Functional analysis of retinitis pigmentosa 2 (RP2) protein reveals variable pathogenic potential of disease-associated missense variants

Genetic mutations are frequently associated with diverse phenotypic consequences, which limits the interpretation of the consequence of a variation in patients. Mutations in the retinitis pigmentosa 2 (RP2) gene are associated with X-linked RP, which is a phenotypically heterogenic form of retinal degeneration. The purpose of this study was to assess the functional consequence of disease-associated mutations in the RP2 gene using an in vivo assay. Morpholino-mediated depletion of rp2 in zebrafish resulted in perturbations in photoreceptor development and microphthalmia (small eye). Ultrastructural and immunofluorescence analyses revealed defective photoreceptor outer segment development and lack of expression of photoreceptor-specific proteins. The retinopathy phenotype could be rescued by expressing the wild-type human RP2 protein. Notably, the tested RP2 mutants exhibited variable degrees of rescue of rod versus cone photoreceptor development as well as microphthalmia. Our results suggest that RP2 plays a key role in photoreceptor development and maintenance in zebrafish and that the clinical heterogeneity associated with RP2 mutations may, in part, result from its potentially distinct functional relevance in rod versus cone photoreceptors.     

Estimating Cytomegalovirus Growth Rates by Using Only a Single Point

Calculation of pathogen growth rates is important in understanding the natural history of infection and effects of therapy. However, it is often difficult to estimate pathogen growth because patients are treated immediately upon the detection of infection, leaving only one nonzero untreated reading. Previous approaches have relied on the flawed assumption that pathogen loads just prior to detection are at the assay detection threshold. We have developed a novel method for estimating the pathogen growth rate from a single reading and investigated the initial growth of cytomegalovirus (CMV) in allogeneic hematopoietic stem cell transplant (HSCT) patients. We applied this approach to CMV viral loads measured at least weekly in 122 patients in the 3 months posttransplant. Viral growth rates were estimated by using a modeling approach that accounts for the viral load and the time since the last negative reading. Viral growth rates decreased rapidly within the first week, from 0.72/day (doubling time, 0.96 day) at the point of reactivation to 0.22/day (doubling time, 3.1 days) at 1 week. Results from this method correlated closely with a two-point regression analysis of a subset of 58 patients with detectable subthreshold viral loads immediately prior to overt reactivation. Patients with lymphocyte counts of ≥0.5 × 10⁹/liter had significantly slower viral growth than patients with low lymphocyte counts (0.612/day versus 0.325/day, P < 0.0001). Thus, our novel method of estimating pathogen growth rates reveals a rapid slowing of CMV growth during reactivation in HSCT patients and a significant impact of the lymphocyte count on CMV growth.     

Visual Impairment in the South Indian State of Andhra Pradesh: Andhra Pradesh - Rapid Assessment of Visual Impairment (AP-RAVI) Project

Purpose

To assess the prevalence and causes of visual impairment in urban and rural population aged ≥40 years in the South India state of Andhra Pradesh.

Methods

A population based cross-sectional study was conducted in which 7800 subjects were sampled from two rural and an urban locations. Visual Acuity (VA) was assessed using a tumbling E chart and eye examinations were performed by trained vision technicians. A questionnaire was used to collect personal and demographic information and previous consultation to eye care providers. Blindness and moderate Visual Impairment (VI) was defined as presenting VA <6/60 and <6/18 to 6/60 in the better eye respectively. VI included blindness and moderate VI.

Results

Of the 7800 subjects enumerated, 7378 (94.6%) were examined. Among those examined, 46.4% were male and 61.8% of them had no education. The mean age of those examined (51.7 years; standard deviation 10.9 years) was similar to those not examined (52.8 years; standard deviation 9.9 years) (p = 0.048). Age and gender adjusted prevalence of VI was 14.3% (95% CI: 13.5–15.0). Refractive errors were the leading cause of VI accounting for 47.6% of all VI followed by cataract (43.7%). Together, they contributed to over 91.3% of the total VI. With multiple logistic regression, the odds of having VI increased significantly with increasing age. Those respondents who had no education were twice (95% CI: 1.7–2.5) more likely to have VI compared to those who were educated. VI was associated with rural residence (OR: 1.3; 95% CI: 1.1–1.6). The association between VI and gender was not statistically significant.

Conclusions

The visual impairment remains a public health challenge in Andhra Pradesh, most of which can be addressed with relatively straight forward interventions like cataract surgery and spectacles. The eye care services need to be streamlined to address this challenge.     

Lead optimization of isocytosine-derived xanthine oxidase inhibitors

We report our attempts at improving the oral efficacy of low-nanomolar inhibitors of xanthine oxidase from isocytosine series through chemical modifications. Our lead compound had earlier shown good in vivo efficacy when administered intraperitoneally but not orally. Several modifications are reported here which achieved more than twofold improvement in exposure. A compound with significant improvement in oral efficacy was also obtained.     

Calcium ionophore A23187 reveals calcium related cellular stress as "I-Bodies": an old actor in a new role

Calcimycin (A23187) is an ionophore widely used in studies related to calcium dynamics in cells, but its fluorometric potential to reveal intracellular physiology has not been explored. Exploiting the microenvironment-induced changes in its fluorescence, we show that a brief exposure of cells to non-toxic concentrations (≤3 μM) of the ionophore results in the characteristic organization of the ionophore forming brightly fluorescent cytoplasmic bodies termed “I-Bodies”, which are closely related to stress linked disturbances/changes in calcium homeostasis. “I-Bodies” appear to be Ca²⁺ rich intracellular sites formed during stress-induced release of intracellular Ca²⁺, causing dysfunction and aggregation of mitochondria, providing scaffold for high density packing of A23187. Formation of “I-Bodies” in cells exposed to ionizing radiation and certain anticancer drugs suggest their potential in revealing alterations in calcium signaling and mitochondrial function during (related to) macromolecular damage-induced cell death. The absence of “I-Bodies” in non-malignant cells and their varying numbers in malignant cells with 5 fold increase in fluorescence imply that they can be potential biomarkers of cancer. Thus, “I-Bodies” are novel indicators of endogenous and induced stress linked to disturbances in calcium homeostasis in cells, with a potential to serve as biomarker of cancer.     

7, 8-diacetoxy-4-methylcoumarin induced cell death in human tumor cells is influenced by calreticulin

Calreticulin (CRT), an endoplasmic reticulum resident protein demonstrates transacetylase activity in presence of 7, 8 diacetoxy-4-methyl coumarin (DAMC) in vitro. To investigate the possible role of CRT and DAMC mediated protein acetylation in cells, we investigated the effects of DAMC in tumor cells with different levels of CRT. DAMC was more toxic (clonogenicity, metabolic viability and proliferation) to human glioma cells (BMG-1) expressing low endogenous CRT level as compared to head and neck carcinoma cells (KB) with a high CRT level. The cytotoxicity was accompanied by loss of mitochondrial membrane potential in both the cells, which correlated with corresponding changes in the levels of pro-apoptotic (Bax) and anti-apoptotic (NFkB) regulators. Manipulation of CRT protein level in KB cells by application of small RNA interference enhanced the sensitivity by four folds while over expression of CRT in BMG-1 cells reduced their sensitivity to DAMC by ∼20% strongly suggesting the influence of CRT on DAMC induced cytotoxicity. The partial rescue of CROE cells from DAMC induced toxicity was accompanied by changes in NFkB levels and over all protein acetylation status, besides increase in the NADPH-cytochrome c reductase activity related to its well known antioxidant property. Since CRT is over-expressed in cancer cells, which are generally resistant to radio- and chemotherapy; targeting CRT transacetylase system, may be an attractive approach for increasing the efficacy of anticancer therapies.     

Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression

We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Poster Sessions CP14: Transplantation and Neurological Repair. GDNF and VMC cotransplantation helps in restoration of neurobehavioral function in Parkinson's disease

During transplantation of VMC in Parkinson's disease their degeneration is very high due to lack of trophic support and mismatch conditions. To overcome this problem, Glial cell line‐derived Neurotrophic Factor (GDNF) known to increase the functional viability and regeneration of dopaminergic cells. In the present study an attempt has been made to validate the role of GDNF cotransplanted with fetal VMC in functional restoration in rat model of Parkinson's disease. A significant restoration was observed in apomorphine induced rotation in rats co transplanted with GDNF and VMC (66%) as compare to VMC alone (42%). Apomorphine induced locomotor activity was restored by 67, 38% in cotransplanted and VMC alone transplanted rats, respectively. Level of dopamine and 3,4 dihydroxy‐phenyl acetic acid (DOPAC) in the striatum were significantly restored by 67 and 62, 42 and 33% in cotransplanted and VMC alone transplanted rats, respectively. A significant restoration was observed in striatum dopamine receptors by 69% in rats cotransplanted with VMC & GDNF, and 45% in those transplanted with VMC alone. GDNF alone transplantation did not show significant restoration in either of the parameters. Functional viabilty of dopaminergic neurons was further confirmed by Tyrosine Hydroxylase (TH) immunopositivity in striatal region where a significantly high expression was observed in cotransplanted animals when compared with VMC alone.Results of the present study suggests that cotransplantation of GDNF and VMC may help in better functional restoration in 6‐OHDA lesioned rat model of Parkinson's disease studied at 4 weeks post transplantation.