Techniques for Processing Eyes Implanted With a Retinal Prosthesis for Localized Histopathological Analysis

With the recent development of retinal prostheses, it is important to develop reliable techniques for assessing the safety of these devices in preclinical studies. However, the standard fixation, preparation, and automated histology procedures are not ideal. Here we describe new procedures for evaluating the health of the retina directly adjacent to an implant. Retinal prostheses feature electrode arrays in contact with eye tissue. Previous methods have not been able to spatially localize the ocular tissue adjacent to individual electrodes within the array. In addition, standard histological processing often results in gross artifactual detachment of the retinal layers when assessing implanted eyes. Consequently, it has been difficult to assess localized damage, if present, caused by implantation and stimulation of an implanted electrode array. Therefore, we developed a method for identifying and localizing the ocular tissue adjacent to implanted electrodes using a (color-coded) dye marking scheme, and we modified an eye fixation technique to minimize artifactual retinal detachment. This method also rendered the sclera translucent, enabling localization of individual electrodes and specific parts of an implant. Finally, we used a matched control to increase the power of the histopathological assessments. In summary, this method enables reliable and efficient discrimination and assessment of the retinal cytoarchitecture in an implanted eye.     

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.     

A Fungal Pathogen of Amphibians,Batrachochytrium dendrobatidis,Attenuates in Pathogenicity with In Vitro Passages

Laboratory investigations into the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd), have accelerated recently, given the pathogen’s role in causing the global decline and extinction of amphibians. Studies in which host animals were exposed to Bd have largely assumed that lab-maintained pathogen cultures retained the infective and pathogenic properties of wild isolates. Attenuated pathogenicity is common in artificially maintained cultures of other pathogenic fungi, but to date, it is unknown whether, and to what degree, Bd might change in culture. We compared zoospore production over time in two samples of a single Bd isolate having different passage histories: one maintained in artificial media for more than six years (JEL427-P39), and one recently thawed from cryopreserved stock (JEL427-P9). In a common garden experiment, we then exposed two different amphibian species, Eleutherodactylus coqui and Atelopus zeteki, to both cultures to test whether Bd attenuates in pathogenicity with in vitro passages. The culture with the shorter passage history, JEL427-P9, had significantly greater zoospore densities over time compared to JEL427-P39. This difference in zoospore production was associated with a difference in pathogenicity for a susceptible amphibian species, indicating that fecundity may be an important virulence factor for Bd. In the 130-day experiment, Atelopus zeteki frogs exposed to the JEL427-P9 culture experienced higher average infection intensity and 100% mortality, compared with 60% mortality for frogs exposed to JEL427-P39. This effect was not observed with Eleutherodactylus coqui, which was able to clear infection. We hypothesize that the differences in phenotypic performance observed with Atelopus zeteki are rooted in changes of the Bd genome. Future investigations enabled by this study will focus on the underlying mechanisms of Bd pathogenicity.     

Fluorescent multiplexing of 3D spheroids: Analysis of biomarkers using automated immunohistochemistry staining platform and multispectral imaging

In preclinical cancer studies, three-dimensional (3D) cell spheroids and aggregates are preferred over monolayer cell cultures due to their architectural and functional similarity to solid tumors. We performed a proof-of-concept study to generate physiologically relevant and predictive preclinical models using non–small cell lung adenocarcinoma, and colon and colorectal adenocarcinoma cell line-derived 3D spheroids and aggregates. Distinct panels were designed to determine the expression profiles of frequently studied biomarkers of the two cancer subtypes. The lung adenocarcinoma panel included ALK, EGFR, TTF-1, and CK7 biomarkers, and the colon and colorectal adenocarcinoma panel included BRAF V600E, MSH2, MSH6, and CK20. Recent advances in immunofluorescence (IF) multiplexing and imaging technology enable simultaneous detection and quantification of multiple biomarkers on a single slide. In this study, we performed IF staining of multiple biomarkers per section on formalin-fixed paraffin-embedded 3D spheroids and aggregates. We optimized protocol parameters for automated IF and demonstrated staining concordance with automated chromogenic immunohistochemistry performed with validated protocols. Next, post-acquisition spectral unmixing of the captured fluorescent signals were utilized to delineate four differently stained biomarkers within a single multiplex IF image, followed by automated quantification of the expressed markers. This workflow has the potential to be adapted to preclinical high-throughput screening and drug efficacy studies utilizing 3D spheroids from cancer cell lines and patient-derived organoids. The process allows for cost, time, and resource savings through concurrent staining of several biomarkers on a single slide, the ability to study the interactions of multiple expressed proteins within a single region of interest, and enable quantitative assessment of biomarkers in cancer cells.     

Polymerase manager protein UmuD directly regulates Escherichia coli DNA polymerase III α binding to ssDNA

Replication by Escherichia coli DNA polymerase III is disrupted on encountering DNA damage. Consequently, specialized Y-family DNA polymerases are used to bypass DNA damage. The protein UmuD is extensively involved in modulating cellular responses to DNA damage and may play a role in DNA polymerase exchange for damage tolerance. In the absence of DNA, UmuD interacts with the α subunit of DNA polymerase III at two distinct binding sites, one of which is adjacent to the single-stranded DNA-binding site of α. Here, we use single molecule DNA stretching experiments to demonstrate that UmuD specifically inhibits binding of α to ssDNA. We predict using molecular modeling that UmuD residues D91 and G92 are involved in this interaction and demonstrate that mutation of these residues disrupts the interaction. Our results suggest that competition between UmuD and ssDNA for α binding is a new mechanism for polymerase exchange.     

Discordant risk: Overweight and cardiometabolic risk in Chinese adults

Objective:

Recent US work identified “metabolically healthy overweight” and “metabolically at risk normal weight” individuals. Less is known for modernizing countries with recent increased obesity.

Design and Methods:

Fasting blood samples, anthropometry and blood pressure from 8,233 adults aged 18‐98 in the 2009 nationwide China Health and Nutrition Survey, were used to determine prevalence of overweight (Asian cut point, BMI ≥23 kg/m²) and five risk factors (prediabetes/diabetes (hemoglobin A1c ≥5.7%) inflammation (high‐sensitivity C‐reactive protein (hsCRP) ≥3 mg/l), prehypertension/hypertension (Systolic blood pressure/diastolic blood pressure≥130/85 mm Hg), high triglycerides (≥150 mg/dl), low high‐density lipoprotein cholesterol (<40 (men)/ <50 mg/dl (women)). Sex‐stratified, logistic, and multinomial logistic regression models estimated concurrent obesity and cardiometabolic risk, with and without abdominal obesity, adjusting for age, smoking, alcohol consumption, physical activity, urbanicity, and income.

Results:

Irrespective of urbanicity, 78.3% of the sample had ≥1 elevated cardiometabolic risk factor (normal weight: 33.2% had ≥1 elevated risk factor; overweight: 5.7% had none). At the age of 18‐30 years, 47.4% had no elevated risk factors, which dropped to 6% by the age 70, largely due to age‐related increase in hypertension risk (18‐30 years: 11%; >70 years: 73%). Abdominal obesity was highly predictive of metabolic risk, irrespective of overweight (e.g., “metabolically at risk overweight” relative to “metabolically healthy normal weight” (men: relative risk ratio (RRR) = 39.06; 95% confidence interval (CI): 23.47, 65.00; women: RRR = 22.26; 95% CI: 17.49, 28.33)).

Conclusion:

A large proportion of Chinese adults have metabolic abnormalities. High hypertension risk with age, underlies the low prevalence of metabolically healthy overweight. Screening for cardiometabolic‐related outcomes dependent upon overweight will likely miss a large portion of the Chinese at risk population.     

Plant–microbe networks in soil are weakened by century-long use of inorganic fertilizers

Understanding the changes in plant–microbe interactions is critically important for predicting ecosystem functioning in response to human-induced environmental changes such as nitrogen (N) addition. In this study, the effects of a century-long fertilization treatment (> 150 years) on the networks between plants and soil microbial functional communities, detected by GeoChip, in grassland were determined in the Park Grass Experiment at Rothamsted Research, UK. Our results showed that plants and soil microbes have a consistent response to long-term fertilization—both richness and diversity of plants and soil microbes are significantly decreased, as well as microbial functional genes involved in soil carbon (C), nitrogen (N) and phosphorus (P) cycling. The network-based analyses showed that long-term fertilization decreased the complexity of networks between plant and microbial functional communities in terms of node numbers, connectivity, network density and the clustering coefficient. Similarly, within the soil microbial community, the strength of microbial associations was also weakened in response to long-term fertilization. Mantel path analysis showed that soil C and N contents were the main factors affecting the network between plants and microbes. Our results indicate that century-long fertilization weakens the plant–microbe networks, which is important in improving our understanding of grassland ecosystem functions and stability under long-term agriculture management.     

Maternal obesity is associated with younger age at obesity onset in U.S. adolescent offspring followed into adulthood

Objective: The objective was to test the hypothesis that maternal obesity is associated with younger age of offspring's obesity onset. Research Methods and Procedures: We used prospective, nationally representative, longitudinal data collected across Waves I (1995; 12 to 20 years), II (1996; 13 to 20 years), and III (2001; 18 to 28 years) of the National Longitudinal Study of Adolescent Health (N = 14,654; 49% female). Interval regression analysis was used to assess the association between maternal obesity and age at offspring's obesity onset (International Obesity Task Force BMI ≥30 equivalent age‐ and sex‐specific cut‐off points for adolescents and BMI ≥30 for young adults) using self‐reported heights and weights, adjusting for race/ethnicity, sex, parental education, and family income, accounting for complex sampling design. Results: The net effect of having an obese mother varied by race/ethnicity and was associated with a significantly earlier age at obesity onset (p = 0.0001) for whites [β= ?8.1 year, 95% confidence interval (CI), ?9.3; ?6.9)], blacks (β = ?10.8 years, 95% CI, ?12.4; ?9.2), Hispanics (β = ?7.0 years, 95% CI, ?9.2; ?4.8), and Asians (β = ?8.6 years, 95% CI, ?13.3; ?3.9). Earlier obesity onset (<18 years) was associated with increased severity at young adulthood (mean BMI, 36.0 ± 0.3 kg/m²) vs. onset after age 18 (mean BMI, 34.4 ± 0.2 kg/m²; p = 0.0001). There were no sex differences in the association of maternal obesity to age at obesity onset. Conclusions: Having an obese mother was associated with earlier age at obesity onset across all race/ethnic groups, particularly non‐Hispanic blacks. Early obesity onset has important health consequences because of its association with more severe adult obesity.