional)

    Alternate Protocol 3: Automated spot extraction and analysis (Optional; alternative to Protocol 7)

    Support Protocol: Monomeric brightness determination

     
    more » « less
  5. ; ; ; ( , Current Protocols)
    Abstract

    Protein activity is generally functionally integrated and spatially restricted to key locations within the cell. Knocksideways experiments allow researchers to rapidly move proteins to alternate or ectopic regions of the cell and assess the resultant cellular response. Briefly, individual proteins to be tested using this approach must be modified with moieties that dimerize under treatment with rapamycin to promote the experimental spatial relocalizations. CRISPR technology enables researchers to engineer modified protein directly in cells while preserving proper protein levels because the engineered protein will be expressed from endogenous promoters. Here we provide straightforward instructions to engineer tagged, rapamycin‐relocalizable proteins in cells. The protocol is described in the context of our work with the microtubule depolymerizer MCAK/Kif2C, but it is easily adaptable to other genes and alternate tags such as degrons, optogenetic constructs, and other experimentally useful modifications. Off‐target effects are minimized by testing for the most efficient target site using a split‐GFP construct. This protocol involves no proprietary kits, only plasmids available from repositories (such as addgene.org). Validation, relocalization, and some example novel discoveries obtained working with endogenous protein levels are described. A graduate student with access to a fluorescence microscope should be able to prepare engineered cells with spatially controllable endogenous protein using this protocol. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.

    Basic Protocol 1: Choosing a target site for gene modification

    Basic Protocol 2: Design of gRNA(s) for targeted gene modification

    Basic Protocol 3: Split‐GFP test for target efficiency

    Basic Protocol 4: Design of the recombination template and analytical primers

    Support Protocol 1: Design of primers for analytical PCR

    Basic Protocol 5: Transfection, isolation, and validation of engineered cells

    Support Protocol 2: Stable transfection of engineered cells with binding partners

     
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email