CCNE1 amplification is synthetic lethal with PKMYT1 kinase inhibition.

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.

Influence of unilateral eyelid spasms and botulinum toxin treatment on intraocular pressure measured by transpalpebral tonometer.

PURPOSE: Eyelid spasms might be associated with elevated intraocular pressure (IOP) in hemifacial spasm (HFS) patients. IOP assessment using a Goldmann applanation tonometer (GAT) is often compromised by eyelid spasms. This study aimed to assess the effect of HFS on IOP measurements using the transpalpebral tonometer Diaton® before and after treatment with botulinum toxin type A (BTX-A) and compared Diaton® and GAT measurements after treatment with BTX-A. METHODS: IOP measurements were obtained with Diaton® in 27 patients with moderate-to-severe HFS before and after treatment with BTX-A. After treatment, the IOP was also measured using GAT and the results were compared with the ones measured with a Diaton®. The patients underwent automated perimetry, OCT, and pachymetry for screening to glaucoma. RESULTS: Mean IOP with Diaton® was 11 ± 3.42 mmHg before treatment in the affected eye and 9 ± 2.98 mmHg in the contralateral eye. This difference was statistically significant (P = 0.012). However, after treatment with BTX-A, no interocular difference was found in IOP obtained with Diaton® (P = 0.204) or GAT (P = 0.971). Comparison between GAT and Diaton® measurements showed no significant differences after BTX-A treatment between the affected (P = 0.212) and contralateral eye (P = 0.971). CONCLUSIONS: A significant reduction in IOP measurements on the affected side of HFS patients was observed after treatment with BTX-A, demonstrating that eyelid spasms may increase the IOP. No significant difference was observed between Diaton® and GAT measurements after the application of BTX-A. No differences were found in automated perimetry, OCT, and CCT when comparing affected eyes with contralateral eyes.

A Multi-Objective Demand Response Optimization Model for Scheduling Loads in a Home Energy Management System.

Demand Response (DR) aims to motivate end consumers to change their energy consumption patterns in response to changes in electricity prices or when the reliability of the electrical power system (EPS) is compromised. Most of the proposals found in the literature only aim at reducing the cost for end consumers. However, this article proposes a home energy management system (HEMS) that aims to schedule the use of each home appliance based on the price of electricity in real-time (RTP) and on the consumer satisfaction/comfort level in order to guarantee the stability and the safety of the EPS. Thus, this paper presents a multi-objective DR optimization model which was formulated as a multi-objective nonlinear programming problem subjected to a set of constraints and was solved using the Non-Dominated Sorted Genetic Algorithm (NSGA-II), in order to determine the scheduling of home appliances for the time horizon. The multi-objective DR optimization model not only to minimize the cost of electricity consumption but also to reduce the level of inconvenience for residential consumers. Moreover, a priori, it is expected to obtain a more uniform demand with fewer peaks in the system and, potentially, achieving a more reliable and safer EPS operation. Thus, the energy management controller (EMC) within the HEMS determines an optimized schedule for each home appliance through the multi-objective DR model presented in this article, and ensures a more economic scenario for end consumers. In this paper, a performance evaluation of HEMS in 15 Brazilian families between 1 January and 31 December 2016 is presented with different electric energy consumption patterns in the cities of Belém-PA, Teresina-PI, Cuiabá-MT, Florianópolis-SC and São Paulo-SP, with three families per city, located in the regions north, northeast, central west, south and the southeast of Brazil, respectively. In addition, a total of 425 home appliances were used in the simulations. The results show that the HEMS achieved reductions in the cost of electricity for all the Scenarios used while minimally affecting the satisfaction/comfort of the end consumers as well as taking into account all the restrictions. The largest reduction in the total cost of electricity occurred for the couple without children, resident in the city of Teresina-PI; with a drop from US$ 99.31 to US$ 90.72 totaling 8.65% savings in the electricity bill. Therefore, the results confirm that the proposed HEMS effectively improves the operating efficiency of home appliances and reduces electricity costs for end consumers.

Rate of elongation by RNA polymerase II is associated with specific gene features and epigenetic modifications.

The rate of transcription elongation plays an important role in the timing of expression of full-length transcripts as well as in the regulation of alternative splicing. In this study, we coupled Bru-seq technology with 5,6-dichlorobenzimidazole 1-ß-D-ribofuranoside (DRB) to estimate the elongation rates of over 2000 individual genes in human cells. This technique, BruDRB-seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with rapid elongation rates showed higher densities of H3K79me2 and H4K20me1 histone marks compared to slower elongating genes. Furthermore, high elongation rates had a positive correlation with gene length, low complexity DNA sequence, and distance from the nearest active transcription unit. Features that negatively correlated with elongation rate included the density of exons, long terminal repeats, GC content of the gene, and DNA methylation density in the bodies of genes. Our results suggest that some static gene features influence transcription elongation rates and that cells may alter elongation rates by epigenetic regulation. The BruDRB-seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation.

DNA repair and recovery of RNA synthesis following exposure to ultraviolet light are delayed in long genes.

The kinetics of DNA repair and RNA synthesis recovery in human cells following UV-irradiation were assessed using nascent RNA Bru-seq and quantitative long PCR. It was found that UV light inhibited transcription elongation and that recovery of RNA synthesis occurred as a wave in the 5'-3' direction with slow recovery and TC-NER at the 3' end of long genes. RNA synthesis resumed fully at the 3'-end of genes after a 24 h recovery in wild-type fibroblasts, but not in cells deficient in transcription-coupled nucleotide excision repair (TC-NER) or global genomic NER (GG-NER). Different transcription recovery profiles were found for individual genes but these differences did not fully correlate to differences in DNA repair of these genes. Our study gives the first genome-wide view of how UV-induced lesions affect transcription and how the recovery of RNA synthesis of large genes are particularly delayed by the apparent lack of resumption of transcription by arrested polymerases.

Coordinated regulation of synthesis and stability of RNA during the acute TNF-induced proinflammatory response.

Steady-state gene expression is a coordination of synthesis and decay of RNA through epigenetic regulation, transcription factors, micro RNAs (miRNAs), and RNA-binding proteins. Here, we present bromouride labeling and sequencing (Bru-Seq) and bromouridine pulse-chase and sequencing (BruChase-Seq) to assess genome-wide changes to RNA synthesis and stability in human fibroblasts at homeostasis and after exposure to the proinflammatory tumor necrosis factor (TNF). The inflammatory response in human cells involves rapid and dramatic changes in gene expression, and the Bru-Seq and BruChase-Seq techniques revealed a coordinated and complex regulation of gene expression both at the transcriptional and posttranscriptional levels. The combinatory analysis of both RNA synthesis and stability using Bru-Seq and BruChase-Seq allows for a much deeper understanding of mechanisms of gene regulation than afforded by the analysis of steady-state total RNA and should be useful in many biological settings.

Use of Bru-Seq and BruChase-Seq for genome-wide assessment of the synthesis and stability of RNA.

Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.

Loss of the DNA Repair Gene RNase H2 Identifies a Unique Subset of DDR-Deficient Leiomyosarcomas.

Targeting the DNA damage response (DDR) pathway is an emerging therapeutic approach for leiomyosarcoma (LMS), and loss of RNase H2, a DDR pathway member, is a potentially actionable alteration for DDR-targeted treatments. Therefore, we designed a protein- and genomic-based RNase H2 screening assay to determine its prevalence and prognostic significance. Using a selective RNase H2 antibody on a pan-tumor microarray (TMA), RNase H2 loss was more common in LMS (11.5%, 9/78) than across all tumors (3.8%, 32/843). In a separate LMS cohort, RNase H2 deficiency was confirmed in uterine LMS (U-LMS, 21%, 23/108) and soft-tissue LMS (ST-LMS; 30%, 39/102). In the TCGA database, RNASEH2B homozygous deletions (HomDels) were found in 6% (5/80) of LMS cases, with a higher proportion in U-LMS (15%; 4/27) compared with ST-LMS (2%; 1/53). Using the SNiPDx targeted-NGS sequencing assay to detect biallelic loss of function in select DDR-related genes, we found RNASEH2B HomDels in 54% (19/35) of U-LMS cases with RNase H2 loss by IHC, and 7% (3/43) HomDels in RNase H2 intact cases. No RNASEH2B HomDels were detected in ST-LMS. In U-LMS patient cohort (n = 109), no significant overall survival difference was seen in patients with RNase H2 loss versus intact, or RNASEH2B HomDel (n = 12) versus Non-HomDel (n = 37). The overall diagnostic accuracy, sensitivity, and specificity of RNase H2 IHC for detecting RNA-SEH2B HomDels in U-LMS was 76%, 93%, and 71%, respectively, and it is being developed for future predictive biomarker driven clinical trials targeting DDR in U-LMS.

Outcomes of early versus delayed trabeculotomy for primary congenital glaucoma.

PURPOSE: In patients with primary congenital glaucoma (PCG), elevated intraocular pressure (IOP) causes abnormal eye growth. This study compared the outcomes of children with PCG who underwent ab externo trabeculotomy (TROC) at age ≤ 6 months (early TROC) and of those who underwent TROC at age > 6 months (delayed TROC). METHODS: Intraocular pressure, horizontal corneal diameter (HCD), central corneal thickness (CCT) and axial length (AL) were compared before TROC and at 1-, 3-, 6- and 12-month follow-up visits between the groups of children who underwent TROC until or after 6 months of age. The ALs of these groups were also compared with the ALs of healthy age-matched eyes examined under the same conditions. RESULTS: Trabeculotomy was performed in 43 children: 18 (33 eyes) aged 6 months (group 1) and 25 (37 eyes) aged >6 months (group 2); the mean ages were 86.56 ± 53.64 and 504.48 ± 448.14 days, respectively. The mean pre- and 12-month postoperative IOP values were 15.97 ± 4.78/16.62 ± 4.85 and 9.77 ± 2.88/10.93 ± 4.83 mmHg, respectively. Delayed TROC was associated with abnormal AL in 31 (88.6%) out of 37 eyes, while after early TROC, only 13 (41.9%) out of 33 eyes had abnormal AL (chi-square, 8.00; p = 0.03). In multivariable analysis, each 1-mmHg increase in preoperative IOP was associated with a 0.25-mmHg increase at 12 months (p = 0.04). On average, the mean IOP of the delayed TROC group was higher than that of the early TROC group by 3.72 mmHg at postoperative month 12 (95% CI = 0.44-6.99; p = 0.02). CONCLUSION: Compared with delayed TROC, early TROC is associated with reduced IOP and substantially reduced incidence of abnormal AL at postoperative month 12.

Trends in the epidemiological and microbiological profiles of infectious keratitis in southeastern Brazil.

PURPOSE: To investigate the antibiotic susceptibility as well as the clinical, epidemiological, and microbiological profiles of microbial keratitis. METHODS: This was a longitudinal retrospective study, and we retrospectively reviewed medical and laboratory records from 2015 to 2019. RESULTS: In total, 380 pathogens (321 bacteria and 59 fungi) were isolated from the corneas of 352 patients. Staphylococcus species (45%) were most abundant within the organisms that were isolated, followed by Pseudomonas (18.4%), fungi (15.5%), Streptococcus (7.9%), and Serratia species (3.2%). The isolated gram-positive bacteria were not resistant to amikacin or vancomycin, although 14.8% of the gram-positive isolates were resistant to ciprofloxacin (p<0.05). All the gram-negative isolates were susceptible to amikacin. Male patients represented 62.8% of the 129 cases with accessible clinical data. The mean age of the patients was 53.17 ± 21 years. The time to presentation (from onset of symptoms) was 14.9 ± 19.4 days (median: 7 days). Large ulcers (>5 mm in any dimension) were present in 49.6% (64 eyes) of the cases. The duration of treatment was 49 ± 45.9 days (median: 38 days). Direct ocular trauma was reported by 48 (37.2%) patients, and 15 patients (11.6%) reported using contact lenses. For 72 (55.8%) patients, topical treatment had been previously prescribed, and 16 (12.4%) patients reported using other classes of drugs. Hospitalizations were required for 79 (61.2%) patients, and in terms of major complications, 53 (41.1%) patients had corneal perforations. A total of 40 patients (31%) underwent tectonic penetrating keratoplasty, and 28 (21.7%) developed secondary glaucoma. A progression to endophthalmitis occurred in 8 (6.2%) patients, with 50% of those patients' (3.1% of the total) endophthalmitis evolving to evisceration. The patients' microbial keratitis was largely treated empirically, with 94 (72.9%) patients prescribed moxifloxacin and 56 (43.4%) prescribed ciprofloxacin before receiving their culture results. CONCLUSIONS: For the most part, our hospital treated patients with severe microbial keratitis. Despite identifying gram-positive bacteria in most of the isolates, we also frequently identified gram-negative rods and fungi. Our susceptibility results support prescribing a combination of vancomycin and amikacin as an effective empirical therapeutic regimen to treat microbial keratitis.

Traboulsi syndrome without features of Marfan syndrome caused by a novel homozygous ASPH variant associated with a heterozygous FBN1 variant.

BACKGROUND: Traboulsi syndrome is a rare disease clinically characterized by facial dysmorphism, abnormal spontaneous filtering blebs, ectopia lentis (EL) and multiple anterior segment abnormalities. MATERIAL AND METHODS: An 18-year-old female was referred to the Emergency Service of Hospital São Geraldo (HSG) claiming decreased right eye (RE) visual acuity associated with ocular pain that was noticed approximately 2 months earlier. She underwent a complete ophthalmic and physical examination including hands, ankle, wrist and chest X-ray, abdominal ultrasound, echocardiogram and genetic analysis (whole-exome sequencing). RESULTS: The ophthalmic examination revealed a high myopia with spherical equivalent of - 9.50 D and best corrected visual acuity (BCVA) of 20/60 in RE and - 9.25 D with BCVA of 20/30 in the left eye (LE). Slit-lamp examination showed normal conjunctiva in both eyes (BE) and a superior-temporal cystic lesion in RE and nasal in LE; the flat anterior chamber in BE with the transparent crystalline lens touches the central corneal endothelium in the RE. Fundoscopy suggested glaucoma as the cup/disc ratio was 0.7, although the intraocular pressure (IOP) was 10 mmHg in BE without medication. Validation of data from whole exome demonstrated a novel splicing homozygous pathogenic variant (PV) (c.1765-1G>A) of the ASPH gene as well as a heterozygous variant of unknown significance (VUS) of the FBN1 gene (c.6832C>T). CONCLUSION: We here report a novel splice-affecting homozygous pathogenic variant in the ASPH gene that was detected in a Brazilian patient with clinical features of Traboulsi syndrome.

Detection of Biallelic Loss of DNA Repair Genes in Formalin-Fixed, Paraffin-Embedded Tumor Samples Using a Novel Tumor-Only Sequencing Panel.

Patient selection for synthetic lethal-based cancer therapy may be improved by assessment of gene-specific loss of heterozygosity (LOH) and biallelic loss of function (LOF). This report describes SyNthetic lethal Interactions for Precision Diagnostics (SNiPDx), a targeted next-generation sequencing (NGS) panel for detection of LOH and biallelic LOF alterations in 26 target genes focused on DNA damage response pathways, in tumor-only formalin-fixed, paraffin-embedded (FFPE) samples. NGS was performed across all exons of these 26 genes and encompassed a total of 7632 genome-wide single-nucleotide polymorphisms on genomic DNA from 80 FFPE solid tumor samples. The Fraction and Allele-Specific Copy Number Estimates from Tumor Sequencing algorithm was optimized to assess tumor purity and copy number based on heterozygous single-nucleotide polymorphisms. SNiPDx demonstrated high sensitivity (95%) and specificity (91%) for LOH detection compared with whole genome sequencing. Positive agreement with local NGS-based testing in the detection of genetic alterations was 95%. SNiPDx detected 93% of biallelic ATM LOF mutations, 100% of ATM single-nucleotide variants and small insertions/deletions, and 100% of all ATM LOH status events identified by orthogonal NGS-based testing. SNiPDx is a novel, clinically feasible test for analysis of allelic status in FFPE tumor samples, which demonstrated high accuracy when compared with other NGS-based approaches in clinical use.

Twenty-four hours intraocular pressure in keratoconic eyes assessed by applanation tonometry and Tono-Pen AVIA.

AIM: To assess intraocular pressure (IOP) during the daily curve of intraocular pressure (DCPo) in keratoconic eyes and compare Goldmann applanation tonometer (GAT), without and with astigmatism correction (nGAT and cGAT) and Tono-Pen AVIA (TPA) assessment methods. METHODS: Thirty-nine keratoconic eyes of 24 patients were assessed. DCPo was evaluated with five IOP measurements; four were performed with a GAT (nGAT and cGAT), and a Tono-Pen AVIA (TPA) at various times throughout the day. RESULTS: Mean IOP DCPo values (mm Hg) were: nGAT, 9.9±2.6; cGAT, 11.3±2.6; TPA 12.3±3.1. Mean IOP DCPo differences (mm Hg) and Spearman's correlation coefficients were as follows: cGATc-nGAT, 1.32±1.31, r s=0.879 (P<0.01); cGAT-TPA, -1.02±2.08, r s=0.723 (P<0.01); and nGAT-TPA, -2.35±2.23, r s=0.730 (P<0.01). Bland-Altman analysis for agreement between cGAT-TPA and nGAT-TPA mean IOP DCPo measurements revealed a mean difference of 1.02 (95%CI, 0.35-1.70) and 2.35 (95%CI, 1.62-3.07) mm Hg, respectively. Regression analysis yielded the following equation: TPA IOP=5.49+0.775×cGAT-0.015×ACD-0.299×corneal astig matism, which allowed us to infer TPA IOP values from other parameters. CONCLUSION: In keratoconic eyes, IOP peaks of DCPo measurements are identified at 6 a.m., independent of the tonometer. The mean DCPo values are: TPA>cGAT>nGAT. IOP TPA measures are predictive of cGAT values, adjusted according to anterior chamber depth and corneal astigmatism.

Guiding ATR and PARP inhibitor combinationswith chemogenomic screens.

Combinations of ataxia telangiectasia- and Rad3-related kinase inhibitors (ATRis) and poly(ADP-ribose) polymerase inhibitors (PARPis) synergistically kill tumor cells through modulation of complementary DNA repair pathways, but their tolerability is limited by hematological toxicities. To address this, we performed a genome-wide CRISPR-Cas9 screen to identify genetic alterations that hypersensitize cells to a combination of the ATRi RP-3500 with PARPi, including deficiency in RNase H2, RAD51 paralog mutations, or the "alternative lengthening of telomeres" telomere maintenance mechanism. We show that RP-3500 and PARPi combinations kill cells carrying these genetic alterations at doses sub-therapeutic as single agents. We also demonstrate the mechanism of combination hypersensitivity in RNase H2-deficient cells, where we observe an irreversible replication catastrophe, allowing us to design a highly efficacious and tolerable in vivo dosing schedule. We present a comprehensive dataset to inform development of ATRi and PARPi combinations and an experimental framework applicable to other drug combination strategies.

RP-3500: A Novel, Potent, and Selective ATR Inhibitor that is Effective in Preclinical Models as a Monotherapy and in Combination with PARP Inhibitors.

Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.

The transcriptomic responses of the eastern oyster, Crassostrea virginica, to environmental conditions.

Understanding the mechanisms by which organisms adapt to environmental conditions is a fundamental question for ecology and evolution. In this study, we evaluate changes in gene expression of a marine mollusc, the eastern oyster Crassostrea virginica, associated with the physico-chemical conditions and the levels of metals and other contaminants in their environment. The results indicate that transcript signatures can effectively disentangle the complex interactive gene expression responses to the environment and are also capable of disentangling the complex dynamic effects of environmental factors on gene expression. In this context, the mapping of environment to gene and gene to environment is reciprocal and mutually reinforcing. In general, the response of transcripts to the environment is driven by major factors known to affect oyster physiology such as temperature, pH, salinity, and dissolved oxygen, with pollutant levels playing a relatively small role, at least within the range of concentrations found in the studied oyster habitats. Further, the two environmental factors that dominate these effects (temperature and pH) interact in a dynamic and nonlinear fashion to impact gene expression. Transcriptomic data obtained in our study provide insights into the mechanisms of physiological responses to temperature and pH in oysters that are consistent with the known effects of these factors on physiological functions of ectotherms and indicate important linkages between transcriptomics and physiological outcomes. Should these linkages hold in further studies and in other organisms, they may provide a novel integrated approach for assessing the impacts of climate change, ocean acidification and anthropogenic contaminants on aquatic organisms via relatively inexpensive microarray platforms.

Evolution combined with genomic study elucidates genetic bases of isobutanol tolerance in Escherichia coli.

BACKGROUND: Isobutanol is a promising next-generation biofuel with demonstrated high yield microbial production, but the toxicity of this molecule reduces fermentation volumetric productivity and final titer. Organic solvent tolerance is a complex, multigenic phenotype that has been recalcitrant to rational engineering approaches. We apply experimental evolution followed by genome resequencing and a gene expression study to elucidate genetic bases of adaptation to exogenous isobutanol stress. RESULTS: The adaptations acquired in our evolved lineages exhibit antagonistic pleiotropy between minimal and rich medium, and appear to be specific to the effects of longer chain alcohols. By examining genotypic adaptation in multiple independent lineages, we find evidence of parallel evolution in marC, hfq, mdh, acrAB, gatYZABCD, and rph genes. Many isobutanol tolerant lineages show reduced RpoS activity, perhaps related to mutations in hfq or acrAB. Consistent with the complex, multigenic nature of solvent tolerance, we observe adaptations in a diversity of cellular processes. Many adaptations appear to involve epistasis between different mutations, implying a rugged fitness landscape for isobutanol tolerance. We observe a trend of evolution targeting post-transcriptional regulation and high centrality nodes of biochemical networks. Collectively, the genotypic adaptations we observe suggest mechanisms of adaptation to isobutanol stress based on remodeling the cell envelope and surprisingly, stress response attenuation. CONCLUSIONS: We have discovered a set of genotypic adaptations that confer increased tolerance to exogenous isobutanol stress. Our results are immediately useful to further efforts to engineer more isobutanol tolerant host strains of E. coli for isobutanol production. We suggest that rpoS and post-transcriptional regulators, such as hfq, RNA helicases, and sRNAs may be interesting mutagenesis targets for future global phenotype engineering.

Comparison of Tono-Pen Avia and Handheld Applanation Tonometry in Primary Congenital Glaucoma.

PRCIS: Tono-Pen AVIA (TPA) intraocular pressure (IOP) values are different from those taken with handheld Goldmann applanation tonometer (GAT) in primary congenital glaucoma (PCG). These differences indicate both tonometers cannot be used interchangeably for measuring IOP in PCG. PURPOSE: The aim was to compare IOP measurements obtained using TPA and a handheld version of GAT in children with PCG. MATERIALS AND METHODS: Forty-two eyes from 23 patients were evaluated for central corneal thickness (CCT), axial length, biomicroscopy and IOP measurement with TPA and a handheld GAT under inhalation anesthesia. After 1 eye from each patient was randomized, paired the Student t-test and the Pearson correlation were used for analysis. Generalized linear mixed model was used to estimate the difference between tonometers. RESULTS: Mean age of children was 28.3±20.5 months. Mean axial length was 24.89±3.33 mm and mean CCT was 605.9±81.0 µm. Mean IOP was 22.1±9.6 for TPA and 14.0±4.5 mm Hg for GAT. There was a significant difference of 8.1±6.9 mm Hg between TPA IOP and GAT IOP (P<0.001). Each 6 months increase in age was associated with 1.32 mm Hg reduction in the difference between tonometers (P=0.002) and each 1 mm Hg higher of mean GAT IOP was associated with -0.73 mm Hg in the difference between TPA and GAT (P=0.002). Also, for every 20 µm increase in CCT an increase of 1.16 mm Hg in the difference between both devices was expected (P=0.003), after adjustment for potentially confounding variables. CONCLUSION: There is a significant difference between TPA IOP and GAT IOP in PCG. The difference between TPA and GAT in PCG is influenced by CCT, age and GAT IOP value.

Transcriptome analysis of cadmium exposure in kidney fibroblast cells of the North Atlantic Right Whale (Eubalaena glacialis).

An 8X15k oligonucleotide microarray was developed consisting of 2334 Eubalaena glacialis probes and 2166 Tursiops truncatus probes and used to measure the effects, at transcriptomic level, of cadmium exposure in right whale kidney fibroblast cells. Cells were exposed to three concentrations (1 µM, 0.1 µM, and 0.01 µM) of cadmium chloride (CdCl2) for three exposure times (1, 4, and 24 h). Cells exposed to 1 µM CdCl2 for 4 h and 24 h showed upregulated genes involved in protection from metal toxicity and oxidative stress, protein renaturation, apoptosis inhibition, as well as several regulators of cellular processes. Downregulated genes represented a suite of functions including cell proliferation, transcription regulation, actin polymerization, and stress fiber synthesis. The collection of differentially expressed genes in this study support proposed mechanisms of cadmium-induced apoptosis such as ubiquitin proteasome system disruption, Ca2+ homeostasis interference, mitochondrial membrane potential collapse, reactive oxygen species (ROS) production, and cell cycle arrest. The results also have confirmed the right whale microarray as a reproducible tool in measuring differentiated gene expression that could be a valuable asset for transcriptome analysis of other baleen whales and potential health assessment protocols.

Correlation between retinal nerve fiber layer thickness and IOP variation in glaucoma suspects and patients with primary open-angle glaucoma.

PURPOSE: To analyze the relationship between retinal nerve fiber layer thickness (RNFLT) and intraocular pressure (IOP) variation in glaucoma suspects (GS) and patients with primary open-angle glaucoma (POAG). METHODS: Thirty-one GS and 34 POAG patients underwent ophthalmologic examination and 24-h IOP measurements. GS had IOPs ranging from 19 to 24 mmHg and/or suspicious appearance of the optic nerve. POAG patients had reproducible abnormal visual fields. We only included patients who presented with short-term IOP fluctuation >6 mm Hg (∆IOP). Only one eye per patient was included through a randomized process. Peripapillary RNFLT was assessed by spectral-domain optical coherence tomography. We correlated RNFLT with IOP parameters. RESULTS: Mean IOP was similar between GS and POAG groups (15.6 ± 3.47 vs 15.6 ± 2.83 mmHg, p = 0.90) as was IOP peak at 6 AM (21.7 ± 3.85 vs 21.3 ± 3.80 mmHg, p = 0.68). Statistically significant negative correlations were found in POAG group between IOP at 6 AM and RNFLT in global (rs = -0.543; p < 0.001), inferior (rs = -0.540; p < 0.001), superior (rs = -0.405; p = 0.009), and nasal quadrants (rs = -0.561; p < 0.001). Negative correlations were also found between ∆IOP and RNFLT in global (rs = -0.591; p < 0.001), and all other sectors (p < 0.05). In GS IOP at 6 AM correlated only with inferior quadrant (rs = -0.307; p = 0.047). CONCLUSION: IOP at 6 AM and ∆IOP had negative correlations with RNFLT quadrants in POAG. In GS this correlation occurred between IOP at 6 AM and inferior quadrant. These findings may indicate potential risk factors for glaucoma progression.