Naama Geva-Zatorsky

Harvard Medical School | HMS · Department of Microbiology and Immunobiology

Aims: Oxidative modifications of cysteine thiols regulate various physiological processes including inflammatory responses. The thioredoxin system plays a key role in thiol redox control. The aim of this study was to characterize the dynamic cysteine proteome of human macrophages upon activation by the prototypical pro-inflammatory agent, bacterial...

We leverage electroosmotic-flow generation in porous media in combination with a hydrophobic air gap to create a controllable valve capable of operating in either finite dosing or continuous flow mode, enabling the implementation of multi-step assays on paper-based devices. The hydrophobic air gap between two paper pads creates a barrier keeping th...

This work presents the use of electroosmotic flow generation in porous media in combination with a hydrophobic air gap to create a controllable valve capable of operating in either finite dosing or continuous flow mode, enabling the implementation of multi-step biochemical assays on paper-based devices. A hierarchical superhydrophobic surface place...

Background Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. These potentially limb-threatening ulcers are challenging to treat due to impaired wound healing characterizing diabetic patients and the complex microbial environment of these ulcers.AimTo analyze the microbiome of IDFU in association with clinical outco...

Background Infected diabetic foot ulcers (IDFU) are a major complication of diabetes mellitus. These potentially limb-threatening ulcers are challenging to treat due to the impairment of wound healing in diabetic patients and the complex microbial environment characterizing these ulcers. Our aim was to analyze the microbiome of IDFU in association...

Patterns of diurnal activity differ substantially between individuals, with early risers and late sleepers being examples of opposite chronotypes. Growing evidence suggests that the late chronotype significantly impacts the risk of developing mood disorders, obesity, diabetes, and other chronic diseases. Despite the vast potential of utilizing chro...

Gut bacteria were shown to exert pivotal effects on health and disease. However, mechanistic studies of gut bacterial communities are limited due to the lack of technologies for real-time studies on live bacteria. Here, we developed COMBInatorial cliCK-chemistry (COMBICK) labeling on human gut-derived bacteria, both aerobic and anaerobic strains, t...

Background: Idiopathic intracranial hypertension syndrome (IIH) is most common among obese women. Weight loss is an important factor in improving papilledema. Over the last decade, growing evidence has identified gut microbiota as a potential factor in the pathophysiology of obesity. Accordingly, we investigated whether the gut microbiome is modif...

Our gut bacteria influence multiple physiological process including the immune response of our body. In this article, we describe a method for labeling anaerobic gut bacteria with a fluorescent marker that reflects light if it is illuminated, just like light reflectors on our bikes. This marker can be identified by a special microscope. Using this...

When we think about bacteria, we often think first about disease. While there are many bacteria that cause diseases, there are also many other bacteria that are useful for human health. The human body has an enormous number of these good bacteria, which work together and with the immune system to protect us against diseases. In this article, we wil...

A variety of species of bacteria are known to colonize human tumours1–11, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment12–14. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antige...

Patterns of diurnal activity differ substantially between individuals, with early risers and late sleepers being examples of extreme chronotypes. Growing evidence suggests that the late chronotype significantly impacts the risk of developing mood disorders, obesity, diabetes, and other chronic diseases. Despite the vast potential of utilizing chron...

The gut microbiota and the immune system have co‐evolved to interact and cooperate in many ways. A recent study characterizing the immunomodulatory effects of over 60 different human‐derived gut microbes across phyla showed that bacteria‐induced immunomodulations are not dictated by the bacterial phylogeny. Yet, it remains unclear whether strains f...

The COVID-19 pandemic has the potential to affect the human microbiome in infected and uninfected individuals, having a substantial impact on human health over the long term. This pandemic intersects with a decades-long decline in microbial diversity and ancestral microbes due to hygiene, antibiotics, and urban living (the hygiene hypothesis). High...

SARS-CoV-2 has quickly spread all around the globe causing illness and wide damages. Most countries were unprepared for such a rapid spread and crisis. This led to various strategies for effective control of the new pandemic. A key aspect in all countries was to effectively test the population for the virus. Most countries chose a lockdown strategy...

The metabolic pathways encoded by the human gut microbiome constantly interact with host gene products through numerous bioactive molecules1. Primary bile acids (BAs) are synthesized within hepatocytes and released into the duodenum to facilitate absorption of lipids or fat-soluble vitamins2. Some BAs (approximately 5%) escape into the colon, where...

Animals exist as biodiverse composite organisms that include microbial residents, eukaryotic cells, and organs that collectively form a human being. Through an interdependent relationship and an inherent ability to transmit and reciprocate stimuli in a bidirectional way, a human body or the holobiont secures growth, health, and reproduction. As suc...

Within the human gut reside diverse microbes coexisting with the host in a mutually advantageous relationship. Evidence has revealed the pivotal role of the gut microbiota in shaping the immune system. To date, only a few of these microbes have been shown to modulate specific immune parameters. Herein, we broadly identify the immunomodulatory effec...

Significance Th17 cells accumulate in the gut, where they mediate barrier defenses and repair but can also provoke inflammatory disease. In mice, segmented filamentous bacteria (SFB) is sufficient to induce Th17 cells in the gut, but functionally analogous microbes in humans have not been defined. Here, we identified Bifidobacterium adolescentis as...

Adaptive co-evolution has formed a complex network of inextricable bonds between the resident microbes and the mammalian host. These symbiotic microbes are now believed to have critical impacts on human health. One of the most common gut symbionts, Bacteroides fragilis, produces an outersurface polysaccharide (PSA) that possesses immunomodulatory e...

Significance Mechanisms of competition are not well-studied in the mammalian gut microbiota, especially among abundant species of this ecosystem. Theoretical models predict that antagonistic mechanisms should profoundly affect member fitness, yet identification and experimental analyses of such factors are few. Here we show that the abundant gut sp...

s: CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY Just as the organs within an individual must communicate to function as a whole, the interactions of the microbiota with the host define local and systemic physiology. Within the intestine, pattern r...

The intestine is densely populated by anaerobic commensal bacteria. These microorganisms shape immune system development, but understanding of host-commensal interactions is hampered by a lack of tools for studying the anaerobic intestinal environment. We applied metabolic oligosaccharide engineering and bioorthogonal click chemistry to label vario...

T regulatory cells that express the transcription factor Foxp3 (Foxp3(+) Treg) promote tissue homeostasis in several settings. We now report that symbiotic members of the human gut microbiota induce a distinct Treg population in the mouse colon, which constrains immuno-inflammatory responses. This induction-which we find to map to a broad, but spec...

Author Summary Inferring the function of proteins and the role they play in cellular processes is essential for our understanding of cell biology, genetics and biology in general. Standard genetic approaches use large perturbations to cells such as gene knockout, knockdown or over expression of genes. Such methods are powerful, but have the drawbac...

Regulation of proteins across the cell cycle is a basic process in cell biology. It has been difficult to study this globally in human cells due to lack of methods to accurately follow protein levels and localizations over time. Estimates based on global mRNA measurements suggest that only a few percent of human genes have cell-cycle dependent mRNA...

The Supplementary table S1 includes the full dataset of protein profiles and scores. (XLSX)

The online Supplementary material includes additional information about the methods and results presented above. (DOCX)

Bootstrapping approach was used to determine the threshold for cycling genes. Data from four different proteins, each with 48 repeat movies was used to generate histograms of experimental deviation between profiles. For each protein, 4 different movies were chosen randomly from the set of 48 and a profile was generated. A score was calculated (90th...

Single cell trajectories display linear profiles in the case of YB1 and quadratic profiles in the case of PRC1. Each single cell trajectory denoting fluorescent levels measured over the cell cycle was fit to a polynomial function of first and second degree. Plotted are the pearson correlation coefficient values (R2) of the experimental data and the...

Analysis of cell-cycle dependency of protein level using the median pixel. On the left, the median average profile is shown when using the median pixel. On the right, Bootstrapping approach was used to determine the threshold for cycling genes based on median pixel. Similar analysis to the analysis descibed in Figure S1 was done here for protein pr...

Normalization of partial tracks of single cells. This example illustrates how partial tracks of a single cell that divided 8 hours after the beginning of the movie to 2 daughter cells were used. The partial tracks were normalized to the average cell cycle (21 hours) and overlaid on the cell-cycle profile accordingly. (TIF)

Protein removal has a central role in numerous cellular processes. Obtaining systematic measurements of multiple protein removal rates is necessary to understand the principles that govern these processes, but it is currently a major technical challenge. To address this, we developed 'bleach-chase', a noninvasive method for measuring the half-lives...

Cells remove proteins by two processes: degradation and dilution due to cell growth. The balance between these basic processes is poorly understood. We addressed this by developing an accurate and noninvasive method for measuring protein half-lives, called "bleach-chase," that is applicable to fluorescently tagged proteins. Assaying 100 proteins in...

A key circuit in the response of cells to damage is the p53-mdm2 feedback loop. This circuit shows sustained, noisy oscillations in individual human cells following DNA breaks. Here, we apply an engineering approach known as systems identification to quantify the in vivo interactions in the circuit on the basis of accurate measurements of its power...

Drugs and drug combinations have complex biological effects on cells and organisms. Little is known about how drugs affect protein dynamics that determine these effects. Here, we use a dynamic proteomics approach to accurately follow 15 protein levels in human cells in response to 13 different drugs. We find that protein dynamics in response to com...

Recent advances allow tracking the levels and locations of a thousand proteins in individual living human cells over time using a library of annotated reporter cell clones (LARC). This library was created by Cohen et al. to study the proteome dynamics of a human lung carcinoma cell-line treated with an anti-cancer drug. Here, we report the Dynamic...

Immunoblots of YB1, PRC1, and ANLN with anti-GFP. Estimated molecular weight of each protein without yellow fluorescent tag is: YB1 ∼36 kDa, PRC1 ∼71 kDa and ANLN ∼124 kDa. YFP ∼27 kDa. (1.85 MB TIF)

Coefficient of Variance (CV) of PRC1 and YB1. CV of PRC1 (blue) and YB1 (red) of protein levls across cells synchronized to the beginning of protein accumulation. Inset denotes the CV of the first 4 hours. (1.35 MB TIF)

Correlation across consecutive cell cycles. (A,B) cells were tracked for two consecutive cell cycles. Shown is the correlation matrix of each time point in first cell cycle and all time points in the second cell cycle for PRC1 and YB1 respectively. Note that PRC1 doesn't retain any memory across cell cycles while YB1 shows relatively high correlati...

(0.61 MB PDF)

Time-lapse movie of transmitted light images of the clone with YFP CD-tagged PRC1. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (1.93 MB AVI)

Time-lapse movie of yellow fluorescence images of the clone with YFP CD-tagged YB1. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (3.61 MB AVI)

Protein distribution within cellular compartments during cell cycle. (A,B) Mean of total fluorescent levels in different compartments of the cell (cyan - cytoplasm, magenta - nucleus and black- whole cell) for PRC1 and YB1. (C,D) Mean of mean fluorescent levels in different compartments of the cell (cyan - cytoplasm, magenta - nucleus) for PRC1 and...

Cell cycle dependent behavior of ANLN. (A) Mean of total fluorescent levels in different compartments of the cell (cyan - cytoplasm, magenta - nucleus and black- whole cell). (B) Mean of mean fluorescent levels in different compartments of the cell (cyan - cytoplasm, magenta - nucleus). (C) The same as Legend of figure S5A just for ANLN. (0.10 MB T...

Time-lapse movie of yellow fluorescence images overlaid on transmitted light images of the clone with YFP CD-tagged ANLN. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (1.76 MB AVI)

Cell cycle dependent behavior of ANLN. (A) Example of one cell automatically tracked through the cell cycle. Each image is an overlay of fluorescent tagged protein on phase contrast image. Images are ordered according to the fraction of the time elapsed between two division events. The cells are automatically centered. The percentage of elapsed cel...

Time-lapse movie of yellow fluorescence images overlaid on transmitted light images of the clone with YFP CD-tagged YB1. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (2.39 MB AVI)

Time-lapse movie of transmitted light images of the clone with YFP CD-tagged ANLN. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (1.60 MB AVI)

Time-lapse movie of yellow fluorescence images of the clone with YFP CD-tagged ANLN. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (1.12 MB AVI)

Time-lapse movie of yellow fluorescence images of the clone with YFP CD-tagged PRC1. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (2.82 MB AVI)

Time-lapse movie of yellow fluorescence images overlaid on transmitted light images of the clone with YFP CD-tagged PRC1. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (2.03 MB AVI)

Time-lapse movie of transmitted light images of the clone with YFP CD-tagged YB1. Movie duration is 46 hours. (time-lapse: 1 frame per 20 minutes). (2.25 MB AVI)

A current challenge in biology is to understand the dynamics of protein circuits in living human cells. Can one define and test equations for the dynamics and variability of a protein over time? Here, we address this experimentally and theoretically, by means of accurate time-resolved measurements of endogenously tagged proteins in individual human...

Why do seemingly identical cells respond differently to a drug? To address this, we studied the dynamics and variability of the protein response of human cancer cells to a chemotherapy drug, camptothecin. We present a dynamic-proteomics approach that measures the levels and locations of nearly 1000 different endogenously tagged proteins in individu...

We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation p...

Protein expression is a stochastic process that leads to phenotypic variation among cells. The cell-cell distribution of protein levels in microorganisms has been well characterized but little is known about such variability in human cells. Here, we studied the variability of protein levels in human cells, as well as the temporal dynamics of this v...

We examined cell cycle-dependent changes in the proteome of human cells by systematically measuring protein dynamics in individual living cells. We used time-lapse microscopy to measure the dynamics of a random subset of 20 nuclear proteins, each tagged with yellow fluorescent protein (YFP) at its endogenous chromosomal location. We synchronized th...

Supplementary Figures and Information

Supplementary Movie

Understanding the dynamics and variability of protein circuitry requires accurate measurements in living cells as well as theoretical models. To address this, we employed one of the best-studied protein circuits in human cells, the negative feedback loop between the tumor suppressor p53 and the oncogene Mdm2. We measured the dynamics of fluorescent...

The tumor suppressor p53, one of the most intensely investigated proteins, is usually studied by experiments that are averaged over cell populations, potentially masking the dynamic behavior in individual cells. We present a system for following, in individual living cells, the dynamics of p53 and its negative regulator Mdm2 (refs. 1,4-7): this sys...