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This article from Country Living features a family cottage in Alabama that embraces color without being kitschy. The cottage, which was renovated by the previous owners, was a bit cramped for the Finger family, so they decided to add more space with a thoughtful addition. The old and new spaces are united with a warm, earthy palette, creating a sense of warmth that was important to the couple. The article also highlights various design elements and features of the cottage, such as the trim, patterned decor, kitchen, dining space, living room, primary suite, and guest room. [Extracted from the article]

Parental interpartner conflict is a highly prevalent form of family risk that is stressful for adolescents with ramifications for their sleep. Multiple studies have demonstrated that adolescents from high-conflict homes are at risk for sleep problems. Building on this literature, we conducted novel analyses and investigated whether exposure to interpartner conflict in adolescence predicts sleep problems in the subsequent developmental period of emerging adulthood.

This article from Country Living magazine tells the story of Justin Reis and Mark Schwindenhammer, two old-house enthusiasts who fell in love with a historic home in Hampton, Connecticut. Despite its faded frescoes, uneven floorboards, and wavy windowpanes, the couple saw the beauty in these details and decided to make the house their primary home. They focused on preserving the existing character of the house and researched its previous female owners to stay true to its spirit. The couple also transformed the property's three acres into a dreamy English-inspired garden with chicken coops, a woodworking shop, and a cutting garden. [Extracted from the article]

Lili Liu and Blake Civiello have transformed a collection of seaside cottages, fishing shacks, and roadside sheds in Brunswick, Maine into their unconventional home. The property, known as Gurnet Village, has a rich history and has been preserved by the lobsterman's family who originally built it over 100 years ago. The couple has made minimal changes to the buildings, maintaining their rustic charm and connection to the working waterfront. They have also incorporated found objects and antiques to enhance the character of the space. The couple's summer kitchen and guest cottage provide a unique and immersive experience of living on the coast of Maine. [Extracted from the article]

This article from Country Living magazine discusses the renovation of an 1890s riverfront cottage in South Carolina's Low Country. The cottage was restored by interior designer Jackye Lanham and architect Stan Dixon, who aimed to preserve the original architecture while adding modern updates. The renovated cottage seamlessly blends old and new elements, with a focus on creating ample space for gathering and relaxation. The article highlights the cottage's serene surroundings, open design, and comfortable furnishings. [Extracted from the article]

This article from Country Living magazine discusses the renovation of a small cottage on a farm in rural Ohio. The cottage, built in the 1920s, was in a dilapidated condition but was saved by the Dutton family who wanted to preserve its character. The renovation process involved uncovering hidden gems, such as original heart-pine floorboards, and embracing imperfections to create a lived-in feel. The cottage now features a muted and moody color palette, natural light, and salvaged materials. The article also highlights specific design elements, such as Shaker cabinetry and a reclaimed claw-foot tub. The cottage's exterior is inspired by the surrounding goldenrod wildflowers, and the rebuilt porch invites visitors to enjoy the sunset. [Extracted from the article]

A Minnesota family decided to build a larger garage to accommodate their outdoor gear, but it turned into a multifunctional space called the "sports hut." The garage includes ample storage for outdoor gear, a home gym with a yoga studio, and a lounge area for the family and friends to hang out. The space features a live-edge wood slab table, salvaged French doors, and a massive stone fireplace. The design of the sports hut mimics classic cabins and is surrounded by a 3.5-acre lakeside landscape in Buffalo, Minnesota. [Extracted from the article]

-Homeowner Stefanie Turner midwestern mindset THE MORE THE MERRIER The four sisters share two sets of bunk beds in their upstairs bedroom, where Stefanie puts up a miniature tree every year. Stefanie and Scott Turner's Michigan farmhouse might look plucked out of a snow globe now, but it hasn't always been so picturesque. Current MOOD Clare Christmas from Coast to Coast Heartland Holiday Grab a pop and a scotcheroo or two and kick back with homeowner Stefanie Turner as she chats about some good ol' Midwestern merry making. [Extracted from the article]

The laundry room is her favorite spot in the house, where Bambi enjoys arranging freshly snipped garden flowers into pitchers of all varieties. Bambi transformed a blank nook next to the stairs by installing an electric firebox surrounded by an antique mantel and shelves on either side. CHARM TACTIC Antiquing Treasures at Every Turn Bambi's treasure trove of thrifted pieces and antique finds spills over to the mudroom in an ever-rotating display. [Extracted from the article]

We compared three cell isolation and two proteomic sample preparation methods for single-cell and near-single-cell analysis. Whole blood was used to quantify hemoglobin (Hb) and glycated-Hb (gly-Hb) in erythrocytes using targeted mass spectrometry and stable isotope-labeled standard peptides. Each method differed in cell isolation and sample preparation as follows: 1) FACS and automated preparation in one-pot for trace samples (autoPOTS); 2) limited dilution via microscopy and a novel rapid one-pot sample preparation method that circumvented the need for the solid-phase extraction, low-volume liquid handling instrumentation and humidified incubation chamber; and 3) CellenONE-based cell isolation and the same one-pot sample preparation method used for limited dilution. Only the CellenONE device routinely isolated single-cells from which Hb was measured to be 540–660 amol per red blood cell (RBC), which was comparable to the calculated SI reference range for mean corpuscular hemoglobin (390–540 amol/RBC). FACSAria sorter and limited dilution could routinely isolate single-digit cell numbers, to reliably quantify CMV-Hb heterogeneity. Finally, we observed that repeated measures, using 5–25 RBCs obtained from N= 10 blood donors, could be used as an alternative and more efficient strategy than single RBC analysis to measure protein heterogeneity, which revealed multimodal distribution, unique for each individual.

Sarah Anderson's family cabin on the outskirts of Amery, Wisconsin, is less than an hour's drive north from her home, but that doesn't mean the former vintage dealer won't make a pit stop or two before she gets there. THIS PAGE: In the great room, Sarah updated an $8 pair of Cesca-style chairs found at Goodwill with a 1950s Hudson Bay blanket that belonged to her grandfather. LEFT: Sarah and Jamie Anderson accented their home's black siding with color-matched board bands and contrasting trim (Acacia by Sherwin-Williams). [Extracted from the article]

Analyzing proteins from single cells by tandem mass spectrometry (MS) has recently become technically feasible. While such analysis has the potential to accurately quantify thousands of proteins across thousands of single cells, the accuracy and reproducibility of the results may be undermined by numerous factors affecting experimental design, sample preparation, data acquisition and data analysis. We expect that broadly accepted community guidelines and standardized metrics will enhance rigor, data quality and alignment between laboratories. Here we propose best practices, quality controls and data-reporting recommendations to assist in the broad adoption of reliable quantitative workflows for single-cell proteomics. Resources and discussion forums are available at https://single-cell.net/guidelines.

At first, the Wattses left the ceiling white, "but it didn't feel cozy enough, so we painted it the next year", says Stephenie. Stephenie and Chase Watts - with their kids, Hampton (7) and Klein (4) - updated their cute-as-a-button 1639 cedar shake cottage by painting the door a happy hue. Cottage Capsule CHAR ACTER IN EVERY CRANNY The original beams in the kitchen and adjacent dining room (far right) put the ceiling height at a mere 6 feet. [Extracted from the article]

Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1deficiency ameliorated hepatocellular PMR induced with TNF plus d-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia–reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia–reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.

"We wanted it to feel like something you would see in a candy shop or European café", says Anthony. redefining rustic TONE-ONTONE TEXTURE Blush tile of Anthony's own design (Zio and Sons; cletile.com) adds a dusting of light color in the kitchen, where exposed piping and rustic antiques bring primitive country elements together. For a lighter Scandinavian feel, Anthony sanded away their original sealant, then used a water-based clear-coat finish. redefining rustic STREAMLINED FURNISHINGS Straight-back hickory chairs surround a Shaker-style table in the dining area. "Half of the kitchen has new custom millwork, but I wanted to work in antiques", says Anthony. [Extracted from the article]

"Plaids and tartans have a magical quality of pulling all the colors in a room together", says Erick. IF HOMES EMBRACED PUNCTUATION, designer Erick Espinoza's would be peppered with exclamation points - and, perhaps, the occasional question mark. IN WITH THE BOLD: STATEMENT FURNITURE Homeowner Erick Espinoza's guests are greeted by a playful pairing of a folk art windmill whirligig with a wood-turned spinning wheel armchair accented with sewing bobbins. [Extracted from the article]

Carved timbers, rough-cut stone, and walls of windows reflect a combination of Jill's Scandinavian heritage and Jeff's Irish roots. LAKE LURE Perfect-Size Porch Early on, the Murphys considered a four-season porch, but they quickly nixed the idea in favor of the breezes and sounds of the waves that come courtesy of a screened-in porch. Black Cabot Stain The black-stained siding of Jill and Jeff Murphy's low-slung cottage is crisply contrasted with white trim and topped with a cedar shake roof, echoing buildings in Norway and Denmark. [Extracted from the article]

The cabin's cozy size, with ceilings just shy of 6 feet in some spaces, appeals especially to the Albers's 5-yearold son, Otto. That's what Abby and Joe Albers learned when they renovated their 800-square-foot cabin in Pentwater, Michigan. On the porch of their cottage, Abby, Joe, and son Otto are a five-minute bike ride from the beach. [Extracted from the article]

But the crowning glory is the barn's hayloft, which was transformed into the ultimate party room with game tables, four beds, and a bar. "It all just feels really natural and well-loved, like the barn itself", Lori says. Pendleton blankets and a suspended canoe - which Cait found on Facebook Marketplace and Brian hoisted into place with a pulley - make the dreamy space cozy for both kid sleepovers and girlfriend getaways alike. «weekends in the country» Party-Ready Patio Cait, a book publicist, and Brian, who works in finance, enjoy whiling away Saturdays enjoying the backyard. [Extracted from the article]

Single-cell proteomics (SCP) has great potential to advance biomedical research and personalized medicine. The sensitivity of such measurements increases with low-flow separations (<100 nL/min) due to improved ionization efficiency, but the time required for sample loading, column washing, and regeneration in these systems can lead to low measurement throughput and inefficient utilization of the mass spectrometer. Herein, we developed a two-column liquid chromatography (LC) system that dramatically increases the throughput of label-free SCP using two parallel subsystems to multiplex sample loading, online desalting, analysis, and column regeneration. The integration of MS1-based feature matching increased proteome coverage when short LC gradients were used. The high-throughput LC system was reproducible between the columns, with a 4% difference in median peptide abundance and a median CV of 18% across 100 replicate analyses of a single-cell-sized peptide standard. An average of 621, 774, 952, and 1622 protein groups were identified with total analysis times of 7, 10, 15, and 30 min, corresponding to a measurement throughput of 206, 144, 96, and 48 samples per day, respectively. When applied to single HeLa cells, we identified nearly 1000 protein groups per cell using 30 min cycles and 660 protein groups per cell for 15 min cycles. We explored the possibility of measuring cancer therapeutic targets with a pilot study comparing the K562 and Jurkat leukemia cell lines. This work demonstrates the feasibility of high-throughput label-free single-cell proteomics.

DIY Pallet Sofa For the back patio, Rebecca's husband, Jamie, built a corner sofa from old shipping pallets that Rebecca then topped with bench cushions and a mix of grain sack and gingham pillows. Before Rebecca and Jamie Lovatt moved their family from Northern England to the Suffolk countryside, they made a list of features they didn't want in their home: nothing too old or in need of repair and most certainly not a thatched roof, which requires regular maintenance. COTTAGE CLASSIC Slipcovered Sofas To offset the sitting room's rugged walls, Rebecca selected slipcovered sofas and thick floral draperies backed with striped fabric. [Extracted from the article]

The writing desk once belonged to Provincetown artist William Maynard, who had lived in the house in front of Steve and Colin's cottage. The house needed a rescue, but the couple wanted to thoughtfully maintain its spirit, so Steve and Colin embarked on a two-year restoration, bringing the cottage back to life one heart-pine board and brass light at a time. Buried Treasure VARIED-WIDTH FLOORBOARDS Steve and Colin turned the former parlor on the main level into a guest bedroom. [Extracted from the article]

The goal of proteomics is to identify and quantify the complete set of proteins in a biological sample. Single-cell proteomics specializes in the identification and quantitation of proteins for individual cells, often used to elucidate cellular heterogeneity. The significant reduction in ions introduced into the mass spectrometer for single-cell samples could impact the features of MS2 fragmentation spectra. As all peptide identification software tools have been developed on spectra from bulk samples and the associated ion-rich spectra, the potential for spectral features to change is of great interest. We characterize the differences between single-cell spectra and bulk spectra by examining three fundamental spectral features that are likely to affect peptide identification performance. All features show significant changes in single-cell spectra, including the loss of annotated fragment ions, blurring signal and background peaks due to diminishing ion intensity, and distinct fragmentation pattern, compared to bulk spectra. As each of these features is a foundational part of peptide identification algorithms, it is critical to adjust algorithms to compensate for these losses.

Shannan and Drew crafted an artful sconce from a salvaged corbel, a couple of weathered wood shutters, and a $13 lamp kit. OLD-TRY TACTIC Statement-Making Salvage When the Haupts bought the house, the updated kitchen looked like it belonged in a new-build home; "It didn't talk to the rest of the house", Shannan says. OLD-TRY TACTIC Uncovered Brick Drew and Shannan discovered the bedroom's original brick fireplace hiding behind layers of white plaster. [Extracted from the article]

Magic in the making You would be hard-pressed to conjure a more idyllic setting for the holidays than interior designer Angela Wheeler's dreamy Canadian stone house. Angela and Josh began building their farmhouse nine years ago, with "cozy" as their aesthetic true north. Front Entryway Angela forages cedar and pine clippings from trees on their farm to make swags of garlands throughout her house. [Extracted from the article]

Inside, tree bark covers a sliding door leading into the study, and stacked Tennessee fieldstone frames the living room fireplace. KEEP CALM WITH… Rooms Wrapped in Neutrals Reclaimed timber beams lower the living room's ceiling height for a more intimate gathering spot. "The homeowner requested a rocking chair [for the back porch]", says architect Ken Pursley. [Extracted from the article]

CABIN CRED Knotty Pine Walls "When I walk in here, it reminds me of the great lodges I visited as a kid", says Doug of the original pine walls that cast a golden glow. Nora and Doug O'Leary admit they had ulterior motives when they bought their lakeside cabin in Cable, Wisconsin, seven years ago. CABIN CRED: Help-Yourself Open Shelving While Doug flips hotcakes on the range, a dozen more family members can weave in and out of the kitchen, helping themselves to dishes from the open cabinets. [Extracted from the article]

Low-volume liquid handling capabilities in bioanalytical workflows can dramatically improve sample processing efficiency and reduce reagent costs, yet many commercial nanoliter liquid handlers cost tens of thousands of dollars or more. We have successfully adapted a low-cost and open-source commercial pipetting robot, the Opentrons OT-1, to accurately aspirate and dispense nanoliter volumes. Based on fluorescence measurements, the modified OT-1 was able to reproducibly transfer 50 nL of water with less than 3% measurement error and 5% coefficient of variation (CV). For 15 nL transfers, the volume measurements indicated less than 4% error and 4% CV. We applied this platform to the preparation of low-nanogram proteomic samples for liquid chromatography–mass spectrometry analysis, demonstrating that the modified OT-1 is an effective platform for nanoliter liquid handling. At a total materials cost of less than $6000, including the commercial liquid handler and all modifications, this system is also far less expensive than other platforms with similar capabilities, placing automated nanoliter handling within reach of a far broader scientific community.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Low-volume liquid handling capabilities in bioanalytical workflows can dramatically improve sample processing efficiency and reduce reagent costs, yet many commercial nanoliter liquid handlers cost tens of thousands of dollars or more. We have successfully adapted a low-cost and open-source commercial pipetting robot, the Opentrons OT-1, to accurately aspirate and dispense nanoliter volumes. Based on fluorescence measurements, the modified OT-1 was able to reproducibly transfer 50 nL of water with less than 3% measurement error and 5% coefficient of variation (CV). For 15 nL transfers, the volume measurements indicated less than 4% error and 4% CV. We applied this platform to the preparation of low-nanogram proteomic samples for liquid chromatography–mass spectrometry analysis, demonstrating that the modified OT-1 is an effective platform for nanoliter liquid handling. At a total materials cost of less than $6000, including the commercial liquid handler and all modifications, this system is also far less expensive than other platforms with similar capabilities, placing automated nanoliter handling within reach of a far broader scientific community.

A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases.

Peptide separations that combine high sensitivity, robustness, peak capacity, and throughput are essential for extending bottom-up proteomics to smaller samples including single cells. To this end, we have developed a multicolumn nanoLC system with offline gradient generation. One binary pump generates gradients in an accelerated fashion to support multiple analytical columns, and a single trap column interfaces with all analytical columns to reduce required maintenance and simplify troubleshooting. A high degree of parallelization is possible, as one sample undergoes separation while the next sample plus its corresponding mobile phase gradient are transferred into the storage loop and a third sample is loaded into a sample loop. Selective offline elution from the trap column into the sample loop prevents salts and hydrophobic species from entering the analytical column, thus greatly enhancing column lifetime and system robustness. With this design, samples can be analyzed as fast as every 20 min at a flow rate of just 40 nL/min with close to 100% MS utilization time and continuously for as long as several months without column replacement. We utilized the system to analyze the proteomes of single cells from a multiple myeloma cell line upon treatment with the immunomodulatory imide drug lenalidomide.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/zdependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4–5-fold increases in reporter signal compared to conventional SPS-MS3approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4–5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

To better understand disease conditions and environmental perturbations, multiomic studies combining proteomic, lipidomic, and metabolomic analyses are vastly increasing in popularity. In a multiomic study, a single sample is typically extracted in multiple ways, and various analyses are performed using different instruments, most often based upon mass spectrometry (MS). Thus, one sample becomes many measurements, making high throughput and reproducible evaluations a necessity. One way to address the numerous samples and varying instrumental conditions is to utilize a flow injection analysis (FIA) system for rapid sample injections. While some FIA systems have been created to address these challenges, many have limitations such as costly consumables, low pressure capabilities, limited pressure monitoring, and fixed flow rates. To address these limitations, we created an automated, customizable FIA system capable of operating at a range of flow rates (∼50 nL/min to 500 μL/min) to accommodate both low- and high-flow MS ionization sources. This system also functions at varying analytical throughputs from 24 to 1200 samples per day to enable different MS analysis approaches. Applications ranging from native protein analyses to molecular library construction were performed using the FIA system, and results showed a highly robust and reproducible platform capable of providing consistent performance over many days without carryover, as long as washing buffers specific to each molecular analysis were utilized.

Post-traumatic stress disorder (PTSD) is associated with osteopenia, osteoporosis and increased fracture risk in the clinical population. Yet, the development of preclinical models to study PTSD-induced bone loss remains limited. In this study, we present a previously unreported model of PTSD in adult female C57BL/6 mice, by employing inescapable foot shock and social isolation, that demonstrates high face and construct validity. A subset of mice exposed to this paradigm (i.e. PTSD mice) display long–term alterations in behavioral and inflammatory indices. Using three–dimensional morphometric calculations, cyclic reference point indentation (cRPI) testing and histological analyses, we find that PTSD mice exhibit loss of trabecular bone, altered bone material quality, and aberrant changes in bone tissue architecture and cellular activity. This adult murine model of PTSD exhibits clinically relevant changes in bone physiology and provides a valuable tool for investigating the cellular and molecular mechanisms underlying PTSD-induced bone loss.

Recent advances in sample preparation and analysis have enabled direct profiling of protein expression in single mammalian cells and other trace samples. Several techniques to prepare and analyze low-input samples employ custom fluidics for nanoliter sample processing and manual sample injection onto a specialized separation column. While being effective, these highly specialized systems require significant expertise to fabricate and operate, which has greatly limited implementation in most proteomic laboratories. Here, we report a fully automated platform termed autoPOTS (automated preparation in one pot for trace samples) that uses only commercially available instrumentation for sample processing and analysis. An unmodified, low-cost commercial robotic pipetting platform was utilized for one-pot sample preparation. We used low-volume 384-well plates and periodically added water or buffer to the microwells to compensate for limited evaporation during sample incubation. Prepared samples were analyzed directly from the well plate with a commercial autosampler that was modified with a 10-port valve for compatibility with 30 μm i.d. nanoLC columns. We used autoPOTS to analyze 1–500 HeLa cells and observed only a moderate reduction in peptide coverage for 150 cells and a 24% reduction in coverage for single cells compared to our previously developed nanoPOTS platform. To evaluate clinical feasibility, we identified an average of 1095 protein groups from ∼130 sorted B or T lymphocytes. We anticipate that the straightforward implementation of autoPOTS will make it an attractive option for low-input and single-cell proteomics in many laboratories.

Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells.

Using a multimethod approach, we examined relations between dating aggression and sleep in emerging adulthood. Participants were 108 dating couples from diverse ethnic and educational backgrounds (49% were cohabiting). Women and men self-reported on the frequency to which they perpetrated psychological and physical aggression toward their partners. Actigraphs were worn for seven consecutive nights and measured sleep duration, sleep efficiency, and long wake episodes. Participants also self-reported on their cognitive and somatic presleep arousal. After controlling for many covariates, actor–partner interdependence models revealed that women and men who perpetrated higher levels of aggression were at greater risk for disruption in many actigraphy and self-reported sleep domains. Partner effects also emerged and indicated that an individual’s perpetration of aggression related to greater disruption in their partner’s sleep. Findings build on this scant literature and demonstrate relations between a prevalent social stressor and a fundamental facet of emerging adults’ health.

Single-cell proteomics can provide critical biological insight into the cellular heterogeneity that is masked by bulk-scale analysis. We have developed a nanoPOTS (nanodroplet processing in one pot for trace samples) platform and demonstrated its broad applicability for single-cell proteomics. However, because of nanoliter-scale sample volumes, the nanoPOTS platform is not compatible with automated LC-MS systems, which significantly limits sample throughput and robustness. To address this challenge, we have developed a nanoPOTS autosampler allowing fully automated sample injection from nanowells to LC-MS systems. We also developed a sample drying, extraction, and loading workflow to enable reproducible and reliable sample injection. The sequential analysis of 20 samples containing 10 ng tryptic peptides demonstrated high reproducibility with correlation coefficients of >0.995 between any two samples. The nanoPOTS autosampler can provide analysis throughput of 9.6, 16, and 24 single cells per day using 120, 60, and 30 min LC gradients, respectively. As a demonstration for single-cell proteomics, the autosampler was first applied to profiling protein expression in single MCF10A cells using a label-free approach. At a throughput of 24 single cells per day, an average of 256 proteins was identified from each cell and the number was increased to 731 when the Match Between Runs algorithm of MaxQuant was used. Using a multiplexed isobaric labeling approach (TMT-11plex), ∼77 single cells could be analyzed per day. We analyzed 152 cells from three acute myeloid leukemia cell lines, resulting in a total of 2558 identified proteins with 1465 proteins quantifiable (70% valid values) across the 152 cells. These data showed quantitative single-cell proteomics can cluster cells to distinct groups and reveal functionally distinct differences.

In many areas of application, key objectives of chemical separation and analysis are to minimize the sample quantity while maximizing the chemical information obtained. Increasing measurement sensitivity is especially critical for proteomics research, especially when processing trace samples and where multiple measurements are desired. A rich collection of technologies has been developed, but the resulting sensitivity remains insufficient for achieving in-depth coverage of proteomic samples as small as single cells. Here, we combine picoliter-scale liquid chromatography (picoLC) with mass spectrometry (MS) to address this issue. The picoLC employs a 2-μm-i.d. open tubular column to reduce the sample input needed to greatly increase the sensitivity achieved using electrospray ionization (ESI) with MS. With this picoLC-MS system, we show that we can identify ∼1000 proteins reliably using only 75 pg of tryptic peptides, representing a 10–100-fold sensitivity improvement compared with the state-of-the-art liquid chromatography (LC) or capillary electrophoresis (CE)-MS methods. PicoLC-MS extends the limit of separation science and is expected to be a powerful tool for single cell proteomics.

The Centers for Disease Control and Prevention have declared that the United States is amidst a continuing opioid epidemic, with drug overdose–related death tripling between 1999 and 2014. Among the 47,055 overdose-related deaths that occurred in 2014, 28,647 (60.9%) of them involved an opioid.

Single-cell proteomics can provide unique insights into biological processes by resolving heterogeneity that is obscured by bulk measurements. Gains in the overall sensitivity and proteome coverage through improvements in sample processing and analysis increase the information content obtained from each cell, particularly for less abundant proteins. Here we report on improved single-cell proteome coverage through the combination of the previously developed nanoPOTS platform with further miniaturization of liquid chromatography (LC) separations and implementation of an ultrasensitive latest generation mass spectrometer. Following nanoPOTS sample preparation, protein digests from single cells were separated using a 20 μm i.d. in-house-packed nanoLC column. Separated peptides were ionized using an etched fused-silica emitter capable of stable operation at the ∼20 nL/min flow rate provided by the LC separation. Ultrasensitive LC–MS analysis was achieved using the Orbitrap Eclipse Tribrid mass spectrometer. An average of 362 protein groups were identified by tandem mass spectrometry (MS/MS) from single HeLa cells, and 874 protein groups were identified using the Match Between Runs feature of MaxQuant. This represents an >70% increase in label-free proteome coverage for single cells relative to previous efforts using larger bore (30 μm i.d.) LC columns coupled to a previous-generation Orbitrap Fusion Lumos mass spectrometer.

Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ∼1 600 proteins with a median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2 300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.

The functional role of CD8+ lymphocytes in tuberculosis remains poorly understood. We depleted innate and/or adaptive CD8+ lymphocytes in macaques and showed that loss of all CD8α+ cells (using anti-CD8α antibody) significantly impaired early control of Mycobacterium tuberculosis (Mtb) infection, leading to increased granulomas, lung inflammation, and bacterial burden. Analysis of barcoded Mtb from infected macaques demonstrated that depletion of all CD8+ lymphocytes allowed increased establishment of Mtb in lungs and dissemination within lungs and to lymph nodes, while depletion of only adaptive CD8+ T cells (with anti-CD8β antibody) worsened bacterial control in lymph nodes. Flow cytometry and single-cell RNA sequencing revealed polyfunctional cytotoxic CD8+ lymphocytes in control granulomas, while CD8-depleted animals were unexpectedly enriched in CD4 and γδ T cells adopting incomplete cytotoxic signatures. Ligand-receptor analyses identified IL-15 signaling in granulomas as a driver of cytotoxic T cells. These data support that CD8+ lymphocytes are required for early protection against Mtb and suggest polyfunctional cytotoxic responses as a vaccine target.