The gene therapy for corneal pathology with novel nonsense cystinosis mouse lines created by CRISPR Gene Editing.

PURPOSE: Cystinosis is an autosomal recessive lysosomal storage disease (LSDs) caused by mutations in the gene encoding cystinosin (CTNS) that leads to cystine crystal accumulation in the lysosome that compromises cellular functions resulting in tissue damage and organ failure, especially in kidneys and eyes. However, the underlying molecular mechanism of its pathogenesis remains elusive. Two novel mice lines created via CRISPR are used to examine the pathogenesis of cystinosis in the kidney and cornea and the treatment efficacy of corneal pathology using self-complimentary Adeno-associated viral (scAAV-CTNS) vector. METHODS: The CRISPR technique generated two novel cystinotic mouse lines, Ctnsis1 (an insertional mutation) and Ctnsis2 (a nonsense mutation). Immune histochemistry, renal functions test and HRT2 in vivo confocal microscopy were used to evaluate the age-related renal pathogenesis and treatment efficacy of the scAAV-CTNS virus in corneal pathology. RESULTS: Both mutations lead to the production of truncated Ctns proteins. Ctnsis1 and Ctnsis 2 mice exhibit the characteristic of cystinotic corneal crystal phenotype at four-week-old. Treatment with the scAAV-CTNS viral vector decreased the corneal crystals in the treated mice cornea. Ctnsis 1 show renal abnormalities manifested by increased urine volume, reduced urine osmolality, and the loss of response to Desmopressin (dDAVP) at 22-month-old but Ctnsis2 don't manifest renal pathology up to 2 years of age. CONCLUSIONS: Both Ctnsis1 and Ctnsis2 mice exhibit phenotypes resembling human intermediate nephropathic and ocular cystinosis, respectively. scAAV-CTNS viral vectors reduce the corneal cystine crystals and have a great potential as a therapeutic strategy for treating patients suffering from cystinosis.

CHOROIDAL MELANOMA IN PHAKOMATOSIS PIGMENTOVASCULARIS WITH OVERLAPPING STURGE-WEBER SYNDROME AND KLIPPEL-TRENAUNAY SYNDROME.

PURPOSE: To present the rare occurrence of choroidal melanoma in an adult patient with phakomatosis pigmentovascularis and an overlap of Sturge-Weber syndrome and Klippel-Trenaunay syndrome. METHODS: Observational case report. RESULTS: A 75-year-old White woman with nevus flammeus involving the left forehead, periorbital area, cheek, chin, upper limb, and trunk, along with hemihypertrophy of the left side of the face and left upper limb, presented for evaluation of an intraocular mass in the left eye. Anterior examination of the left eye showed diffuse episcleral and iris melanocytosis. Fundus examination of the left eye showed diffuse choroidal melanocytosis and an elevated choroidal lesion. B-scan ultrasonography demonstrated a hollow lesion, and the patient was diagnosed with choroidal melanoma in the left eye in the setting of phakomatosis pigmentovascularis with overlap of Sturge-Weber syndrome and Klippel-Trenaunay syndrome. Fine-needle aspiration biopsy confirmed the diagnosis, and Iodine 125 plaque radiotherapy was performed. CONCLUSION: Individuals with clinical features suggestive of phakomatosis pigmentovascularis, Sturge-Weber syndrome, or Klippel-Trenaunay syndrome should undergo a complete ophthalmological evaluation for the presence of ocular melanocytosis and uveal melanoma.

Massive spontaneous subconjunctival hemorrhage in a patient on therapeutic warfarin: A case report.

PURPOSE: To describe a case of massive spontaneous subconjunctival hemorrhage in a patient taking warfarin with a therapeutic international normalized ratio (INR). OBSERVATIONS: Massive circumferential hemorrhagic chemosis, extensive periorbital and facial ecchymosis, and active arterial extravasation in the subconjunctiva which required cessation and reversal of anticoagulation. Findings gradually resolved over several months after discharge. CONCLUSIONS AND IMPORTANCE: While subconjunctival hemorrhage, even in anticoagulated patients, is usually benign, rare examples of severe presentations exist. We present, to our knowledge, the first documented case of a subconjunctival hemorrhage necessitating cessation and reversal of anticoagulation in the setting of a therapeutic INR.

Bird feathers are potential biomonitors for airborne elemental carbon.

Birds can serve as effective biomonitors of air pollution, yet few studies have quantified external particulate matter accumulation on bird feathers. Biomonitoring of airborne elemental carbon (EC) is of critical significance because EC is a component of particulate matter with adverse effects on air quality and human health. To assess their effectiveness for use in EC monitoring, we compared EC accumulation on bird feathers at two sites that differed in vehicular traffic volume in an urban environment within the Dallas-Fort Worth Metropolitan Area, USA. Moulted flight feathers from domestic chickens were experimentally exposed to ambient EC pollution for 5 days in two urban microenvironments 1.5 km distant from each other that differed in traffic volume--adjacent to an interstate highway and a university campus bus stop. Feathers near the highway accumulated approximately eight times more EC (307 ± 34 µg m-2 day-1), on average, than feathers near the bus stop (40 ± 9 µg m-2 day-1). These findings indicate that EC accumulation on feathers varies over short distances within urban areas and that bird feathers potentially can be used for biomonitoring airborne EC.

Nanoparticle mediated increased insulin-like growth factor 1 expression enhances human placenta syncytium function.

INTRODUCTION: Placental dysfunction is an underlying cause of many major obstetric diseases and treatment options for complications like fetal growth restriction (FGR) are limited .We previously demonstrated nanoparticle delivery of the human insulin-like growth factor 1 (hIGF1) transgene under control of the trophoblast-specific PLAC1 promoter maintains normal fetal growth in a surgically-induced FGR mouse model. However, uptake by human placental syncytiotrophoblast has yet to be determined. METHODS: An ex vivo human placenta perfusion model, term placenta villous fragments, and other in vitro syncytiotrophoblast models were used to determine nanoparticle uptake, transgene expression, and functional responses under oxidative stress conditions. RESULTS: In the ex vivo perfusion, fluorescence from a Texas-Red conjugated nanoparticle increased in maternal perfusate upon nanoparticle addition and declined by the conclusion of the experiment (P < 0.001. Fluorescent histology confirmed localization in the syncytiotrophoblasts. No Texas-Red fluorescence was detected in the fetal perfusate. Transgene expression of hIGF1 in differentiated BeWo cells, isolated primary trophoblasts and fragments was increased compared to untreated (55,000-fold, P = 0.0003; 95-fold, P = 0.003; 400-fold, P < 0.001, respectively). Functionally, increased hIGF1 expression in villous fragments resulted in translocation of glucose transporter 1 to the syncytiotrophoblast cell membrane and under conditions of oxidative stress in BeWo cells, protected against increased cell death (P < 0.01) and decreased mitochondrial activity (P < 0.01). CONCLUSION: The current study confirms that our nanoparticle is capable of uptake in human placental syncytium which results in enhanced transgene expression, functional changes to cellular activity and protection against increased oxidative stress.

Clinical Features, Metastasis, and Survival in Patients Younger Than 21 Years With Posterior Uveal Melanoma.

Importance: Given the rarity of posterior uveal melanoma in patients younger than 21 years, reporting clinical experience in this area has relevance. Objective: To describe the baseline clinical features, treatment, and clinical course of a group of patients younger than 21 years who have primary posterior uveal melanoma. Design, Setting, and Participants: This retrospective descriptive case series of patients younger than 21 years who have a primary choroidal or ciliochoroidal melanoma was conducted at a single-center subspecialty referral practice. Patients in the relevant age group who were treated in a single practice between July 1980 and December 2013 were included; clinical data collected through December 2017 were captured to permit adequate follow-up time in all cases. Main Outcomes and Measures: Conventional descriptive statistics of relevant clinical variables (eg, demographic, tumor, treatment, and outcome variables) of each patient were recorded. Actuarial metastasis-free and overall survival curves were computed and plotted, as was a postdetection survival curve of patients who developed metastasis during available follow-up. Results: Of 2265 patients with posterior uveal melanoma encountered by the authors during the study interval, 18 (0.8%) were younger than 21 years when diagnosed and treated. Ten were female and 8 male, and the mean (SD) age was 16.6 (4.2) years. Through available follow-up, 8 of these patients had developed metastatic uveal melanoma (44%). All 8 died of metastasis. Actuarial survival analysis showed that the cumulative probability of metastatic death in this group exceeded 50%. The median overall survival time after treatment of the primary intraocular tumor was 11.9 (95% CI, 7.3-16.5) years. The median survival time after detection of metastasis in the 8 patients who developed metastasis was 2.3 months (95% CI, 0.0-5.2) months. Conclusions and Relevance: Posterior uveal melanoma in patients younger than 21 years appears to have a similar if not worse prognosis than patients with PUM in the population overall. Owing to the later onset of metastasis observed, patients younger than 21 years should continue to have surveillance tests for more than 10 years after treatment.

Posterior uveal melanoma in adolescents and children: current perspectives.

Recognizing that <1% of all uveal melanomas occur in young persons, and that very few clinicians encounter more than a few such cases over an extended career, we felt that a retrospective review of literature and sharing of our clinical experience would be appropriate to remind readers about this age subgroup of patients with posterior uveal melanoma. This interest stems from the increase in reported cases of uveal melanoma in younger individuals and recent advances in the field.

Connexin43 phosphorylation at S368 is acute during S and G2/M and in response to protein kinase C activation.

Phorbol esters such as 12-O-tetradeconylphorbol-13-acetate (TPA) activate protein kinase C, increase Connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. Previous work has implicated protein kinase C (PKC) in the direct phosphorylation of Cx43 at S368, which results in a change in single channel behavior that contributes to a decrease in intercellular communication. We have examined Cx43 phosphorylation in several cell lines with an antibody specific for phosphorylated S368. We show that this antibody detects Cx43 only when it is phosphorylated at S368 and, consistent with previous results, TPA treatment causes a dramatic increase in phosphorylation at S368. However, in some cell types, the increased phosphorylation at S368 did not cause a detectable shift in migration as compared with the nonphosphorylated Cx43. Immunofluorescence showed increased S368 immunolabeling in cytoplasmic and plasma membrane structures in response to TPA. Immunoblot analysis of synchronized cells showed increased phosphorylation at S368 during S and G2/M phases of the cell cycle. S-phase cells contained more total Cx43 but assembled fewer functional gap junctional channels than G0-phase cells. Since M-phase cells also communicate poorly and contain few assembled gap junctions, phosphorylation at S368 appears to be negatively correlated with gap junction assembly. Thus, both gap junctional communication and S368 phosphorylation change during S phase and G2/M, implying that phosphorylation at S368 might play a role in key cell-cycle events.