Histone oxidation as a new mechanism of metabolic control over gene expression.

The emergence of aerobic respiration created unprecedented bioenergetic advantages, while imposing the need to protect critical genetic information from reactive byproducts of oxidative metabolism (i.e., reactive oxygen species, ROS). The evolution of histone proteins fulfilled the need to shield DNA from these potentially damaging toxins, while providing the means to compact and structure massive eukaryotic genomes. To date, several metabolism-linked histone post-translational modifications (PTMs) have been shown to regulate chromatin structure and gene expression. However, whether and how PTMs enacted by metabolically produced ROS regulate adaptive chromatin remodeling remain relatively unexplored. Here, we review novel mechanistic insights into the interactions of ROS with histones and their consequences for the control of gene expression regulation, cellular plasticity, and behavior.

Quantification of gastric mucosal microcirculation as a surrogate marker of portal hypertension by spatially resolved subdiffuse reflectance spectroscopy in diagnosis of cirrhosis: a proof-of-concept study.

BACKGROUND AND AIMS: Portal pressure can be used to identify patients with chronic liver disease who have progressed to cirrhosis. Portal pressure can also provide accurate prognostication for patients with cirrhosis. However, there are no practical means for assessment of portal pressure. Although it is well established that the gastric mucosal blood supply increases in patients with cirrhosis, this has been difficult to quantify reproducibly. Our group has developed a novel spectroscopic technology called spatially resolved subdiffuse reflectance spectroscopy (SRSRS), which enables quantification of mucosal microcirculation. We aim to ascertain if quantification of the gastric mucosal microcirculation with SRSRS correlates with clinical evidence of portal hypertension. METHODS: Patients undergoing EGD for clinical indications had 10 measurements taken in the endoscopically normal gastric fundus via SRSRS probe to assess the microcirculation. Cases were defined as patients with cirrhosis (n = 18), and controls were those without evidence of liver disease (n = 18); this was corroborated with transient elastography. RESULTS: The blood volume fraction (P = .06) and subdiffuse reflectance (P = .02) from a shallow depth in the gastric fundus were higher in patients with cirrhosis than those without. These markers were combined to yield an overall optical marker that can differentiate patients with cirrhosis from controls with a sensitivity of 72% and specificity of 94% (area under receiver operating curve, 0.82). CONCLUSIONS: Spectroscopic quantification of gastric fundal mucosal microcirculation is a promising surrogate of clinical correlates of portal hypertension. This approach may represent a less-intrusive surrogate biomarker for liver disease prognostication and potentially response to therapy.

Early Upper Aerodigestive Tract Cancer Detection Using Electron Microscopy to Reveal Chromatin Packing Alterations in Buccal Mucosa Cells.

A profound characteristic of field cancerization is alterations in chromatin packing. This study aimed to quantify these alterations using electron microscopy image analysis of buccal mucosa cells of laryngeal, esophageal, and lung cancer patients. Analysis was done on normal-appearing mucosa, believed to be within the cancerization field, and not tumor itself. Large-scale electron microscopy (nanotomy) images were acquired of cancer patients and controls. Within the nuclei, the chromatin packing of euchromatin and heterochromatin was characterized. Furthermore, the chromatin organization was quantified through chromatin packing density scaling. A significant difference was found between the cancer and control groups in the chromatin packing density scaling parameter for length scales below the optical diffraction limit (200 nm) in both the euchromatin (p = 0.002) and the heterochromatin (p = 0.006). The chromatin packing scaling analysis also indicated that the chromatin organization of cancer patients deviated significantly from the control group. They might allow for novel strategies for cancer risk stratification and diagnosis with high sensitivity. This could aid clinicians in personalizing screening strategies for high-risk patients and follow-up strategies for treated cancer patients.

Dynamic Crowding Regulates Transcription.

The nuclear environment is highly crowded by biological macromolecules, including chromatin and mobile proteins, which alter the kinetics and efficiency of transcriptional machinery. These alterations have been described, both theoretically and experimentally, for steady-state crowding densities; however, temporal changes in crowding density ("dynamic crowding") have yet to be integrated with gene expression. Dynamic crowding is pertinent to nuclear biology because processes such as chromatin translocation and protein diffusion lend to highly mobile biological crowders. Therefore, to capture such dynamic crowding and investigate its influence on transcription, we employ a three-pronged, systems-molecular approach. A system of chemical reactions represents the transcription pathway, the rates of which are determined by molecular-scale simulations; Brownian dynamics and Monte Carlo simulations quantify protein diffusion and DNA-protein binding affinity, dependent on macromolecular density. Altogether, this approach shows that transcription depends critically on dynamic crowding as the gene expression resultant from dynamic crowding can be profoundly different than that of steady-state crowding. In fact, expression levels can display both amplification and suppression and are notably different for genes or gene populations with different chemical and structural properties. These properties can be exploited to impose circadian expression, which is asymmetric and varies in strength, or to explain expression in cells under biomechanical stress. Therefore, this work demonstrates that dynamic crowding nontrivially alters transcription kinetics and presents dynamic crowding within the bulk nuclear nanoenvironment as a novel regulatory framework for gene expression.

Uncovering the role of Symbiodiniaceae assemblage composition and abundance in coral bleaching response by minimizing sampling and evolutionary biases.

BACKGROUND: Biodiversity and productivity of coral-reef ecosystems depend upon reef-building corals and their associations with endosymbiotic Symbiodiniaceae, which offer diverse functional capabilities to their hosts. The number of unique symbiotic partners (richness) and relative abundances (evenness) have been hypothesized to affect host response to climate change induced thermal stress. Symbiodiniaceae assemblages with many unique phylotypes may provide greater physiological flexibility or form less stable symbioses; assemblages with low abundance phylotypes may allow corals to retain thermotolerant symbionts or represent associations with less-suitable symbionts. RESULTS: Here we demonstrate that true richness of Symbiodiniaceae phylotype assemblages is generally not discoverable from direct enumeration of unique phylotypes in association records and that cross host-species comparisons are biased by sampling and evolutionary patterns among species. These biases can be minimized through rarefaction of richness (rarefied-richness) and evenness (Probability of Interspecific Encounter, PIE), and analyses that account for phylogenetic patterns. These standardized metrics were calculated for individual Symbiodiniaceae assemblages composed of 377 unique ITS2 phylotypes associated with 123 coral species. Rarefied-richness minimized correlations with sampling effort, while maintaining important underlying characteristics across host bathymetry and geography. Phylogenetic comparative methods reveal significant increases in coral bleaching and mortality associated with increasing Symbiodiniaceae assemblage richness and evenness at the level of host species. CONCLUSIONS: These results indicate that the potential flexibility afforded by assemblages characterized by many phylotypes present at similar relative abundances does not result in decreased bleaching risk and point to the need to characterize the overall functional and genetic diversity of Symbiodiniaceae assemblages to quantify their effect on host fitness under climate change.

Characterizing chromatin packing scaling in whole nuclei using interferometric microscopy.

Chromatin is the macromolecular assembly containing the cell's genetic information, and its architectural conformation facilitates accessibility to activation sites and thus gene expression. We have developed an analytical framework to quantify chromatin structure with spectral microscopy. Chromatin structure can be described as a mass fractal, with packing scaling D up to specific genomic length scales. Considering various system geometries, we established a model to measure D with the interferometric technique partial wave spectroscopy (PWS) and validated the analysis using finite difference time domain to simulate the PWS system. Calculations of D were consistent with ground truth electron microscopy measurements, enabling a high-throughput, label-free approach to quantifying chromatin structure in the nanometer length scale regime.

Evidence for possible association of vitamin D status with cytokine storm and unregulated inflammation in COVID-19 patients.

OBJECTIVES: We present evidence for a possible role of Vitamin D (VitD) deficiency in unregulated cytokine production and inflammation leading to complications in COVID-19 patients. DESIGN: The time-adjusted case mortality ratio (T-CMR) was estimated as the ratio of deceased patients on day N to the confirmed cases on day N-8. The adaptive average of T-CMR (A-CMR) was calculated as a metric of COVID-19 associated mortality. A model based on positivity change (PC) and an estimated prevalence of COVID-19 was used to determine countries with similar screening strategies. A possible association of A-CMR with the mean concentration of 25-hydroxyvitamin D (25(OH)D) in elderly individuals in countries with similar screening strategy was investigated. We considered high C-reactive protein (CRP) in severe COVID-19 patients (CRP ≥ 1 mg/dL) as a surrogate of a cytokine storm. We considered high-sensitivity CRP (hs-CRP) in healthy subjects as hs-CRP ≥ 0.2 mg/dL. RESULTS: A link between 25(OH)D and A-CMR in countries with similar screening strategy is evidence for VitD's possible role in reducing unregulated cytokine production and inflammation among patients with severe COVID-19. We observed an odds ratio (OR) of 1.8 with 95% confidence interval (95% CI) (1.2 to 2.6) and an OR of 1.9 with 95% CI (1.4 to 2.7) for hs-CRP in VitD deficient elderly from low-income families and high-income families, respectively. COVID-19 patient-level data show an OR of 3.4 with 95% CI (2.15 to 5.4) for high CRP in severe COVID-19 patients. CONCLUSION: We conclude that future studies on VitD's role in reducing cytokine storm and COVID-19 mortality are warranted.

Superresolution intrinsic fluorescence imaging of chromatin utilizing native, unmodified nucleic acids for contrast.

Visualizing the nanoscale intracellular structures formed by nucleic acids, such as chromatin, in nonperturbed, structurally and dynamically complex cellular systems, will help expand our understanding of biological processes and open the next frontier for biological discovery. Traditional superresolution techniques to visualize subdiffractional macromolecular structures formed by nucleic acids require exogenous labels that may perturb cell function and change the very molecular processes they intend to study, especially at the extremely high label densities required for superresolution. However, despite tremendous interest and demonstrated need, label-free optical superresolution imaging of nucleotide topology under native nonperturbing conditions has never been possible. Here we investigate a photoswitching process of native nucleotides and present the demonstration of subdiffraction-resolution imaging of cellular structures using intrinsic contrast from unmodified DNA based on the principle of single-molecule photon localization microscopy (PLM). Using DNA-PLM, we achieved nanoscopic imaging of interphase nuclei and mitotic chromosomes, allowing a quantitative analysis of the DNA occupancy level and a subdiffractional analysis of the chromosomal organization. This study may pave a new way for label-free superresolution nanoscopic imaging of macromolecular structures with nucleotide topologies and could contribute to the development of new DNA-based contrast agents for superresolution imaging.

Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy.

The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.

In vivo broadband visible light optical coherence tomography probe enables inverse spectroscopic analysis.

We report the design and characterization of a 6 mm outer diameter pull-back circumferential scanning visible optical coherence tomography probe. The probe's large visible bandwidth (500-695 nm) allowed for inverse spectroscopic analysis and an axial resolution of ∼1.1 µm in tissue. We verify spectral imaging capabilities by measuring microsphere backscattering spectra and demonstrate in vivo spatial nanoscale characterization of tissue.

Sub-10-nm imaging of nucleic acids using spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON).

Elucidating chromatin structure in vitro requires resolution below 10 nm to visualize the mononucleosome has been an ongoing challenge. In this work, we achieve sub-10-nm imaging of nucleic acids via spectroscopic intrinsic-contrast photon-localization optical nanoscopy (SICLON) without the use of external labels. SICLON leverages two key innovations: using endogenous nucleotides as the emission source and a custom-made imaging system that can simultaneously record the position and optical spectra of emitting molecules. With a novel spectral regression algorithm that identifies the spectroscopic fingerprints of neighboring molecules that were previously indistinguishable, we demonstrate the utility of SICLON by visualizing unlabeled poly-nucleotides and linear single-stranded DNA fibers with a resolution of 6.2 nm.

Early increase in blood supply (EIBS) is associated with tumor risk in the Azoxymethane model of colon cancer.

BACKGROUND: The present study aimed to investigate the role of blood supply in early tumorigenesis in colorectal cancer. We leveraged the renin angiotensin system (RAS) to alter colonic blood supply and determine the effect on tumor initiation and progression. METHODS: To test the effect of blood supply on tumorigenesis, 53 male A/J mice were randomly assigned to one of three RAS modulation groups and one of two AOM treatments. The RAS modulation groups were I) water (RAS-unmodulated) as a control group, II) angiotensin-II and III) the angiotensin receptor blocker, Losartan. The mice in each group were then randomly split into either the saline control condition or the AOM-treated condition in which tumors were induced with a standard protocol of serial azoxymethane (AOM) injections. To monitor microvascular changes in the rectal mucosa during the study, we used confocal laser endomicroscopy (CLE) with FITC-Dextran for in-vivo imaging of vessels and polarization-gated spectroscopy (PGS) to quantify rectal hemoglobin concentration ([Hb]) and blood vessel radius (BVR). RESULTS: At 12 weeks post-AOM injections and before tumor formation, CLE images revealed many traditional hallmarks of angiogenesis including vessel dilation, loss of co-planarity, irregularity, and vessel sprouting in the pericryptal capillaries of the rectal mucosa in AOM-Water tumor bearing mice. PGS measurements at the same time-point showed increased rectal [Hb] and decreased BVR. At later time points, CLE images showed pronounced angiogenic features including irregular networks throughout the colon. Notably, the AOM-Losartan mice had significantly lower tumor multiplicity and did not exhibit the same angiogenic features observed with CLE, or the increase in [Hb] or decrease in BVR measured with PGS. The AOM-AngII mice did not have any significant trends. CONCLUSION: In-vivo PGS measurements of rectal colonic blood supply as well as CLE imaging revealed angiogenic disruptions to the capillary network prior to tumor formation. Losartan demonstrated an effective way to mitigate the changes to blood supply during tumorigenesis and reduce tumor multiplicity. These effects can be used in future studies to understand the early vessel changes observed.

The effects of chemical fixation on the cellular nanostructure.

Chemical fixation is nearly indispensable in the biological sciences, especially in circumstances where cryo-fixation is not applicable. While universally employed for the preservation of cell organization, chemical fixatives often introduce artifacts that can confound identification of true structures. Since biological research is increasingly probing ever-finer details of the cellular architecture, it is critical to understand the nanoscale transformation of the cellular organization due to fixation both systematically and quantitatively. In this work, we employed Partial Wave Spectroscopic (PWS) Microscopy, a nanoscale sensitive and label-free live cell spectroscopic-imaging technique, to analyze the effects of the fixation process through three commonly used fixation protocols for cells in vitro. In each method investigated, we detected dramatic difference in both nuclear and cytoplasmic nanoarchitecture between live and fixed states. But significantly, despite the alterations in cellular nanoscale organizations after chemical fixation, the population differences in chromatin structure (e.g. induced by a specific chemotherapeutic agent) remains. In conclusion, we demonstrated that the nanoscale cellular arrangement observed in fixed cells was fundamentally divorced from that in live cells, thus the quantitative analysis is only meaningful on the population level. This finding highlights the importance of live cell imaging techniques with nanoscale sensitivity or cryo-fixation in the interrogation of cellular structure, to complement more traditional chemical fixation methods.

Spectral biomarkers for chemoprevention of colonic neoplasia: a placebo-controlled double-blinded trial with aspirin.

OBJECTIVE: A major impediment to translating chemoprevention to clinical practice has been lack of intermediate biomarkers. We previously reported that rectal interrogation with low-coherence enhanced backscattering spectroscopy (LEBS) detected microarchitectural manifestations of field carcinogenesis. We now wanted to ascertain if reversion of two LEBS markers spectral slope (SPEC) and fractal dimension (FRAC) could serve as a marker for chemopreventive efficacy. DESIGN: We conducted a multicentre, prospective, randomised, double-blind placebo-controlled, clinical trial in subjects with a history of colonic neoplasia who manifested altered SPEC/FRAC in histologically normal colonic mucosa. Subjects (n=79) were randomised to 325 mg aspirin or placebo. The primary endpoint changed in FRAC and SPEC spectral markers after 3 months. Mucosal levels of prostaglandin E2 (PGE2) and UDP-glucuronosyltransferase (UGT)1A6 genotypes were planned secondary endpoints. RESULTS: At 3 months, the aspirin group manifested alterations in SPEC (48.9%, p=0.055) and FRAC (55.4%, p=0.200) with the direction towards non-neoplastic status. As a measure of aspirin's pharmacological efficacy, we assessed changes in rectal PGE2 levels and noted that it correlated with SPEC and FRAC alterations (R=-0.55, p=0.01 and R=0.57, p=0.009, respectively) whereas there was no significant correlation in placebo specimens. While UGT1A6 subgroup analysis did not achieve statistical significance, the changes in SPEC and FRAC to a less neoplastic direction occurred only in the variant consonant with epidemiological evidence of chemoprevention. CONCLUSIONS: We provide the first proof of concept, albeit somewhat underpowered, that spectral markers reversion mirrors antineoplastic efficacy providing a potential modality for titration of agent type/dose to optimise chemopreventive strategies in clinical practice. TRIAL NUMBER: NCT00468910.

Stochastic fluorescence switching of nucleic acids under visible light illumination.

We report detailed characterizations of stochastic fluorescence switching of unmodified nucleic acids under visible light illumination. Although the fluorescent emission from nucleic acids under the visible light illumination has long been overlooked due to their apparent low absorption cross section, our quantitative characterizations reveal the high quantum yield and high photon count in individual fluorescence emission events of nucleic acids at physiological concentrations. Owing to these characteristics, the stochastic fluorescence switching of nucleic acids could be comparable to that of some of the most potent exogenous fluorescence probes for localization-based super-resolution imaging. Therefore, utilizing the principle of single-molecule photon-localization microscopy, native nucleic acids could be ideal candidates for optical label-free super-resolution imaging.

Bleaching response of coral species in the context of assemblage response.

Caribbean coral reefs are declining due to a mosaic of local and global stresses, including climate change-induced thermal stress. Species and assemblage responses differ due to factors that are not easily identifiable or quantifiable. We calculated a novel species-specific metric of coral bleaching response, taxon-α and -ß, which relates the response of a species to that of its assemblages for 16 species over 18 assemblages. By contextualizing species responses within the response of their assemblages, the effects of environmental factors are removed and intrinsic differences among taxa are revealed. Most corals experience either a saturation response, overly-sensitive to weak stress (α > 0) but under-responsive compared to assemblage bleaching (ß < 1), or a threshold response, insensitive to weak stress (α < 0) but over-responsive compared to assemblage bleaching (ß > 1). This metric may help reveal key factors of bleaching susceptibility and identify species as targets for conservation.

Measuring the Autocorrelation Function of Nanoscale Three-Dimensional Density Distribution in Individual Cells Using Scanning Transmission Electron Microscopy, Atomic Force Microscopy, and a New Deconvolution Algorithm.

Essentially all biological processes are highly dependent on the nanoscale architecture of the cellular components where these processes take place. Statistical measures, such as the autocorrelation function (ACF) of the three-dimensional (3D) mass-density distribution, are widely used to characterize cellular nanostructure. However, conventional methods of reconstruction of the deterministic 3D mass-density distribution, from which these statistical measures can be calculated, have been inadequate for thick biological structures, such as whole cells, due to the conflict between the need for nanoscale resolution and its inverse relationship with thickness after conventional tomographic reconstruction. To tackle the problem, we have developed a robust method to calculate the ACF of the 3D mass-density distribution without tomography. Assuming the biological mass distribution is isotropic, our method allows for accurate statistical characterization of the 3D mass-density distribution by ACF with two data sets: a single projection image by scanning transmission electron microscopy and a thickness map by atomic force microscopy. Here we present validation of the ACF reconstruction algorithm, as well as its application to calculate the statistics of the 3D distribution of mass-density in a region containing the nucleus of an entire mammalian cell. This method may provide important insights into architectural changes that accompany cellular processes.

Coral bleaching response index: a new tool to standardize and compare susceptibility to thermal bleaching.

As coral bleaching events become more frequent and intense, our ability to predict and mitigate future events depends upon our capacity to interpret patterns within previous episodes. Responses to thermal stress vary among coral species; however the diversity of coral assemblages, environmental conditions, assessment protocols, and severity criteria applied in the global effort to document bleaching patterns creates challenges for the development of a systemic metric of taxon-specific response. Here, we describe and validate a novel framework to standardize bleaching response records and estimate their measurement uncertainties. Taxon-specific bleaching and mortality records (2036) of 374 coral taxa (during 1982-2006) at 316 sites were standardized to average percent tissue area affected and a taxon-specific bleaching response index (taxon-BRI) was calculated by averaging taxon-specific response over all sites where a taxon was present. Differential bleaching among corals was widely variable (mean taxon-BRI = 25.06 ± 18.44%, ±SE). Coral response may differ because holobionts are biologically different (intrinsic factors), they were exposed to different environmental conditions (extrinsic factors), or inconsistencies in reporting (measurement uncertainty). We found that both extrinsic and intrinsic factors have comparable influence within a given site and event (60% and 40% of bleaching response variance of all records explained, respectively). However, when responses of individual taxa are averaged across sites to obtain taxon-BRI, differential response was primarily driven by intrinsic differences among taxa (65% of taxon-BRI variance explained), not conditions across sites (6% explained), nor measurement uncertainty (29% explained). Thus, taxon-BRI is a robust metric of intrinsic susceptibility of coral taxa. Taxon-BRI provides a broadly applicable framework for standardization and error estimation for disparate historical records and collection of novel data, allowing for unprecedented accuracy in parameterization of mechanistic and predictive models and conservation plans.