Imaging red blood cell dynamics by quantitative phase microscopy

Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease.     

An acute model for atrial fibrillation arising from a peripheral atrial site: evidence for primary and secondary triggers

Background: We previously demonstrated that acetylcholine (Ach) injected into cardiac ganglionated plexi (GP) causes pulmonary vein (PV) ectopy initiating atrial fibrillation (AF).
Objective: To determine the effects of Ach applied at non-PV sites.
Methods: Overall, 54 dogs were anesthetized with Na-pentobarbital. A right and left thoracotomy allowed the placement of multielectrode catheters to record from the superior PVs, mid portion of the atrium and the atrial appendages (AA). A monophasic action potential (MAP) was recorded from the AA. Ach (1, 10, 100 mM) was applied sequentially to the AA.
Results: In 19 of 26 animals, Ach 100 mM on the right (n = 15) or left (n = 4) AA induced focal, sustained AF (≥10 minutes) with rapid regular firing (cycle length = 37 ± 7 ms) at the AA. A clamp with teeth placed across the AA caused arrest in the AA. However, AF was sustained only when PV sites adjacent to the GP manifested complex fractionated atrial electrograms (CFAE). Clamping the AA prior to Ach (100 mM) application resulted in focal AF arising at the PVs but not at the AA. When a clamp without teeth was applied prior to Ach application, no AF at either AA or PV site could be induced.
Conclusion: Isolation of the focal AF at the AA (primary trigger) by clamping caused cessation of activity in the AA, but AF continued due to secondary triggers arising from PVs. The possible mechanism(s) responsible for these findings are discussed, and various ancillary experiments (n = 28) were added to help elucidate mechanisms.     

Quantification of (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene adducts in human serum albumin by laser-induced fluorescence: implications for the in vivo metabolism of benzo[a]pyrene.

The ubiquitous environmental carcinogen benzo[a]pyrene (BaP) is metabolized in vivo in humans to its ultimate carcinogenic form of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE). Mouse skin tumorigenicity studies indicate that the (7R,8S,9S,10R) enantiomer of BPDE, (7R,8S)-dihydroxy-(9S,10R)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7R,8S,9S,10R)-BPDE], is a potent tumor initiator, whereas the (7S,8R,9R,10S) enantiomer of BPDE, (7S,8R)-dihydroxy-(9R,10S)-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(7S,8R,9R,10S)-BPDE], may act as a tumor promoter. In vitro experiments have shown that human liver microsomes are capable of metabolizing BaP to both the (7R,8S,9S,10R) and (7S,8R,9R,10S) enantiomers of BPDE. However, the metabolism of BaP to (7S,8R,9R,10S)-BPDE has not been demonstrated in humans in vivo. The adducts formed between human serum albumin (HSA) and the (7S,8R,9R,10R) and (7R,8S,9S,10R) enantiomers of BPDE have been described previously. (7S,8R,9R,10S)-BPDE forms a stable adduct at histidine146 of HSA, whereas (7R,8S,9R,10R)-BPDE forms a relatively unstable ester adduct at aspartate187 or glutamate188 of HSA. Using high-performance liquid chromatography with laser-induced fluorescence (LIF) detector, we quantified the level of (7S,8R,9R,10S)-BPDE adducts at histidine146 in HSA isolated from 63 healthy males who were population control subjects for an ongoing case-control study of bladder cancer. By design, roughly half of the participants were lifelong nonsmokers (n = 35), whereas the remaining 28 participants were current smokers of varying intensities. HP-BPDE adducts were detected in 60 of the 63 samples (95%) by HPLC-LIF. Adduct levels ranged from undetectable (<0.04 fmol/mg HSA) to 0.77 fmol/mg HSA. The samples had a mean and median (7S,8R,9R,10S)-BPDE-HSA adduct level of 0.22 and 0.16 fmol of adduct/mg albumin, respectively. Mean adduct levels did not differ between smokers and nonsmokers (P = 0.72). Occupational exposure to polycyclic aromatic hydrocarbons was unrelated to adduct level (P = 0.62). Intake frequencies of two food items showed statistically significant associations with adduct levels. Consumption of sweet potatoes was negatively related to adduct level (P = 0.029), whereas intake of grapefruit juice was positively related to adduct level (P = 0.045). None of the three indices of residential ambient air pollution under study showed a statistically significant association with adduct levels.     

Quantitative dispersion microscopy

Refractive index dispersion is an intrinsic optical property and a useful source of contrast in biological imaging studies. In this report, we present the first dispersion phase imaging of living eukaryotic cells. We have developed quantitative dispersion microscopy based on the principle of quantitative phase microscopy. The dual-wavelength quantitative phase microscope makes phase measurements at 310 nm and 400 nm wavelengths to quantify dispersion (refractive index increment ratio) of live cells. The measured dispersion of living HeLa cells is found to be around 1.088, which agrees well with that measured directly for protein solutions using total internal reflection. This technique, together with the dry mass and morphology measurements provided by quantitative phase microscopy, could prove to be a useful tool for distinguishing different types of biomaterials and studying spatial inhomogeneities of biological samples.     

Evidence of association of APOE with age-related macular degeneration: a pooled analysis of 15 studies

Age-related macular degeneration (AMD) is the most common cause of incurable visual impairment in high-income countries. Previous studies report inconsistent associations between AMD and apolipoprotein E (APOE), a lipid transport protein involved in low-density cholesterol modulation. Potential interaction between APOE and sex, and smoking status has been reported. We present a pooled analysis (n = 21,160) demonstrating associations between late AMD and APOε4 (odds ratio [OR] = 0.72 per haplotype; confidence interval [CI]: 0.65-0.74; P = 4.41×10(-11) ) and APOε2 (OR = 1.83 for homozygote carriers; CI: 1.04-3.23; P = 0.04), following adjustment for age group and sex within each study and smoking status. No evidence of interaction between APOE and sex or smoking was found. Ever smokers had significant increased risk relative to never smokers for both neovascular (OR = 1.54; CI: 1.38-1.72; P = 2.8×10(-15) ) and atrophic (OR = 1.38; CI: 1.18-1.61; P = 3.37×10(-5) ) AMD but not early AMD (OR = 0.94; CI: 0.86-1.03; P = 0.16), implicating smoking as a major contributing factor to disease progression from early signs to the visually disabling late forms. Extended haplotype analysis incorporating rs405509 did not identify additional risks beyond ε2 and ε4 haplotypes. Our expanded analysis substantially improves our understanding of the association between the APOE locus and AMD. It further provides evidence supporting the role of cholesterol modulation, and low-density cholesterol specifically, in AMD disease etiology.     

Rheumatologists on the road: a subspecialist's role in caring for the homebound

Objective

By 2030, the number of permanently homebound individuals in the US will increase by 50% to reach 2 million. However, no medicine subspecialty consult services exist for this rising subset of the population. This pilot program establishes a rheumatology consult service for the Mount Sinai Visiting Doctors, the largest primary care academic home visit program in the nation serving more than 1,000 patients in New York City. Our service addresses the unmet need for homebound patients with rheumatic diseases, and secondarily provides an educational opportunity for trainees in community‐based rheumatology.

Methods

Using an electronic medical record, home‐based primary care physicians sent consult requests to the Rheumatology Division. Initial assessments were made using the Routine Assessment of Patient Index Data 3 (RAPID3) questionnaire.

Results

Over 12 months, 57 home visits were made: 31 new consults and 26 followup visits. Reasons for referral included medical management of a known connective tissue disease, question of inflammatory arthritis, and procedures. The demographics for new consults were as follows: 94% women, 45% Hispanic, and 80% between ages 60 and 101 years. Thirty‐nine percent of patients had rheumatoid arthritis. Treatment interventions included addition of a disease‐modifying antirheumatic drug in 11 patients, 11 procedures, nonpharmacologic management in 8 patients, and a change in the dose of the existing medication in 5 patients. At the initial evaluation, the average RAPID3 scores for patients reflected high severity of disease.

Conclusion

The number of consults and the severity of disease seen highlight the importance of a rheumatologist's role in the community, especially because the number of homebound patients will dramatically increase in the future.