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GNAO1-associated disorders are ultra-rare autosomal dominant conditions, which can manifest, depending on the exact pathogenic variant in GNAO1, as a spectrum of neurological phenotypes, including epileptic encephalopathy, developmental delay with movement disorders, or late-onset dystonia. There are currently no effective treatments available, apart from symptomatic options. In this work, we suggest harnessing personalized RNA therapy to treat GNAO1 patients and focus specifically on a recurrent pathogenic variant (E246K). We systemically screened allele-specific antisense oligonucleotides (ASOs) targeting the mutated allele to identify a potent and specific sequence using both reporter-based platforms and a patient-derived cellular model. We show that reduction of mutated GNAO1 in vitro by knockout or by ASO has a beneficial functional outcome, which can be measured by cAMP accumulation and gene expression changes. We established a Gnao1-E246K mouse model that shows a neurological phenotype, which partially recapitulates the human condition. Due to sequence similarity, the mouse can be treated with the selected ASO to test treatment efficacy in animal models, as shown in vitro using murine neural progenitor cells. Our results demonstrate a beneficial effect for the reduction of mutated GNAO1 by ASO in patient-derived models, demonstrating its feasibility as a therapeutic approach.

Introduction: Peroxisomal D-bifunctional protein (DBP) deficiency is an autosomal recessive disorder historically described as a Zellweger-like syndrome comprising neonatal seizures, retinopathy, hearing loss, dysmorphic features, and other complications. The HSD17B4 gene encodes DBP which is essential for oxidation of peroxisomal substrates. We describe 4 patients - 2 unrelated female girls and 2 monozygotic twin sisters - with DBP deficiency and phenotypic diversity. Patient reports: Patient 1 presented neonatally with hypotonia and seizures, and later on developed global developmental delay and regression, sensorineural hearing loss, nystagmus and cortical blindness. The brain MRI demonstrated bilateral peri-sylvian polymicrogyria. Whole exome sequencing revealed 2 mutations in the HSD17B4 gene (c.752G>A, p.(Arg251Gln); c.868 + 1delG).Patient 2 presented with hypotonia, motor delay, and sensorineural hearing loss in infancy, considerable developmental regression during her fourth year, nystagmus, and peripheral neuropathy. Brain MRI demonstrated cerebellar atrophy and abnormal basal ganglia and white matter signal, which appeared after the age of two years. Whole exome sequencing revealed 2 mutations in the HSD17B4 gene (c.14 T>G, p.(Leu5Arg); c.752G>A, p.(Arg251Gln)).Patients 3 and 4, two female monozygotic twins, presented with hypotonia, developmental delay, and macrocephaly from birth, and later on also sensorineural hearing loss, infantile spasms and hypsarrhythmia, and adrenal insufficiency. Brain MRI demonstrated delayed myelination, and an assay of peroxisomal beta oxidation suggested DBP deficiency. Sequencing of the HSD17B4 gene revealed the same 2 mutations as in patient 1. Discussion: We describe 4 patients with variable and diverse clinical picture of DBP deficiency and particularly emphasize the clinical, biochemical, and neuroimaging characteristics. Interestingly, the clinical phenotype varied even between patients with the exact two mutations in the HSD17B4 gene. In addition, in two of the three patients in whom levels of VLCFA including phytanic acid were measured, the levels were within normal limits. This is expanding further the clinical spectrum of this disorder, which should be considered in the differential diagnosis of every patient with hypotonia and developmental delay especially if accompanied by polymicrogyria, seizures, sensorineural hearing loss, or adrenal insufficiency regardless of their VLCFA profile.

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited. Consequently, DDR factor accumulation often serves as a surrogate for damage, without reporting the actual inducing structure. This limitation has practical implications given the importance of the response to DNA reactive drugs such as those used in cancer therapy. These include interstrand crosslink (ICL) forming compounds which are frequently employed clinically. Among them are the psoralens, natural products that form ICLs upon photoactivation and applied therapeutically since antiquity. However, despite multiple attempts, antibodies against psoralen ICLs have not been developed. To overcome this limitation, we developed a psoralen tagged with an antigen for which there are commercial antibodies. In this report we describe our application of the tagged psoralen in imaging experiments, and the unexpected discoveries they revealed., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Published by Elsevier B.V.)

Introduction: Despite widespread implementation of the electronic health record (EHR) in practice, the EHR curriculum in pharmacy schools is slow to emerge. This study used a single assessment tool to evaluate pharmacy students' perceived readiness to use an EHR after completion of two different curricula., Methods: Pharmacy students at the University of Nebraska Medical Center (UNMC) and the University of California San Diego (UCSD) pharmacy schools voluntarily participated. A 14-item survey was administered asking students to rate their comfort on several different EHR skills. Baseline data on non-academic EHR exposure was also collected., Results: Seventy students at UNMC and 69 students at UCSD participated in the survey (27.2% and 28.5% response rate, respectively). Gender and academic year were similar between institutions. Overall, students were more comfortable finding information compared to entering new information in the EHR. Students were most comfortable reviewing laboratory information, progress notes, and medication lists and less comfortable performing medication reconciliation and identifying clinical errors. There were no differences between institutions for overall comfort. Students with at least one month of extracurricular EHR experience rated themselves as more comfortable, but not all of those differences were statistically significant., Conclusions: This study demonstrated that two different EHR curricula in two different pharmacy schools can result in similar student confidence. The information and assessment tool from this study can be a helpful starting point for other schools to determine student preparedness to work with the EHR as well as provide information to inform EHR curricula design and assessment., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020. Published by Elsevier Inc.)

Objective: This study aimed to investigate the neurobiologic correlates of two distinct clusters of agitation symptoms to identify the unique biologic substrates underlying agitated behaviors., Methods: Eighty-eight outpatients with mild to moderate Alzheimer disease (AD) were recruited from the VA Greater Los Angeles Healthcare System Geropsychiatry Outpatient Program. A cross-sectional investigation was conducted of the relationship between cerebral glucose metabolism measured via 18 F-fluorodeoxyglucose positron emission tomography and agitated symptoms from the Neuropsychiatric Inventory (NPI) in patients with AD. Two empirically derived clusters of agitation symptoms were investigated: an Agitation factor comprising agitation/aggression and irritability/lability items of the NPI, and a Behavioral Dyscontrol factor comprising elation/euphoria, disinhibition, aberrant motor behavior, sleep, and appetite items of the NPI. Mean cerebral metabolism for patients who scored positively on each of the two factors was compared with mean cerebral metabolism for those who did not., Results: Patients with AD who scored positively on the Agitation factor showed reduced glucose metabolism of the right temporal, right frontal, and bilateral cingulate cortex. In contrast, the Behavioral Dyscontrol factor did not show specific neurobiologic correlates., Conclusion: Symptoms encompassed within the Agitation factor have distinct neurobiologic underpinnings. The precipitants, course, and outcomes related to these symptoms may be unique from other neuropsychiatric symptoms characteristic of AD. Special attention to treatment of agitated behaviors involving anger, aggressiveness, hostility, and irritability/emotional lability is warranted, because they appear to reflect a clinically relevant symptom cluster with unique underlying neurobiologic correlates., (Published by Elsevier Inc.)

Introduction: Prognostic markers for progression of polycystic kidney disease (PKD) are limited. We evaluated the potential of early para-[(18)F]fluorohippurate ([(18)F]PFH) positron emission tomography (PET) renography to predict future progression of PKD in Han:SPRD rats with slowly progressive autosomal dominant PKD., Materials and Methods: Male and female heterozygous (Cy/+) and normal littermate (+/+) Han:SPRD rats underwent [(18)F]PFH PET renography and blood sampling to measure serum creatinine (S-Cr) and serum urea nitrogen (SUN) concentrations at 6 and 26 wk of age. T2 and T20 values, which represent the percent of the injected dose of [(18)F]PFH in kidneys at 2 and 20 min after injection, were determined from imaging data. T20/T2 ratio was assessed as a prognostic marker. Rats were euthanized after renography at 26 wk of age, and kidney weight/body weight ratios (KW/BW%) were determined as a measure of PKD progression., Results: Male and female Cy/+ rats are known to manifest PKD of different severity, male Cy/+ rats display much more severe PKD than female rats. S-Cr and SUN concentrations did not differ between +/+ and Cy/+ rats and between female and male Cy/+ rats at 6 wk of age, but they were higher at 26 wk of age and male rats displayed higher values than female rats, which indicates inability of S-Cr and SUN to measure disease severity at an early stage. T20/T2 ratios were higher for Cy/+ than +/+ rats at 6 wk of age. Importantly, male Cy/+ rats displayed higher T20/T2 ratios than female Cy/+ rats. T20/T2 ratios obtained at 6 wk of age correlated well with S-Cr, SUN, and KW/BW% values obtained at 26 wk of age., Conclusions: This study indicates that T20/T2 ratio derived from [(18)F]PFH PET renography at an early age could be useful as a novel prognostic marker to predict future disease severity in a rat model of ADPKD., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Objective: The objective of this study was to investigate a new fluorine-18 labeled hippurate, m-cyano-p-[(18)F]fluorohippurate ([(18)F]CNPFH), as a potential radiopharmaceutical for evaluating renal function by PET., Methods: [(18)F]CNPFH was synthesized by a direct one-step nucleophilic aromatic substitution using an (18)F-for-[N(CH(3))(3)](+)-reaction. In vivo stability was determined by HPLC analysis of urine collected from a healthy rat at 30min p.i. of [(18)F]CNPFH. The plasma protein binding (PPB) and erythrocyte uptake of [(18)F]CNPFH were determined using blood collected from healthy rats at 5min p.i. Biodistribution studies were conducted in healthy rats at 10min and 1h p.i. of [(18)F]CNPFH. Dynamic PET/CT imaging data were acquired in normal rats. For comparison, the same rats underwent an identical imaging study using the previously reported p-[(18)F]fluorohippurate ([(18)F]PFH) renal agent., Results: [(18)F]CNPFH demonstrated high in vivo stability with no metabolic degradation. The in vivo PPB and erythrocyte uptake of [(18)F]CNPFH were found to be comparable to those of [(18)F]PFH. Biodistribution and dynamic PET/CT imaging studies revealed a rapid clearance of [(18)F]CNPFH primarily through the renal-urinary pathway. However, unlike [(18)F]PFH, a minor (about 12%) fraction was eliminated via the hepatobiliary route. The PET-derived [(18)F]CNPFH renograms revealed an average time-to-peak (T(max)) of 3.2±0.4min which was similar to [(18)F]PFH, but the average time-to-half-maximal activity (11.4±2.8min) was found to be higher than that of [(18)F]PFH (7.1±1.3min)., Conclusions: Our in vivo results indicate that [(18)F]CNPFH has renogram characteristics similar to those of [(18)F]PFH, however, the unexpected hepatobiliary elimination is adding undesirable background signal in the PET images., (Copyright © 2012 Elsevier Inc. All rights reserved.)

DNA polymerase zeta is believed to be an essential constituent of DNA damage tolerance, comprising several pathways that allow the replication of DNA templates containing unrepaired damage. We wanted to better define the role of polymerase zeta in DNA damage tolerance in mammalian cells. To this aim we have investigated replication of ultraviolet light-damaged DNA templates in mouse embryonic fibroblasts deficient for Rev3, the catalytic subunit of polymerase zeta. We found that Rev3 is important for a post-replication repair pathway of helix-distorting [6-4]pyrimidine-pyrimidone photoproducts and, to a lesser extent, of cyclobutane pyrimidine dimers. Unlike its partner Rev1, Rev3 appears not to be involved in an immediate translesion synthesis pathway at a stalled replication fork. The deficiency of Rev3(-/-) MEFs in post-replication repair of different photoproducts contributes to the extreme sensitivity of these cells to UV light.

Fullerenes are soccer ball-shaped molecules composed of carbon atoms, and, when derivatized with functional groups, they become soluble and can act as photosensitizers. Antimicrobial photodynamic therapy combines a nontoxic photosensitizer with harmless visible light to generate reactive oxygen species that kill microbial cells. We have compared the antimicrobial activity of six functionalized C(60) compounds with one, two, or three hydrophilic or cationic groups in combination with white light against gram-positive bacteria, gram-negative bacteria, and fungi. After a 10 min incubation, the bis- and tris-cationic fullerenes were highly active in killing all tested microbes (4-6 logs) under conditions in which mammalian cells were comparatively unharmed. These compounds performed significantly better than a widely used antimicrobial photosensitizer, toluidine blue O. The high selectivity and efficacy exhibited by these photosensitizers encourage further testing for antimicrobial applications.

Bombesin (BBN), a 14 amino acid peptide, is an analogue of human gastrin releasing peptide (GRP) that binds to GRP receptors (GRPr) with high affinity and specificity. The GRPr is over expressed on a variety of human cancer cells including prostate, breast, lung, and pancreatic cancers. The specific aim of this study was to identify a BBN analogue that can be radiolabeled with (177)Lu and maintains high specificity for GRPr positive prostate cancer tumors in vivo. A preselected synthetic sequence via solid phase peptide synthesis (SPPS) was designed to produce a DOTA-BBN (DOTA = 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid) conjugate with the following general structure: DOTA-X-Q-W-A-V-G-H-L-M-(NH(2)), where the spacer group, X = omega-NH(2)(CH(2))(7)COOH (8-Aoc). The BBN-construct was purified by reversed phase-HPLC (RP-HPLC). Electrospray Mass Spectrometry (ES-MS) was used to characterize both metallated and non-metallated BBN-conjugates. The new DOTA-conjugate was metallated with (177)Lu(III)Cl(3) or non-radioactive Lu(III)Cl(3). The (177)Lu(III)- and non-radiolabeled Lu(III)-conjugates exhibit the same retention times under identical RP-HPLC conditions. The (177)Lu-DOTA-8-Aoc-BBN[7-14]NH(2) conjugate was found to exhibit optimal pharmacokinetic properties in CF-1 normal mice. In vitro and in vivo models demonstrated the ability of the (177)Lu-DOTA-8-Aoc-BBN[7-14]NH(2) conjugate to specifically target GRP receptors expressed on PC-3 human prostate cancer cells.

A diamido-dihydroxymethylenephosphine (N(2)P(2)) bifunction chelating agent (BFCA) was shown to form well-defined (99m)Tc- and (188)Re-chelate structures. The 4, 4-bis [bis-hydroxymethyl-phosphonyl-propylcarbonmoyl]-butyric acid bifunctional chelating agent (N(2)P(2)-BFCA) formed stable complexes with (99m)Tc and (188)Re in >95% yield with high radiochemical purity (RCP). The biodistribution of the (99m)Tc- and (188)Re-N(2)P(2)-BFCAs after intravenous injection studied in normal mice showed the activity was excreted primarily via renal-urinary pathway indicating their use for labeling peptides with (99m)Tc and (188)Re.

In vitro competitive binding studies of In-DOTA-NCS-6-Ahx-Phe(19)-ST[1-19] vs. 125I-Tyr(5)-6-Ahx-Phe(19)-ST[1-19] with guanylate cyclase -C (GC-C) receptors on human colon cancer LS-180 cells revealed an IC(50) value of 7.7 +/- 0.1.6 nM. The in vitro cellular residualization studies of the 111In-DOTA-NCS-ST peptide and GC-C receptor mediated stimulated cGMP production with LS-180 cells demonstrates that this peptide selectively binds to LS-180 cells in an agonistic fashion. In vivo biodistribution studies in LS-180 tumor bearing SCID mice demonstrates that the 111In-DOTA-NCS-ST peptide targets the tumor with a specific uptake of 0.94 +/- 0.31%ID/g at 1 hr p.i. and approximately 23% was retained by the tumor at 4 hrs p.i. The radioactivity cleared rapidly from the blood stream with 84.5 +/- 3.4%ID at 1h p.i. found in the urine. High activity in urine and kidney, and minimal activity in liver and intestines, demonstrates preferential clearance of the radioactivity through the renal/urinary pathway. The specific in vitro and in vivo accumulation of the radioactivity by LS-180 human colonic cancer cells highlights the potential of radiometallated-DOTA-ST analogs as diagnostic/therapeutic radiopharmaceuticals.

Enhanced tyrosine kinase activity due to aberrant or overexpression of receptor and/or non-receptor tyrosine kinases has been implicated in a variety of human malignancies including cutaneous neoplasms. Epidermal growth factor receptor (EGFR)-mediated tyrosine phosphorylation may be a primary indicator of signal transduction regulating cell growth and proliferation. Recent studies have shown that skin tumor promoters such as phorbol ester and ultraviolet B radiation activate EGFR in mouse skin as well as in cell culture. Similarly, oxidative stress, which is implicated in skin tumor promotion, also activates EGFR-mediated cell signaling. Since this signaling pathway has been suggested to be involved in skin tumor promotion, its impairment by antioxidants may lead to an efficient way for skin cancer prevention and therapy. Recently, we showed that silymarin, a flavonoid antioxidant, affords exceptionally high to complete protection against several skin tumor promoters caused tumor promotion in mouse skin. Employing human epidermoid carcinoma cells A431 that contain overexpressed EGFR, in this study, we assessed whether the anti-skin tumor promoting effects of silymarin are due to its inhibitory effect on EGFR activation and down stream signaling pathway leading to perturbations in cell cycle progression. Treatment of cells with silymarin resulted in a significant inhibition of ligand-induced activation of EGFR with no change in its protein levels. Silymarin treatment also resulted in a significant decrease in tyrosine phosphorylation of Shc, an immediate downstream target of EGFR, but no change in the protein levels of Shc. The inhibition of EGFR activation by silymarin was associated with a highly significant to complete inhibition of EGFR intrinsic kinase activity. Cells treated with silymarin also showed a significant G2-M arrest in cell cycle progression, and a highly significant inhibition of DNA synthesis and cell growth in a dose-dependent manner. Taken together, these results suggest that skin cancer chemopreventive effects of silymarin are mediated via impairment of EGFR signaling which ultimately leads to perturbation in cell cycle progression resulting in the inhibition of proliferation and induction of growth arrest., (Copyright 1998 Academic Press.)