Application of the rainbow trout derived intestinal cell line (RTgutGC) for ecotoxicological studies: molecular and cellular responses following exposure to copper.

There is an acknowledged need for in vitro fish intestinal model to help understand dietary exposure to chemicals in the aquatic environment. The presence and use of such models is however largely restrictive due to technical difficulties in the culturing of enterocytes in general and the availability of appropriate established cell lines in particular. In this study, the rainbow trout (Oncorhynchus mykiss) intestinal derived cell line (RTgutGC) was used as a surrogate for the "gut sac" method. To facilitate comparison, RTgutGC cells were grown as monolayers (double-seeded) on permeable Transwell supports leading to a two-compartment intestinal model consisting of polarised epithelium. This two-compartment model divides the system into an upper apical (lumen) and a lower basolateral (portal blood) compartment. In our studies, these cells stained weakly for mucosubstances, expressed the tight junction protein ZO-1 in addition to E-cadherin and revealed the presence of polarised epithelium in addition to microvilli protrusions. The cells also revealed a comparable transepithelial electrical resistance (TEER) to the in vivo situation. Importantly, the cell line tolerated apical saline (1:1 ratio) thus mimicking the intact organ to allow assessment of uptake of compounds across the intestine. Following an exposure over 72 h, our study demonstrated that the RTgutGC cell line under sub-lethal concentrations of copper sulphate (Cu) and modified saline solutions demonstrated uptake of the metal with saturation levels comparable to short term ex situ gut sac preparations. Gene expression analysis revealed no significant influence of pH or time on mRNA expression levels of key stress related genes (i.e. CYP3A, GST, mtA, Pgp and SOD) in the Transwell model. However, significant positive correlations were found between all genes investigated suggesting a co-operative relationship amongst the genes studied. When the outlined characteristics of the cell line are combined with the division of compartments, the RTgutGC double seeded model represents a potential animal replacement model for ecotoxicological studies. Overall, this model could be used to study the effects and predict aquatic gastrointestinal permeability of metals and other environmentally relevant contaminants in a cost effective and high throughput manner

Эффективность и переносимость фиксированной комбинации травопроста 0,004% и тимолола 0,5% в лечении первичной открытоугольной глаукомы или офтальмогипертензии при недостаточной эффективности монотерапии бета-блокаторами

PURPOSE: To evaluate the efficacy and tolerability of travoprost 0.004%/timolol 0.5% fixed-dose combination (TTFC) in patients with open-angle glaucoma (OAG) or ocular hypertension (OHT) inadequately controlled on betablocker monotherapy. METHODS: In this phase IV, open-label study, 156 patients on beta-blocker monotherapy with mean intraocular pressure (IOP) between 18 and 32 mmHg were randomized (no washout period) to receive TTFC for 8 weeks (TTFC group) or to continue beta-blocker monotherapy for 4 weeks followed by TTFC for the remaining 4 weeks (beta-blocker group). RESULTS: The mean IOP at baseline in the TTFC and betablocker groups was 22.5±2.5 mmHg and 22.2±2.3 mmHg, respectively, and at weeks 4 and 8, was 16.7±3.1 mmHg and 16.1±3.1 mmHg, respectively, in TTFC group and 21.1±3.1 mmHg and 16.1±2.8 mmHg, respectively, in the beta-blocker group. There was a significant least squares mean difference between TTFC and beta-blocker in 8 a.m. IOP at week 4 (−4.6 mmHg; ????0.0001). Both treatments were well tolerated. CONCLUSION: Superior IOP control was achieved with TTFC in patients with OAG or OHT previously uncontrolled with beta-blockers. No new safety findings were identified. ЦЕЛЬ. Оценка эффективности и переносимости фиксированной комбинации травопроста 0,004% и тимолола 0,5% у пациентов с первичной открытоугольной глаукомой (ПОУГ) и офтальмогипертензией в случае недостаточной гипотензивной эффективности монотерапии бета-блокаторами. МЕТОДЫ. В открытом исследовании IV фазы приняли участие 156 пациентов, получавших монотерапию бета- блокаторами, средний уровень внутриглазного давления (ВГД) у которых составил от 18 до 32 мм рт.ст. Эти пациенты были подвергнуты рандомизации (без периода вымывания) для назначения фиксированной комбинации травопрост/тимолол в течение 8 недель или продолжения монотерапии бета-блокаторами в течение 4 недель с последующим переходом на фиксированную комбинацию в течение еще 4 недель. РЕЗУЛЬТАТЫ. Исходно средний уровень ВГД составил 22,5±2,5 мм рт.ст. в группе фиксированной комбинации и 22,2±2,3 мм рт.ст. в группе бета-блокаторов. Через 4 недели средний уровень ВГД равнялся 16,7±3,1 и 21,1±3,1 мм рт.ст. соответственно, а через 8 недель — 16,1±3,1 и 16,1±2,8 мм рт.ст. соответственно. При измерении ВГД в 8 часов утра через 4 недели имели место статистически достоверные отличия среднего Ро между группой фиксированной комбинации и группой бета-блокаторов (на 4,6 мм рт.ст., р<0,0001). Оба варианта лечения хорошо переносились. ЗАКЛЮЧЕНИЕ. Фиксированная комбинация траво- прост/тимолол позволяет лучше контролировать ВГД у пациентов с ПОУГ или офтальмогипертензией при недостаточной эффективности бета-блокаторов. Ранее неизвестных побочных явлений не выявлено. КЛЮЧЕВЫЕ СЛОВА: первичная открытоугольная глау- кома, травопрост, тимолол.>< 0,0001). Оба варианта лечения хорошо переносились. ЗАКЛЮЧЕНИЕ. Фиксированная комбинация травопрост/тимолол позволяет лучше контролировать ВГД у пациентов с ПОУГ или офтальмогипертензией при недостаточной эффективности бета-блокаторов. Ранее неизвестных побочных явлений не выявлено.

Biochemical warfare on the reef : the role of glutathione transferases in consumer tolerance of dietary prostaglandins

© 2010 The Authors. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS ONE 5 (2010): e8537, doi:10.1371/journal.pone.0008537.Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla. Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A2 (PGA2) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA2) were also the most potent inhibitors. In vivo estimates of PGA2 concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA2 and operating at or near physiological capacity. The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer's gorgonian diet. This generalist's GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.Financial support for this work was provided by the Ocean Life Institute Tropical Research Initiative Grant (WHOI) to KEW and MEH; the Robert H. Cole Endowed Ocean Ventures Fund (WHOI) to KEW; the National Undersea Research Center - Program Development Proposal (CMRC-03PRMN0103A) to KEW; Walter A. and Hope Noyes Smith, and a National Science Foundation Graduate Research Fellowship to KEW

Protein Dynamics in Complex DNA Lesions.

A single mutagen can generate multiple different types of DNA lesions. How different repair pathways cooperate in complex DNA lesions, however, remains largely unclear. Here we measured, clustered, and modeled the kinetics of recruitment and dissociation of 70 DNA repair proteins to laser-induced DNA damage sites in HeLa cells. The precise timescale of protein recruitment reveals that error-prone translesion polymerases are considerably delayed compared to error-free polymerases. We show that this is ensured by the delayed recruitment of RAD18 to double-strand break sites. The time benefit of error-free polymerases disappears when PARP inhibition significantly delays PCNA recruitment. Moreover, removal of PCNA from complex DNA damage sites correlates with RPA loading during 5'-DNA end resection. Our systematic study of the dynamics of DNA repair proteins in complex DNA lesions reveals the multifaceted coordination between the repair pathways and provides a kinetics-based resource to study genomic instability and anticancer drug impact

HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain.

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions

HspB8 prevents aberrant phase transitions of FUS by chaperoning its folded RNA binding domain

Aberrant liquid-to-solid phase transitions of biomolecular condensates have been linked to various neurodegenerative diseases. However, the underlying molecular interactions that drive aging remain enigmatic. Here, we develop quantitative time-resolved crosslinking mass spectrometry to monitor protein interactions and dynamics inside condensates formed by the protein fused in sarcoma (FUS). We identify misfolding of the RNA recognition motif (RRM) of FUS as a key driver of condensate ageing. We demonstrate that the small heat shock protein HspB8 partitions into FUS condensates via its intrinsically disordered domain and prevents condensate hardening via condensate-specific interactions that are mediated by its α-crystallin domain (αCD). These αCD-mediated interactions are altered in a disease-associated mutant of HspB8, which abrogates the ability of HspB8 to prevent condensate hardening. We propose that stabilizing aggregation-prone folded RNA-binding domains inside condensates by molecular chaperones may be a general mechanism to prevent aberrant phase transitions