Individuals with and without normal tension glaucoma exhibit comparable performance on tests of cognitive function.

AIM: To evaluate aspects of cognition impacted by individuals with and without normal tension glaucoma. METHODS: Fifty normal tension glaucoma (NTG) and 50 control patients ≥50y of age were recruited from the UCSF Department of Ophthalmology. Demographic data and glaucoma parameters were extracted from electronic medical records for both groups. Tests of executive function [Executive Abilities: Measures and Instruments for Neurobehavioral Evaluation and Research (EXAMINER)] and learning and memory [California Verbal Learning Test-Second Edition (CVLT-II)] were administered to both NTG and controls. Race, handedness, best-corrected visual acuity, maximum intraocular pressure, optic nerve cup-to-disc ratio, visual field and optic nerve optical coherence tomography parameters, and a measure of general health (Charlson Comorbidity Index) were compared between NTG and controls as well as within NTG subgroups. Multivariate linear regression was used to compare group performances on the EXAMINER battery and CVLT-II while controlling for age, sex, and years of education. RESULTS: NTG and controls were comparable with respect to age, sex, race, education, handedness, and the Charlson Comorbidity Index (P>0.05 for all). Performance on the EXAMINER composite score and the CVLT-II did not differ between NTG and controls (P>0.05 for both). CONCLUSION: This is the first prospective study in which the cognitive function of subject with NTG were evaluated using a comprehensive, computerized neurocognitive battery. Subjects with NTG do not perform worse than unaffected controls on tests of executive function, learning, and memory. Results do not support the hypothesis that individuals with NTG are at higher risk for cognitive dysfunction and/or dementia.

DNA-Encoded Library Hit Confirmation: Bridging the Gap Between On-DNA and Off-DNA Chemistry.

DNA-encoded library (DEL) technology is a powerful platform for hit identification in academia and the pharmaceutical industry. When conducting off-DNA resynthesis hit confirmation after affinity selection, PCR/sequencing, and data analysis, one typically assumes a "one-to-one" relationship between the DNA tag and the chemical structure of the attached small-molecule it encodes. Because library synthesis often yields a mixture, this approximation increases the risk of overlooking positive discoveries and valuable information. To address this issue, we apply a library synthesis "recipe" strategy for on-DNA resynthesis using a cleavable linker, followed by direct affinity selection mass spectrometry (AS-MS) evaluation and identification of binder(s) from the released small-molecule mixture. We validate and showcase this approach employing the receptor-interacting-protein kinase 2 (RIP2) DEL campaign. We also designed and developed two cleavable linkers to enable this method, a photocleavable linker (nitrophenyl-based) and acid-labile linker (tetrahydropyranyl ether). The strategy provides an effective means of hit identification and rapid determination of key active component(s) of the mixture.

ZBTB20 regulates WNT/CTNNB1 signalling pathway by suppressing PPARG during hepatocellular carcinoma tumourigenesis.

BACKGROUND & AIMS: Zinc finger and BTB domain containing 20 (ZBTB20) has been implicated as a potential oncogene in liver cancer. However, knockout studies have shown it to be a transcriptional repressor of the alpha-foetoprotein (Afp) gene in adult liver, and reduced levels of ZBTB20 allow for upregulation of AFP with increased tumour severity in certain cases of hepatocellular carcinoma (HCC). As there are many discrepancies in the literature regarding its role in liver tumourigenesis, the aim of this study was to elucidate the role of ZBTB20 in HCC tumourigenesis. METHODS: A reverse genetic study using the Sleeping Beauty (SB) transposon system in mice was performed to elucidate the role of ZBTB20 in HCC tumourigenesis. In vitro ZBTB20 gain- and loss-of-function experiments were used to assess the relationship amongst ZBTB20, peroxisome proliferator activated receptor gamma (PPARG) and catenin beta 1 (CTNNB1). RESULTS: Transgenic overexpression of ZBTB20 in hepatocytes and in the context of transformation related protein (T r p53) inactivation induced hepatic hypertrophy, activation of WNT/CTNNB1 signalling, and development of liver tumours. In vitro overexpression and knockout experiments using CRISPR/Cas9 demonstrated the important role for ZBTB20 in downregulating PPARG, resulting in activation of the WNT/CTNNB1 signalling pathway and its downstream effectors in HCC tumourigenesis. CONCLUSIONS: These findings demonstrate a novel interaction between ZBTB20 and PPARG, which leads to activation of the WNT/CTNNB1 signalling pathway in HCC tumourigenesis. LAY SUMMARY: ZBTB20 has been implicated as a potential oncogene in liver cancer. Herein, we uncover its important role in liver cancer development. We show that it interacts with PPARG to upregulate the WNT/CTNNB1 signalling pathway, leading to tumourigenesis.

Sleeping Beauty insertional mutagenesis screen identifies the pro-metastatic roles of CNPY2 and ACTN2 in hepatocellular carcinoma tumor progression.

A forward genetic Sleeping Beauty (SB) insertional mutagenesis screen, followed by high-throughput transcriptome sequencing, was used to identify driver genes responsible for hepatocellular carcinoma (HCC)-associated metastasis. Using RNA-sequencing (RNA-seq) to identify transposon-endogenous transcriptome fusion genes, the phylogenetic lineage between the parental liver tumor and secondary metastasis can be determined to provide mechanistic insight to genetic changes involved in the metastatic evolution process. In the current study, two novel candidate genes were identified to be potentially involved in HCC-associated metastatic progression, canopy FGF signaling regulator 2 (Cnpy2) and actinin alpha 2 (Actn2). Transposon-Cnpy2 fusion transcripts were identified in both primary liver tumors and lung metastases. Its significant association with clinicopathological characteristics and correlated gene enrichment in metastasis-related mechanisms suggest its potential role in modulating local invasion and angiogenesis. Other known driver genes for human HCC that can also promote metastatic progression include epidermal growth factor receptor (Egfr) and RNA imprinted and accumulated in nucleus (Rian). Metabolic pathway related gene carbamoyl phosphate synthetase (Cps1) was identified to play an important role in early HCC development, while cell junction-related pathway gene Rac family small GTPase 1 (Rac1) was identified to take part in both HCC and pro-metastatic progression. Importantly, actinin alpha 2 (Actn2) was identified exclusively in the secondary metastasis site and its role in HCC-related metastatic process was elucidated using in vitro approaches. ACTN2-overexpression in human liver cancer cells displayed enhanced cellular motility and invasion abilities, indicating its possible function in later stage of metastasis, such as extravasation and lung colonization.

BILATERAL ENTEROCOCCUS FAECALIS ENDOPHTHALMITIS WITH MULTIPLE RECURRENCES.

PURPOSE: To describe the first case report of a bilateral recurrent Enterococcus faecalis endophthalmitis postcataract surgery. METHODS: Case report with a description of the timeline, diagnosis, and management of a patient with bilateral recurrent E. faecalis endophthalmitis. RESULTS: An 89-year-old man presented 6 weeks' postcataract surgery with pain, tearing, and blurred vision in the left eye. B-scan ultrasonography revealed vitritis and cultures postvitrectomy grew E. faecalis. There was gradual improvement in vision postintravitreal vancomycin administration. Four years later, the patient experienced another episode of E. faecalis endophthalmitis in the right eye postcataract extraction, followed by several additional episodes in both eyes posttreatment. CONCLUSION: Enterococcus faecalis is a rare but highly virulent cause of endophthalmitis that may remain sequestered in the capsular bag, despite aggressive treatment. Even after recurrent episodes, early vitrectomy and aggressive antibiotic therapy may prove to be effective in preventing vision loss.

Schwann cell-specific Pten inactivation reveals essential role of the sympathetic nervous system activity in adipose tissue development.

There is increasing evidence that the sympathetic nervous system (SNS) plays an important role in adipose tissue development. However, the underlying molecular mechanism(s) associated with this remains unclear. SNS innervation of white adipose tissue (WAT) is believed to be necessary and sufficient to elicit WAT lipolysis. In this current study, mice with Schwann cell (SC)-specific inactivation of phosphatase and tensin homolog (Pten) displayed enlarged inguinal white adipose tissue (iWAT). This serendipitous observation implicates the role of SCs in mediating SNS activity associated with mouse adipose tissue development. Mice with SC-specific Pten inactivation displayed enlarged iWAT. Interestingly, the SNS activity in iWAT of SC-specific Pten-deficient mice was reduced as demonstrated by decreased tyrosine hydroxylase (TH) expression level and neurotransmitters, such as norepinephrine (NE) and histamine (H). The lipolysis related protein, phosphorylated hormone sensitive lipase (pHSL), was also decreased. As expected, AKT-associated signaling pathway was hyperactivated and hypothesized to induce enlarged iWAT in SC-specific Pten-deficient mice. Moreover, preliminary experiments using AKT inhibitor AZD5363 treatment ameliorated the enlarged iWAT condition in SC-specific Pten-deficient mice. Taken together, SCs play an essential role in the regulation of SNS activity in iWAT development via the AKT signaling pathway. This novel role of SCs in SNS function allows for better understanding into the genetic mechanisms of peripheral neuropathy associated obesity.

Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

Discovery of Potent and Selective Inhibitors for ADAMTS-4 through DNA-Encoded Library Technology (ELT).

The aggrecan degrading metalloprotease ADAMTS-4 has been identified as a novel therapeutic target for osteoarthritis. Here, we use DNA-encoded Library Technology (ELT) to identify novel ADAMTS-4 inhibitors from a DNA-encoded triazine library by affinity selection. Structure-activity relationship studies based on the selection information led to the identification of potent and highly selective inhibitors. For example, 4-(((4-(6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)-6-(((4-methylpiperazin-1-yl)methyl)amino)-1,3,5-triazin-2-yl)amino)methyl)-N-ethyl-N-(m-tolyl)benzamide has IC50 of 10 nM against ADAMTS-4, with >1000-fold selectivity over ADAMT-5, MMP-13, TACE, and ADAMTS-13. These inhibitors have no obvious zinc ligand functionality.

A rod-sparing retinopathy in bardet-biedl syndrome.

Bardet-Biedl syndrome is a continuum of disorders characterized by systemic and ocular findings. Retinal abnormalities typically present as diffuse photoreceptor degeneration. Here, we report a novel case that suggests a rod-sparing variant of Bardet-Biedl syndrome.

Prediction of protein-DNA complex mobility in gel-free capillary electrophoresis.

Selection of protein binders from highly diverse combinatorial libraries of DNA-encoded small molecules is a highly promising approach for discovery of small-molecule drug leads. Methods of kinetic capillary electrophoresis provide the high efficiency of partitioning required for such selection but require the knowledge of electrophoretic mobility of the protein-ligand complex. Here we present a theoretical approach for an accurate estimate of the electrophoretic mobility of such complexes. The model is based on a theory of the thin double layer and corresponding expressions used for the mobilities of a rod-like short oligonucleotide and a sphere-like globular protein. The model uses empirical values of mobilities of free protein, free ligand, and electroosmotic flow. The model was tested with a streptavidin-dsDNA complex linked through biotin (small molecule). The deviation of the prediction from the experimental mobility did not exceed 4%, thus confirming that not only is the model adequate but it is also accurate. This model will facilitate reliable use of KCE methods for selection of drug leads from libraries of DNA-encoded small molecules.

2013 update on the management of posterior capsular rupture during cataract surgery.

PURPOSE OF REVIEW: Posterior capsular rupture (PCR) is an infrequent complication of cataract surgery that can lead to significant ocular morbidity and permanent vision loss. In the setting of PCR, the primary objective is the safe and thorough evacuation of vitreous and lens fragments from the anterior segment. The secondary objective is the stable placement of an intraocular lens (IOL) selected for best refractive outcomes. Expedited referral to vitreoretinal specialists is recommended for management of posteriorly dislocated lens material and surveillance for retinal injury. It is the intention of this review to present current guidelines for the management of PCR. RECENT FINDINGS: There are new techniques available to anterior and posterior segment surgeons in the setting of PCR. Endoillumination may facilitate visualization during anterior vitrectomy and the IOL may be used as a pupillary barrier to prevent loss of lens fragments. When secondary procedures are needed, early return to the operating room and small-gauge pars plana techniques may reduce patient morbidity. SUMMARY: When approached carefully and systematically, patients may have good outcomes in the setting of PCR. Recent advancements in instrumentation and technique encourage further study and may lead to new standards of care.

Ethnic variation in optic disc size by fundus photography.

PURPOSE: To compare optic disc size among Caucasian, Chinese, African, Filipino and Hispanic subjects recruited from a university-based general ophthalmology clinic. METHODS: In this retrospective cross-sectional study, optic disc size was calculated from fundus photographs. Differences in age, sex and spherical equivalent among the ethnic groups were examined with either Kruskal--Wallis test or Chi-square test. Ethnic differences in optic disc size were evaluated with linear mixed-effects regression models that adjusted for age, sex, spherical equivalent, glaucoma status, lens status and use of both eyes in the same subject. RESULTS: Five hundred and seventy-four eyes of 305 subjects were analyzed. The mean and standard deviation of the optic disc size were 2.16 ± 0.41 mm(2) for Caucasian, 2.30 ± 0.56 mm(2) for Chinese, 2.31 ± 0.41 mm(2) for Filipino, 2.38 ± 0.40 mm(2) for African and 2.40 ± 0.38 mm(2) for Hispanic subjects. Age and spherical equivalent differed among the ethnic groups by Kruskal--Wallis test (p < 0.05). Sex differed among the ethnic groups by Chi-square test (p < 0.05). Ethnic differences in optic disc size were not observed among African, Hispanic, Filipino and Chinese subjects in linear mixed-effects regression analyses (p > 0.05). However, Caucasian differed from the other ethnicities in linear mixed-effects regression analyses (p < 0.05). CONCLUSIONS: Optic disc size was significantly smaller in Caucasian compared to the other ethnic groups. Optic disc size differences among non-Caucasian ethnic groups were not significant.

Discovery of thieno[3,2-d]pyrimidine-6-carboxamides as potent inhibitors of SIRT1, SIRT2, and SIRT3.

The sirtuins SIRT1, SIRT2, and SIRT3 are NAD(+) dependent deacetylases that are considered potential targets for metabolic, inflammatory, oncologic, and neurodegenerative disorders. Encoded library technology (ELT) was used to affinity screen a 1.2 million heterocycle enriched library of DNA encoded small molecules, which identified pan-inhibitors of SIRT1/2/3 with nanomolar potency (e.g., 11c: IC50 = 3.6, 2.7, and 4.0 nM for SIRT1, SIRT2, and SIRT3, respectively). Subsequent SAR studies to improve physiochemical properties identified the potent drug like analogues 28 and 31. Crystallographic studies of 11c, 28, and 31 bound in the SIRT3 active site revealed that the common carboxamide binds in the nicotinamide C-pocket and the aliphatic portions of the inhibitors extend through the substrate channel, explaining the observable SAR. These pan SIRT1/2/3 inhibitors, representing a novel chemotype, are significantly more potent than currently available inhibitors, which makes them valuable tools for sirtuin research.

Differences in baseline dark and the dark-to-light changes in anterior chamber angle parameters in whites and ethnic Chinese.

PURPOSE: To assess the anterior chamber drainage angle width in the dark and the dark-to-light change (Δ) between Caucasians and Chinese aged 40 years and older. METHODS: The study groups comprised four age- and sex-matched cohorts: American Caucasians, American Chinese, southern mainland Chinese, and northern mainland Chinese. Anterior segment optical coherence tomography (ASOCT) images of the anterior chamber angles were obtained under both light and dark conditions. The parameters analyzed included angle opening distance (AOD), angle recess area (ARA), and trabecular-iris space area (TISA). RESULTS: Data were obtained from 121, 124, 121, and 120 participants who were American Caucasians, American Chinese, and southern and northern mainland Chinese, respectively. In a multiple linear regression analysis, adjusted for age, sex, refractive status, pupil size, lens location, and anterior chamber depth (ACD) and width (ACW), the ethnic Chinese had significantly smaller ARAs (regression coefficient, ß = -0.06, P < 0.001) and TISAs (ß = -0.01, P = 0.039), as well as greater ΔAODs (ß = 0.03, P = 0.009) and ΔTISAs (ß = 0.02, P = 0.029) than did the Caucasians. For the dark-to-light change analysis, the independent associations between angle width and iris thickness (IT) and iris curvature (ICurv) were identified only in the Chinese. CONCLUSIONS: Compared with the Caucasians, the ethnic Chinese had smaller ARA and TISA, but greater dark-to-light changes in AOD and TISA, independent of refractive status and overall ocular anterior segment dimensions.

A phone-counseling smoking-cessation intervention for male Chinese restaurant workers.

We sought to develop a smoking-cessation intervention for male Chinese restaurant workers in New York City that required no seeking out by participants; provided support over a relatively long period of time; and was responsive to participants' cultural backgrounds and daily lives. The resulting intervention consisted of a minimum of 9 proactive phone counseling sessions within a 6-month period for each participant recruited at his worksite. All activities were conducted in Chinese languages. The efficacy of this proactive phone-counseling intervention was assessed in a pretest/posttest design comparing baseline smoking with smoking 6 months after the intervention ended. Of 137 male employees recruited at their restaurants, 101 (median age 40.5) participated in the phone-counseling intervention in 2007-2008, with 75 completing the program with at least 9 counseling calls. We found a linear increase in smoking cessation from 0% at Call 1 to 50.7% at Call 9 for 75 men who completed the program, and we found for all 101 participants a 32.7% intent-to-treat cessation rate for 6 months post-end of program, adjusted to 30.8% by saliva cotinine assessments. The results indicate that combining field outreach with phone counseling over an extended period of time can facilitate smoking cessation for population groups whose environments do not support efforts to quit smoking.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Herpes zoster keratouveitis and inflammatory ocular hypertension 8 years after varicella vaccination.

More than 8 years after varicella vaccination, a healthy 16 year-old boy presented with keratouveitis, severe inflammatory glaucoma in his left eye, and Hutchinson's sign. He was treated with systemic acyclovir, topical steroids, cycloplegics, and glaucoma medications for a full recovery two months after presentation. It is unclear whether the source of herpes zoster which reactivated in this patient represents wild type virus or his previous vaccine strain. Herpes zoster ophthalmicus is very rare in the pediatric population after varicella vaccination but can cause severe inflammatory glaucoma that requires aggressive therapy.

Spontaneous resolution of a traumatic cataract caused by an intralenticular foreign body.

A 49-year-old man presented with an intralenticular metal foreign body incurred while he was sawing wood. The metal chard had violated the lens capsule and was lodged in the cortex of the lens. It was removed using a lens-preservation technique during open-globe repair. Subsequently, a dense posterior cortical cataract developed, which spontaneously resolved over the ensuing months. The cataract had a cruciate configuration with wave-like disruption of the stromal lamellae. To our knowledge, this is the first case of spontaneous resolution of a cataract after capsule violation by an intralenticular foreign body. The unique appearance of the cataract and its unusual resolution led to a new theory of lens injury by shockwave.