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Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.

Single-cell technologies are revolutionizing biology but are today mainly limited to imaging and deep sequencing. However, proteins are the main drivers of cellular function and in-depth characterization of individual cells by mass spectrometry (MS)-based proteomics would thus be highly valuable and complementary. Here, we develop a robust workflow combining miniaturized sample preparation, very low flow-rate chromatography, and a novel trapped ion mobility mass spectrometer, resulting in a more than 10-fold improved sensitivity. We precisely and robustly quantify proteomes and their changes in single, FACS-isolated cells. Arresting cells at defined stages of the cell cycle by drug treatment retrieves expected key regulators. Furthermore, it highlights potential novel ones and allows cell phase prediction. Comparing the variability in more than 430 single-cell proteomes to transcriptome data revealed a stable-core proteome despite perturbation, while the transcriptome appears stochastic. Our technology can readily be applied to ultra-high sensitivity analyses of tissue material, posttranslational modifications, and small molecule studies from small cell counts to gain unprecedented insights into cellular heterogeneity in health and disease.

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2(+)TMPRSS2(+) cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention. An integrated analysis of over 100 single-cell and single-nucleus transcriptomics studies illustrates severe acute respiratory syndrome coronavirus 2 viral entry gene coexpression patterns across different human tissues, and shows association of age, smoking status and sex with viral entry gene expression in respiratory cell populations.

Massively parallel reporter assays (MPRAs) can measure the regulatory function of thousands of DNA sequences in a single experiment. Despite growing popularity, MPRA studies are limited by a lack of a unified framework for analyzing the resulting data. Here we present MPRAnalyze: a statistical framework for analyzing MPRA count data. Our model leverages the unique structure of MPRA data to quantify the function of regulatory sequences, compare sequences' activity across different conditions, and provide necessary flexibility in an evolving field. We demonstrate the accuracy and applicability of MPRAnalyze on simulated and published data and compare it with existing methods.

Massively parallel reporter assays (MPRAs) can measure the regulatory function of thousands of DNA sequences in a single experiment. Despite growing popularity, MPRA studies are limited by a lack of a unified framework for analyzing the resulting data. Here we present MPRAnalyze: a statistical framework for analyzing MPRA count data. Our model leverages the unique structure of MPRA data to quantify the function of regulatory sequences, compare sequences' activity across different conditions, and provide necessary flexibility in an evolving field. We demonstrate the accuracy and applicability of MPRAnalyze on simulated and published data and compare it with existing methods.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, February 2011., This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections., Cataloged from student submitted PDF version of thesis., Includes bibliographical references (p. 174-180)., Despite having a broad set of desirable properties, silicon's potential as a primary constituent in a structural material has not yet been realized because of its extremely low fracture toughness. Motivated by the microstructural design techniques used in toughening inherently brittle ceramic materials, this work aims to develop a silicon-rich alloy with microstructural features that provide for the same types of toughening mechanisms displayed by technical ceramics. In order to add true commercial value to these silicon-based alloys, however, the alloys must be processed using methods more flexible and less expensive than the powder processing routes currently used for engineering ceramics. This thesis will discuss the development of a class of castable silicon-based alloys referred to as silicon-disilicide (Si-XSi2) composites, which naturally form a microstructure composed of a silicon matrix and reinforcing disilicide (XSi2) phase during solidification (where X is a transition metal). Experimental work is performed to characterize the thermal, microstructural, and fracture properties of a specific set of Si-(Cr,V)Si2 alloys which are based on the Si-CrSi2-VSi2 system. First, a reliable thermodynamic description of the Si-CrSi2-VSi2 system is obtained, from which the relevant phase diagram is determined. Comparison between simulated solidification paths and experimentally observed microstructures demonstrates the use of the thermodynamic database to predict the phase evolution of the alloys during processing. Long-crack fracture toughness measurements made through chevron-notched beam (CNB) tests show that the toughness of the composite alloys are over 2-3 times that of unalloyed silicon, with in-situ tests revealing the role of microstructural toughening (via crack deflection and crack bridging) on the enhanced fracture properties. Ball-on-disk experiments reveal an order of magnitude improvement in the wear resistance of the alloys compared to silicon. This enh, by David S. Fischer., Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, February 2011., This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections., Cataloged from student submitted PDF version of thesis., Includes bibliographical references (p. 174-180)., Despite having a broad set of desirable properties, silicon's potential as a primary constituent in a structural material has not yet been realized because of its extremely low fracture toughness. Motivated by the microstructural design techniques used in toughening inherently brittle ceramic materials, this work aims to develop a silicon-rich alloy with microstructural features that provide for the same types of toughening mechanisms displayed by technical ceramics. In order to add true commercial value to these silicon-based alloys, however, the alloys must be processed using methods more flexible and less expensive than the powder processing routes currently used for engineering ceramics. This thesis will discuss the development of a class of castable silicon-based alloys referred to as silicon-disilicide (Si-XSi2) composites, which naturally form a microstructure composed of a silicon matrix and reinforcing disilicide (XSi2) phase during solidification (where X is a transition metal). Experimental work is performed to characterize the thermal, microstructural, and fracture properties of a specific set of Si-(Cr,V)Si2 alloys which are based on the Si-CrSi2-VSi2 system. First, a reliable thermodynamic description of the Si-CrSi2-VSi2 system is obtained, from which the relevant phase diagram is determined. Comparison between simulated solidification paths and experimentally observed microstructures demonstrates the use of the thermodynamic database to predict the phase evolution of the alloys during processing. Long-crack fracture toughness measurements made through chevron-notched beam (CNB) tests show that the toughness of the composite alloys are over 2-3 times that of unalloyed silicon, with in-situ tests revealing the role of microstructural toughening (via crack deflection and crack bridging) on the enhanced fracture properties. Ball-on-disk experiments reveal an order of magnitude improvement in the wear resistance of the alloys compared to silicon. This enh, by David S. Fischer., Ph.D.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2008., Vita., Includes bibliographical references (leaves 112-113)., A fluxless low temperature transient liquid phase (LTTLP) bonding process was studied as a method of producing Cu/Cu joints below 125°C and 75°C using interlayer alloys from the In-Sn and In-Sn-Bi systems. Using thermodynamic models, three different compositions (wt. %) of base alloys were chosen to accomplish this task: 50In-43.6Sn-6.4Bi (Tm = 110°C) and eutectic 50.9In-49.1Sn (Tm = 120°C) alloys were used for bonding at 125°C and a eutectic 48.3In-15.6Sn-36.1Bi (Tm = 60°C) alloy was used for bonding at 75°C. In addition, novel approaches to TLP bonding, including the addition of base material to the interlayer alloy and application of an electroless Ni diffusion barrier layer, were employed in an attempt to optimize this joining method. The LTTLP processes were assessed based on their abilities to produce joints with minimal thickness, high reflow temperatures, and good mechanical properties at room/elevated temperatures. It was found that interlayer alloys containing higher Bi contents produced the thinnest joints, with the 48.3In-15.6Sn-36.1Bi alloy producing joints on the order of 10 gm. Increases in nominal Cu composition of the interlayer alloy tended to form larger joints. Application of the Ni layer was observed to decrease the growth rate of the eutectic In-Sn joints made with 5 wt % Cu additions. Shear tests were performed on the joints at room (25°C) and operating (service) temperatures (100°C). Most of the TLP joints had room temperature shear strengths around 13,000 - 17,000 psi (= 90 - 120 MPa), although increases in strength were observed for eutectic In-Sn joints with 2.5 and 5 wt% Cu additions. At operating temperature, TLP joints made within the In-Sn-Cu system were found to have strengths an order of magnitude higher than those made in the In-Sn-Bi-Cu system., (cont.) Poor mechanical response of the Bicontaining joints was due to the presence of low melting In-Bi IPs present in the reaction zone. Eutectic In-Sn TLP joints made with 2.5 and 5 wt% Cu additions were found to have operational temperature shear strengths of 6,000 - 7,500 psi ( 40 - 50 MPa) and 7,500 - 9,500 psi (= 50 65 MPa), respectively., by David S. Fischer., S.M.