Publications & documents

A Field Experiment on Search Costs and the Formation of Scientific Collaborations

Boudreau K, Brady T, Ganguli I, Gaule P, Guinan EC, Hollenberg T, and Lakhani KR
Scientists typically self-organize into teams, matching with others to collaborate in the production of new knowledge. We present the results of a field experiment conducted at Harvard Medical School to understand the extent to which search costs affect matching among scientific collaborators. We generated exogenous variation in search costs for pairs of potential collaborators by randomly assigning individuals to 90-minute structured information-sharing sessions as part of a grant funding opportunity for biomedical researchers. We estimate that the treatment increases the baseline probability of grant co-application of a given pair of researchers by 75% (increasing the likelihood of a pair collaborating from 0.16 percent to 0.28 percent), with effects higher among those in the same specialization. The findings indicate that matching between scientists is subject to considerable frictions, even in the case of geographically-proximate scientists working in the same institutional context with ample access to common information and funding opportunities.

Acyldepsipeptide Antibiotics Kill Mycobacteria by Preventing the Physiological Functions of the ClpP1P2 Protease

Famulla K, Sass P, Malik I, Akopian T, Kandror O, Alber M, Hinzen B, Ruebsamen-Schaeff H, Kalscheuer R, Goldberg AL, and Brötz-Oesterhelt H
The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.

Analysis of Normal Retinal Nerve Fiber Layer Thickness by Age, Sex, and Race Using Spectral Domain Optical Coherence Tomography

Alasil T, Wang K, Keane PA, Lee H, Baniasadi N, Boer de JF, and Chen TC
Purpose: To determine the effects of age, sex, and race on the retinal nerve fiber layer (RNFL) in the normal human eye as measured by the spectral domain optical coherence tomography (SD-OCT) Spectralis machine (Heidelberg Engineering).

Methods: Peripapillary SD-OCT RNFL thickness measurements were determined in normal subjects seen at a university-based clinic. One randomly selected eye per subject was used for analysis in this cross-sectional study. Multiple regression analysis was applied to assess the effects of age, sex, ethnicity, and mean refractive error on peripapillary RNFL thickness. Results are expressed as means±SD wherever applicable.

Results: The study population consisted of 190 healthy participants from 9 to 86 years of age. Of the 190 participants, 62 (33%) were men, 125 (66%) Caucasians, 26 (14%) African Americans, 14 (7%) Hispanics, 16 (8%) Asians, and 9 (5%) other races. The mean RNFL thickness for the normal population studied was 97.3±9.6 µm. Normal RNFL thickness values follow the ISNT rule with decreasing RNFL thickness values starting from the thickest quadrant inferiorly to the thinnest quadrant temporally: inferior quadrant (126±15.8), superior quadrant (117.2±16.13), nasal quadrant (75±13.9), and temporal quadrant (70.6±10.8 µm). Thinner RNFL measurements were associated with older age (P<0.001); being Caucasian, versus being either Hispanic or Asian (P=0.02 and 0.009, respectively); or being more myopic (P<0.001). For every decade of increased age, mean RNFL thickness measured thinner by approximately 1.5 µm (95% confidence interval, 0.24-0.07). Comparisons between ethnic groups revealed that Caucasians had mean RNFL values (96±9.2 µm) slightly thinner than those of Hispanics (102.9±11 µm; P=0.02) or Asians (100.7±8.5 µm; P=0.009). African Americans RNFL values (99.2±10.2 µm) were not significantly different when compared with Caucasians. There was no relationship between RNFL thickness and sex.

Conclusions: The thickest RNFL measurements were found in the inferior quadrant, followed by the superior, nasal, and temporal quadrants (ISNT rule applied to the RNFL). Thinner RNFL measurements were associated with older age and increasing myopia. Caucasians tend to have thinner RNFL values when compared with Hispanics and Asians. SD-OCT analysis of the normal RNFL showed results similar to time domain OCT studies.

Analysis of Poration-Induced Changes in Cells from Laser-Activated Plasmonic Substrates

Saklayen N, Kalies S, Madrid M, Nuzzo V, Huber M, Shen W, Sinanan-Singh J, Heinemann D, Heisterkamp A, and Mazur E
Laser-exposed plasmonic substrates permeabilize the plasma membrane of cells when in close contact to deliver cell-impermeable cargo. While studies have determined the cargo delivery efficiency and viability of laser-exposed plasmonic substrates, morphological changes in a cell have not been quantified. We porated myoblast C2C12 cells on a plasmonic pyramid array using a 532-nm laser with 850-ps pulse length and time-lapse fluorescence imaging to quantify cellular changes. We obtain a poration efficiency of 80%, viability of 90%, and a pore radius of 20 nm. We quantified area changes in the plasma membrane attached to the substrate (10% decrease), nucleus (5 - 10% decrease), and cytoplasm (5 - 10% decrease) over 1 h after laser treatment. Cytoskeleton fibers show a change of 50% in the alignment, or coherency, of fibers, which stabilizes after 10 mins. We investigate structural and morphological changes due to the poration process to enable the safe development of this technique for therapeutic applications.

Automated Interpretation of Blood Culture Gram Stains Using a Deep Convolutional Neural Network

Smith KP, Kang AD, and Kirby JE
Microscopic interpretation of stained smears is one of the most operator-dependent and time intensive activities in the clinical microbiology laboratory. Here, we investigated application of an automated image acquisition and convolutional neural network (CNN)-based approach for automated Gram stain classification. Using an automated microscopy platform, uncoverslipped slides were scanned with a 40x dry objective, generating images of sufficient resolution for interpretation. We collected 25,488 images from positive blood culture Gram stains prepared during routine clinical workup. These images were used to generate 100,213 crops containing Gram-positive cocci in clusters, Gram-positive cocci in chains/pairs, Gram-negative rods, or background (no cells). These categories were targeted for proof-of-concept development as they are associated with the majority of bloodstream infections. Our CNN model achieved classification accuracy of 94.9% on a test set of image crops. Receiver operating characteristic curve (ROC) analysis indicated a robust ability to differentiate between categories with area under the curve >0.98 for each. After training and validation, we applied the classification algorithm to new images collected from 189 whole slides without human intervention. Sensitivity/specificity was 98.4/75.0% for Gram-positive cocci in chains/pairs; 93.2/97.2% for Gram-positive cocci in clusters; and 96.3/98.1% for Gram-negative rods. Taken together, our data support proof-of-concept for a fully automated classification methodology for blood-culture Gram-stains. Importantly, the algorithm was highly adept at identifying image crops with organisms and could be used to present prescreened, classified crops to technologists to accelerate smear review. This concept could potentially be extended to all Gram stain interpretive activities in the clinical laboratory.

Bayesian Meta-Analysis of Multiple Continuous Treatments with Individual Participant-Level Data: An Application to Antipsychotic Drugs

Spertus JV, M Horvitz-Lemon, and SL Normand
Modeling dose-response relationships of drugs is essential to understanding their safety effects on patients under realistic circumstances. While intention-to-treat analyses of clinical trials provide the effect of assignment to a particular drug and dose, they do not capture observed exposure after factoring in nonadherence and dropout. We develop a Bayesian method to flexibly model the dose-response relationships of binary outcomes with continuous treatment, permitting multiple evidence sources, treatment effect heterogeneity, and nonlinear dose-response curves. In an application, we examine the risk of excessive weight gain for patients with schizophrenia treated with the second-generation antipsychotics paliperidone, risperidone, or olanzapine in 14 clinical trials. We define exposure as total cumulative dose (daily dose × duration) and convert to units equivalent to 100 mg of olanzapine (OLZ doses). Averaging over the sample population of 5891 subjects, the median dose ranged from 0 (placebo randomized participants) to 6.4 OLZ doses (paliperidone randomized participants). We found paliperidone to be least likely to cause excessive weight gain across a range of doses. Compared with 0 OLZ doses, at 5.0 OLZ doses, olanzapine subjects had a 15.6% (95% credible interval: 6.7, 27.1) excess risk of weight gain; corresponding estimates for paliperidone and risperidone were 3.2% (1.5, 5.2) and 14.9% (0.0, 38.7), respectively. Moreover, compared with nonblack participants, black participants had a 6.8% (1.0, 12.4) greater risk of excessive weight gain at 10.0 OLZ doses of paliperidone. Nevertheless, our findings suggest that paliperidone is safer in terms of weight gain risk than risperidone or olanzapine for all participants at low to moderate cumulative OLZ doses.

Biomechanical Characterization of Keratoconus Corneas Ex Vivo with Brillouin Microscopy

Scarcelli G, Besner S, Pineda R, and Yun SH

Loss of corneal strength is a central feature of keratoconus progression. However, it is currently difficult to measure corneal mechanical changes noninvasively. The objective of this study is to evaluate if Brillouin optical microscopy can differentiate the mechanical properties of keratoconic corneas versus healthy corneas ex vivo.

We obtained eight tissue samples from healthy donor corneas used in Descemet's stripping endothelial keratoplasty (DSEK) and 10 advanced keratoconic corneas from patients undergoing deep anterior lamellar keratoplasty (DALK). Within 2 hours after surgery, a confocal Brillouin microscope using a monochromatic laser at 532 nm was used to map the Brillouin frequency shifts of the corneas.

The mean Brillouin shift in the anterior 200 μm of the keratoconic corneas at the cone was measured to be 7.99 ± 0.10 GHz, significantly lower than 8.17 ± 0.06 GHz of the healthy corneas (P < 0.001). The Brillouin shift in the keratoconic corneas decreased with depth from the anterior toward posterior regions with a steeper slope than in the healthy corneas (P < 0.001). Within keratoconic corneas, the Brillouin shift in regions away from the apex of the cone was significantly higher than within the cone region (P < 0.001).

Brillouin measurements revealed notable differences between healthy and keratoconic corneas. Importantly, Brillouin imaging showed that the mechanical loss is primarily concentrated within the area of the keratoconic cone. Outside the cone, the Brillouin shift was comparable with that of healthy corneas. The results demonstrate the potential of Brillouin microscopy for diagnosis and treatment monitoring of keratoconus.

Brillouin Microscopy of Collagen Crosslinking: Noncontact Depth-Dependent Analysis of Corneal Elastic Modulus

Scarcelli G, Kling S, Quijano E, Pineda R, Marcos S, and Yun SH
Corneal collagen crosslinking (CXL) is designed to halt the progression of keratoconus and corneal ectasia by inducing corneal stiffening. However, it currently is difficult to monitor and evaluate CXL outcome objectively due to the lack of suitable methods to characterize corneal mechanical properties. We validated noncontact Brillouin microscopy to quantify corneal mechanical properties before and after CXL.

CXL was performed on fresh porcine eyes using various presoaking times and light doses, with or without epithelial debridement. From Brillouin maps of corneal elastic modulus, stiffness and average modulus of anterior, middle, and posterior stroma were analyzed. Corneal stiffening index (CSI) was introduced as a metric to compare the mechanical efficacy of a given CXL protocol with respect to the standard protocol (30-minute riboflavin presoak, 3 mW/cm² ultraviolet illumination for 30 minutes).

Brillouin corneal stiffness increased significantly (P < 0.001) by epi-off and epi-on CXL. The increase of Brillouin modulus was depth-dependent, indicating that anterior stromal stiffening contributes the most to mechanical outcome. The increase of anterior Brillouin modulus was linearly proportional to the light dose (R² > 0.98). Compared to the standard epi-off procedure, a typical epi-on procedure resulted in a third of stiffness increase in porcine corneas (CSI = 33).

Brillouin microscopy allowed imaging and quantifying CXL-induced mechanical changes without contact in a depth-dependent manner at high spatial resolution. This technique may be useful to evaluate the mechanical outcomes of CXL procedures, to compare different crosslinking agents, and for real-time monitoring of CXL in clinical and experimental settings.

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

Akopian T, Kandror O, Tsu C, Lai JH, Wu W, Liu Y, Zhao P, Park A, Wolf L, Dick LR, Rubin EJ, Bachovchin W, and Goldberg AL
The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X3X2X1-aminomethylcoumarin) and by determining specificity constants (kcat/Km), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X1 position, basic residues or Trp in the X2 position, and Pro ≫ Ala > Trp in the X3 position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.

Combined Noninvasive Metabolic and Spindle Imaging as Potential Tools for Embryo and Oocyte Assessment

Tim Sanchez, Marta Venturas, S. Ali Aghvami, Xingbo Yang, Seth Fraden, Denny Sakkas, and Daniel J. Needleman.
Study question: Is the combined use of fluorescence lifetime imaging microscopy (FLIM)-based metabolic imaging and second harmonic generation (SHG) spindle imaging a feasible and safe approach for noninvasive embryo assessment?

Summary answer: Metabolic imaging can sensitively detect meaningful metabolic changes in embryos, SHG produces high-quality images of spindles and the methods do not significantly impair embryo viability.

What is known already: Proper metabolism is essential for embryo viability. Metabolic imaging is a well-tested method for measuring metabolism of cells and tissues, but it is unclear if it is sensitive enough and safe enough for use in embryo assessment.

Study design, size, duration: This study consisted of time-course experiments and control versus treatment experiments. We monitored the metabolism of 25 mouse oocytes with a noninvasive metabolic imaging system while exposing them to oxamate (cytoplasmic lactate dehydrogenase inhibitor) and rotenone (mitochondrial oxidative phosphorylation inhibitor) in series. Mouse embryos (n = 39) were measured every 2 h from the one-cell stage to blastocyst in order to characterize metabolic changes occurring during pre-implantation development. To assess the safety of FLIM illumination, n = 144 illuminated embryos were implanted into n = 12 mice, and n = 108 nonilluminated embryos were implanted into n = 9 mice.

Participants/materials, setting, methods: Experiments were performed in mouse embryos and oocytes. Samples were monitored with noninvasive, FLIM-based metabolic imaging of nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) autofluorescence. Between NADH cytoplasm, NADH mitochondria and FAD mitochondria, a single metabolic measurement produces up to 12 quantitative parameters for characterizing the metabolic state of an embryo. For safety experiments, live birth rates and pup weights (mean ± SEM) were used as endpoints. For all test conditions, the level of significance was set at P < 0.05.

Main results and the role of chance: Measured FLIM parameters were highly sensitive to metabolic changes due to both metabolic perturbations and embryo development. For oocytes, metabolic parameter values were compared before and after exposure to oxamate and rotenone. The metabolic measurements provided a basis for complete separation of the data sets. For embryos, metabolic parameter values were compared between the first division and morula stages, morula and blastocyst and first division and blastocyst. The metabolic measurements again completely separated the data sets. Exposure of embryos to excessive illumination dosages (24 measurements) had no significant effect on live birth rate (5.1 ± 0.94 pups/mouse for illuminated group; 5.7 ± 1.74 pups/mouse for control group) or pup weights (1.88 ± 0.10 g for illuminated group; 1.89 ± 0.11 g for control group).

Limitations, reasons for caution: The study was performed using a mouse model, so conclusions concerning sensitivity and safety may not generalize to human embryos. A limitation of the live birth data is also that although cages were routinely monitored, we could not preclude that some runt pups may have been eaten.

Wider implications of the findings: Promising proof-of-concept results demonstrate that FLIM with SHG provide detailed biological information that may be valuable for the assessment of embryo and oocyte quality. Live birth experiments support the method's safety, arguing for further studies of the clinical utility of these techniques.

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