Efficient treatment of secondary treated refinery wastewater using sand biofiltration: Removal of hazardous organic pollutants.

This study explored the potential of sand biofiltration for tertiary treatment of real refinery wastewater. The biofilter (2 cm (I.D.) x 15 cm (L)) operated on secondary treated refinery wastewater at flow rate of 1 mL/min had empty bed contact time (EBCT) of 47.12 min for one circulation. Maximum reduction in COD after 4, 8 and 12 times recirculation was 25 %, 52 % and 56 %; while the TOC reduction was 33 %, 43 % and 51 %, respectively, after biofilm development over 30 days. Quantification using two dimensional gas chromatography - time of flight mass spectrometry (GCxGC-TOF MS) revealed that several of the identified target compounds could not be detected in the wastewater after 12 recirculations. After 8 times recirculation, most of the compounds showed very high removal efficiency. For biofiltration over the flow rate range 2-10 mL/min, the reduction in COD and NH4+-N ranged from 62-73 % and 78-86 %, respectively, after 8 times recirculation. The nitrite concentration first increased and subsequently decreased, while the nitrate concentration continuously increased with increase in the number of recirculations. Solid phase micro-extraction (SPME) analysis of the aqueous phase using GCxGC-TOF MS and a semi-quantitative approach indicated that the removal of predominant classes of compounds was greater than 95 % after 8 times recirculation, with maximum reduction occurring in the first pass through the biofilter. Assimilable organic carbon (AOC) reduction was 98 % after 8 times recirculation. Metagenomic analysis revealed that Proteobacteria was the most dominant phylum in the biofilter. Many known polynuclear aromatic hydrocarbon (PAH) degraders, such as Sphingomonadales, Burkholderiales, Rhodobacterales and Rhodospirillales, were found in the biofilter leading to high removal efficiency of hazardous organic pollutants.

Tissue-targeted and localized AAV5-DCN and AAV5-PEDF combination gene therapy abrogates corneal fibrosis and concurrent neovascularization in rabbit eyes in vivo.

PURPOSE: Corneal fibrosis and neovascularization (CNV) after ocular trauma impairs vision. This study tested therapeutic potential of tissue-targeted adeno-associated virus5 (AAV5) mediated decorin (DCN) and pigment epithelium-derived factor (PEDF) combination genes in vivo. METHODS: Corneal fibrosis and CNV were induced in New Zealand White rabbits via chemical trauma. Gene therapy in stroma was delivered 30-min after chemical-trauma via topical AAV5-DCN and AAV5-PEDF application using a cloning cylinder. Clinical eye examinations and multimodal imaging in live rabbits were performed periodically and corneal tissues were collected 9-day and 15-day post euthanasia. Histological, cellular, and molecular and apoptosis assays were used for efficacy, tolerability, and mechanistic studies. RESULTS: The AAV5-DCN and AAV5-PEDF combination gene therapy significantly reduced corneal fibrosis (p < 0.01 or p < 0.001) and CNV (p < 0.001) in therapy-given (chemical-trauma and AAV5-DCN + AAV5-PEDF) rabbit eyes compared to the no-therapy given eyes (chemical-trauma and AAV5-naked vector). Histopathological analyses demonstrated significantly reduced fibrotic α-smooth muscle actin and endothelial lectin expression in therapy-given corneas compared to no-therapy corneas on day-9 (p < 0.001) and day-15 (p < 0.001). Further, therapy-given corneas showed significantly increased Fas-ligand mRNA levels (p < 0.001) and apoptotic cell death in neovessels (p < 0.001) compared to no-therapy corneas. AAV5 delivered 2.69 × 107 copies of DCN and 2.31 × 107 copies of PEDF genes per µg of DNA. AAV5 vector and delivered DCN and PEDF genes found tolerable to the rabbit eyes and caused no significant toxicity to the cornea. CONCLUSION: The combination AAV5-DCN and AAV5-PEDF topical gene therapy effectively reduces corneal fibrosis and CNV with high tolerability in vivo in rabbits. Additional studies are warranted.

Toxicological effects of ocular acrolein exposure to eyelids in rabbits in vivo.

Acrolein is a highly reactive volatile toxic chemical that injures the eyes and many organs. It has been used in wars and terrorism for wounding masses on multiple occasions and is readily accessible commercially. Our earlier studies revealed acrolein's toxicity to the cornea and witnessed damage to other ocular tissues. Eyelids play a vital role in keeping eyes mobile, moist, lubricated, and functional utilizing a range of diverse lipids produced by the Meibomian glands located in the upper and lower eyelids. This study sought to investigate acrolein's toxicity to eyelid tissues by studying the expression of inflammatory and lipid markers in rabbit eyes in vivo utilizing our reported vapor-cap model. The study was approved by the institutional animal care and use committees and followed ARVO guidelines. Twelve New Zealand White Rabbits were divided into 3 groups: Naïve (group 1), 1-min acrolein exposure (group 2), or 3-min acrolein exposure (group 3). The toxicological effects of acrolein on ocular health in live animals were monitored with regular clinical eye exams and intraocular pressure measurements and eyelid tissues post-euthanasia were subjected to H&E and Masson's trichrome histology and qRT-PCR analysis. Clinical eye examinations witnessed severely swollen eyelids, abnormal ocular discharge, chemosis, and elevated intraocular pressure (p < 0.001) in acrolein-exposed eyes. Histological studies supported clinical findings and exhibited noticeable changes in eyelid tissue morphology. Gene expression studies exhibited significantly increased expression of inflammatory and lipid mediators (LOX, PAF, Cox-2, and LTB4; p < 0.001) in acrolein-exposed eyelid tissues compared to naïve eyelid tissues. The results suggest that acrolein exposure to the eyes causes acute damage to eyelids by altering inflammatory and lipid mediators in vivo.

Characterization of C-X-C chemokine receptor type 5 in the cornea and role in the inflammatory response after corneal injury.

C-X-C chemokine receptor type 5 (CXCR5) regulates inflammatory responses in ocular and non-ocular tissues. However, its expression and role in the cornea are still unknown. Here, we report the expression of CXCR5 in human cornea in vitro and mouse corneas in vivo, and its functional role in corneal inflammation using C57BL/6J wild-type (CXCR5+/+) and CXCR5-deficient (CXCR5-/-) mice, topical alkali injury, clinical eye imaging, histology, immunofluorescence, PCR, qRT-PCR, and western blotting. Human corneal epithelial cells, stromal fibroblasts, and endothelial cells demonstrated CXCR5 mRNA and protein expression in PCR, and Western blot analyses, respectively. To study the functional role of CXCR5 in vivo, mice were divided into four groups: Group-1 (CXCR5+/+ alkali injured cornea; n = 30), Group-2 (CXCR5-/- alkali injured cornea; n = 30), Group-3 (CXCR5+/+ naïve cornea; n = 30), and Group-4 (CXCR5-/- naïve cornea; n = 30). Only one eye was wounded with alkali. Clinical corneal evaluation and imaging were performed before and after injury. Mice were euthanized 4 h, 3 days, or 7 days after injury, eyes were excised and used for histology, immunofluorescence, and qRT-PCR. In clinical eye examinations, CXCR5-/- mouse corneas showed ocular health akin to the naïve corneas. Alkali injured CXCR5+/+ mouse corneas showed significantly increased mRNA (p < 0.001) and protein (p < 0.01 or p < 0.0001) levels of the CXCR5 compared to the naïve corneas. Likewise, alkali injured CXCR5-/- mouse corneas showed remarkably amplified inflammation in clinical eye exams in live animals. The histological and molecular analyses of these corneas post euthanasia exhibited markedly augmented inflammatory cells in H&E staining and significant CD11b + cells in immunofluorescence (p < 0.01 or < 0.05); and tumor necrosis factor-alpha (TNFα; p < 0.05), cyclooxygenase 2 (COX-2; p < 0.0001), interleukin (IL)-1ß (p < 0.0001), and IL-6 (p < 0.0001 or < 0.01) mRNA expression compared to the CXCR5+/+ mouse corneas. Interestingly, CXCR5-/- alkali injured corneas also showed altered mRNA expression of fibrotic alpha smooth muscle actin (α-SMA; p > 0.05) and angiogenic vascular endothelial growth factor (VEGF; p < 0.01) compared to the CXCR5+/+ alkali injured corneas. In summary, the CXCR5 gene is expressed in all three major layers of the cornea and appears to influence corneal inflammatory and repair events post-injury in vivo. More studies are warranted to tease the mechanistic role of CXCR5 in corneal inflammation and wound healing.

Concordance of financial disclosures among faculty at the 2018-2020 SAGES annual meetings.

BACKGROUND: Financial relationships with industry may bias educational content delivered by physicians. SAGES strives to mitigate potential bias, relying on physician self-reporting. Retrospective review of relationships is possible using the Open Payments Database (OPD), a public record of industry-reported payments to US physicians. We aimed to evaluate the effectiveness of the SAGES disclosure process by comparing faculty disclosures to SAGES, faculty disclosures within presentations, and OPD records among speakers at the 2018-2020 SAGES meetings. METHODS: We reviewed all presentations from the SAGES 2018-2020 Annual Meetings. For each invited presentation, all slide-disclosed relationships were recorded. For US physicians, we queried the OPD and recorded relationships ≥ $500 USD in the calendar year prior to presentation. We compared the slide-disclosed relationships with OPD-reported relationships and with those provided to SAGES during the faculty disclosure process. We surveyed a sample of the 2020 annual meeting speakers to analyze potential reasons for discordance. RESULTS: From 2018 to 2020, there were 1,355 invited presentations, of which 1,234 (91%) were available for review. Disclosure slides were present in 1,098 (89%), increasing from 86% in 2018 to 93% in 2020. The proportion of speakers with OPD-reported relationships ≥ $500 increased from 54% in 2018 to 66% in 2020. The total value of OPD relationships decreased from $5.9 million (2018) to $3.3 million (2020) with a concomitant decrease in the proportion with high discordance from 9% in 2018 to 5% in 2020. Among the 2020 speakers with high discordance, the most common explanations for discordance were being unaware of payment or payment outside the 12-month timeframe (55%). CONCLUSIONS: Discordance between financial disclosures reported to SAGES and OPD highlight the need for improvements in the faculty disclosure process. SAGES will continue to streamline this process by incorporating faculty review of their OPD disclosures to ensure all educational programs remain free of commercial bias.

Time-dependent in situ structural and cellular aberrations in rabbit cornea in vivo after mustard gas exposure.

An array of corneal pathologies collectively called mustard gas keratopathy (MGK) resulting from ocular exposure to sulfur mustard (SM) gas are the most prevalent chemical warfare injury. MGK involves chronic ocular discomfort that results in vision impairment. The etiology of MGK remains unclear and poorly understood primarily due to a lack of scientific data regarding structural and cellular changes in different layers of the cornea altered by mustard vapor exposure in vivo. The goals of this study were to (a) characterize time-dependent changes in different layers of corneal epithelium, stroma, and endothelium in live animals in situ by employing state-of-the-art multimodal clinical ophthalmic imaging techniques and (b) determine if SM-induced acute changes in corneal cells could be rescued by a topical eye drop (TED) treatment using in an established rabbit in vivo model. Forty-five New Zealand White Rabbit eyes were divided into four groups (Naïve, TED, SM, and SM + TED). Only one eye was exposed to SM (200 mg-min/m3 for 8 min), and each group had three time points with six eyes each (Table-1). TED was topically applied twice a day for seven days. Clinical eye examinations and imaging were performed in live rabbits with stereo, Slit-lamp, HRT-RCM3, and Spectralis microscopy system. Fantes grading, fluorescein staining, Schirmer's tests, and applanation tonometry were conducted to measure corneal haze, ocular surface aberrations, tears, and intraocular pressure respectively. H&E and PSR staining were used for histopathological cellular changes in the cornea. In vivo confocal and OCT imaging revealed significant changes in structural and morphological appearance of corneal epithelium, stroma, and endothelium in vivo in SM-exposed rabbit corneas in a time-dependent manner compared to naïve cornea. Also, SM-exposed eyes showed loss of corneal transparency characterized by increased stromal thickness and light-scattering myofibroblasts or activated keratocytes, representing haze formation in the cornea. Neither naive nor TED-alone treated eyes showed any structural, cellular, and functional abnormalities. Topical TED treatment significantly reduced SM-induced abnormalities in primary corneal layers. We conclude that structural and cellular changes in primary corneal layers are early pathological events contributing to MGK in vivo, and efficient targeting of them with suitable agents has the potential to mitigate SM ocular injury.

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo.

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

Effects of Regular/Dilute Proparacaine Anesthetic Eye Drops in Combination with Ophthalmic Antibiotics on Corneal Wound Healing.

Purpose: Topical, local anesthetic eye drops in conjunction with antibiotics are commonly used to reduce ocular pain and treat patients in emergency clinics; however, their effects on corneal healing are poorly understood. This study examined whether regular or diluted proparacaine eye drops given in combination with common ophthalmic antibiotics affect corneal wound healing parameters using in vitro and in vivo models. Methods: Primary human corneal fibroblasts generated from donor corneas and New Zealand white rabbits were used. Regular (0.5%) and diluted (0.05%) proparacaine eye drops, twice daily for 3 days, were applied to cultures and rabbit eyes, with or without ophthalmic antibiotics (polymyxin B sulfate and trimethoprim). Trypan blue, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and scratch wound assays measured cellular viability, proliferation, and migration, respectively, in vitro. Slit lamp biomicroscopy, tonometry, fluorescein eye test, hematoxylin and eosin (H&E) staining, and 4',6-diamidino-2-phenylindole (DAPI) immunofluorescence were used for in vivo studies. Results: Both regular and diluted proparacaine affected wound healing response in the cornea in vitro and in vivo in a time-dependent manner. Adjunct antibiotic treatments had additive effects characterized by reduced corneal fibroblast viability, proliferation, and migration in vitro and corneal epithelial recovery in vivo. Regular proparacaine with antibiotics showed most pronounced effects on corneal wound healing parameters, and diluted proparacaine without antibiotics had minimal negative effects in vitro and in vivo. Conclusion: Both methods of regular (0.5%) and diluted (0.05%) proparacaine topical application to the cornea are safe, but impede corneal wound healing in vitro and in vivo. Adjunct antibiotic treatments had additive negative effects on corneal wound repair.

Decorin regulates collagen fibrillogenesis during corneal wound healing in mouse in vivo.

A characteristic rigid spatial arrangement of collagen fibrils in the stroma is critical for corneal transparency. This unique organization of collagen fibrils in corneal stroma can be impacted by the presence and interactions of proteoglycans and extracellular matrix (ECM) proteins in a corneal microenvironment. Earlier studies revealed that decorin, a leucine-rich proteoglycan in stroma, regulates keratocyte-collagen matrix assembly and wound healing in the cornea. This study investigated the role of decorin in the regulation of stromal fibrillogenesis and corneal transparency in vivo employing a loss-of-function genetic approach using decorin null (dcn-/-) and wild type (dcn+/+) mice and a standard alkali-injury model. A time-dependent ocular examinations with Slit lamp microscope in live animals assessed corneal clarity, haze, and neovascularization levels in normal and injured eyes. Morphometric changes in normal and injured dcn+/+ and dcn-/- corneas, post-euthanasia, were analyzed with Masson's Trichrome and Periodic Acid-Schiff (PAS) histology evaluations. The ultrastructure changes in all corneas were investigated with transmission electron microscopy (TEM). Injury to eye produced clinically relevant corneal haze and neovascularization in dcn-/- and dcn+/+ mice while corneas of uninjured eyes remained clear and avascular. A clinically significant haze and neovascularization appeared in injured dcn-/- corneas compared to the dcn+/+ corneas at day 21 post-injury and not at early tested times. Histological examinations revealed noticeably abnormal morphology and compromised collagen levels in injured dcn-/- corneas compared to the injured/normal dcn+/+ and uninjured dcn-/- corneas. TEM analysis exhibited remarkably uneven collagen fibrils size and distribution in the stroma with asymmetrical organization and loose packing in injured dcn-/- corneas than injured/normal dcn+/+ and uninjured dcn-/- corneas. The minimum and maximum inter-fibril distances were markedly irregular in injured dcn-/- corneas compared to all other corneas. Together, results of clinical, histological, and ultrastructural investigations in a genetic knockout model suggested that decorin influenced stromal fibrillogenesis and transparency in healing cornea.

Long-Term Safety and Tolerability of BMP7 and HGF Gene Overexpression in Rabbit Cornea.

Purpose: Tissue-targeted localized BMP7+HGF genes delivered into the stroma via nanoparticle effectively treats corneal fibrosis and rehabilitates transparency in vivo without acute toxicity. This study evaluated the long-term safety and tolerability of BMP7+HGF nanomedicine for the eye in vivo. Methods: One eye each of 36 rabbits received balanced salt solution (group 1, naïve; n = 12), naked vector with polyethylenimine-conjugated gold nanoparticles (PEI2-GNP; group 2, naked-vector; n = 12), or BMP7+HGF genes with PEI2-GNP (group 3, BMP7+HGF; n = 12) via a topical delivery technique. Safety and tolerability measurements were performed by clinical biomicroscopy in live rabbits at predetermined time intervals up to 7 months. Corneal tissues were collected at 2 months and 7 months after treatment and subjected to histology, immunofluorescence, and quantitative real-time PCR analyses. Results: Clinical ophthalmic examinations and modified MacDonald-Shadduck scores showed no significant changes in corneal thickness (P = 0.3389), tear flow (P = 0.2121), intraocular pressure (P = 0.9958), epithelial abrasion, or ocular abnormality. Slit-lamp, stereo, confocal, and specular biomicroscopy showed no signs of blepharospasm chemosis, erythema, epiphora, abnormal ocular discharge, or changes in epithelium, stroma, and endothelium after BMP7+HGF therapy for up to 7 months, as compared with control groups. Throughout the 7-month period, no significant changes were recorded in endothelial density (P = 0.9581). Histological and molecular data were well corroborated with the subjective clinical analyses and showed no differences in the naïve, naked-vector, and BMP7+HGF groups. Conclusions: Localized BMP7+HGF therapy is a safe, tolerable, and innovative modality for the treatment of corneal fibrosis. Translational Relevance: Nanoparticle-mediated BMP7+HGF combination gene therapy has the potential to treat corneal fibrosis in vivo without short- or long-term toxicity.

Six-Month In Vivo Safety Profiling of Topical Ocular AAV5-Decorin Gene Transfer.

Purpose: A significant remission of corneal fibrosis and neovascularization in rabbit eye in vivo was observed from a tissue-selective localized adeno-associated virus (AAV)5-Decorin (Dcn) gene therapy. This study sought to investigate 6-month toxicity profiling of this gene therapy for the eye in vivo using a rabbit model. Methods: A small epithelial scrape followed by corneal drying was performed unilaterally in 12 rabbit eyes and either AAV5-Dcn (n = 6) or naked vector (n = 6) was delivered topically using a cloning cylinder technique. Contralateral eyes served as naïve control (n = 6). Safety and tolerability measurements in live rabbits were performed periodically until month 6 using multimodel clinical ophthalmic imaging tools-a slit lamp, stereomicroscope, and HRT3-RCM in vivo confocal microscope. Thereafter, corneas were excised and subjected to hematoxylin and eosin staining, Mason trichome staining, propidium iodide nuclear staining, and quantitative real-time polymerase chain reaction analyses. Results: Clinical eye examinations based on the modified Hackett-McDonald ocular scoring system, and in vivo confocal imaging of the cornea showed no signs of ocular toxicity in rabbit eyes given AAV5-Dcn gene transfer vs control eyes (P > 0.05) through 6 months after treatment. The histologic and molecular analyses showed no significant differences in AAV5-Dcn vs AAV naked or naïve control groups (P > 0.05) and were in accordance with the masked clinical ophthalmic observations showing no abnormalities. Conclusions: Topical tissue-targeted localized AAV5-Dcn gene therapy seems to be safe and nontoxic to the rabbit eye in vivo. Translational Relevance: AAV5-Dcn gene therapy has the potential to treat corneal fibrosis and neovascularization in vivo safely without significant ocular toxicity.

The functional role of decorin in corneal neovascularization in vivo.

Our earlier decorin (Dcn) gene overexpression studies found that the targeted Dcn gene transfer into the cornea inhibited corneal angiogenesis in vivo using a rabbit model. In this study, we tested the hypothesis that anti-angiogenic effects of decorin in the cornea are mediated by alterations in a normal physiologic balance of pro- and anti-angiogenic factors using decorin deficient (Dcn-/-) and wild type (Dcn+/+) mice. Corneal neovascularization (CNV) in Dcn-/- and Dcn+/+ mice was produced with a standard chemical injury technique. The clinical progression of CNV in mice was monitored with stereo- and slit-lamp microscopes, and histopathological hematoxylin and eosin (H&E) staining. Protein and mRNA expression of pro- and anti-angiogenic factors in the cornea were evaluated using immunofluorescence and quantitative real-time PCR, respectively. Slit-lamp clinical eye examinations revealed significantly more CNV in Dcn-/- mice than the Dcn+/+ mice post-injury (p < 0.05) and AAV5-Dcn gene therapy significantly reduced CNV in Dcn-/- mice compered to no AAV5-Dcn gene therapy controls (p < 0.001). H&E-stained corneal sections exhibited morphology with several neovessels in injured corneas of the Dcn-/- mice than the Dcn+/+ mice. Immunofluorescence of corneal sections displayed significantly higher expression of α-smooth muscle actin (α-SMA) and endoglin proteins in Dcn-/- mice than Dcn+/+ mice (p < 0.05). Quantitative real-time PCR found significantly increased mRNA levels of pro-angiogenic factors endoglin (2.53-fold; p < 0.05), Vegf (2.47-fold; p < 0.05), and Pecam (2.14-fold; p < 0.05) and anti-angiogenic factor Vegfr2 (1.56-fold; p < 0.05) in the normal cornea of the Dcn-/- mice than the Dcn+/+ mice. Furthermore, neovascularized Dcn-/- mice corneas showed greater increase in mRNA expression of pro-angiogenic factors endoglin (4.58-fold; p < 0.0001), Vegf (4.16-fold; p < 0.0001), and Pdgf (2.15-fold; p < 0.0001) and reduced expression of anti-angiogenic factors Ang2 (0.12-fold; p < 0.05), Timp1 (0.22-fold; p < 0.05), and Vegfr2 (0.67-fold; p > 0.05) compared to neovascularized Dcn+/+ mice corneas. These gene deficience studies carried with transgenic Dcn-/- mice revealed decorin's role in influencing a physiologic balance between pro-and anti-angiogenic factors in the normal and injured cornea. We infer that the functional deletion of Dcn promotes irregular corneal repair and aggravates CNV.

Glutathione is a potential therapeutic target for acrolein toxicity in the cornea.

Toxic and volatile chemicals are widely used in household products and previously used as warfare agents, causing a public health threat worldwide. This study aimed to evaluate the extent of injury and mechanisms of acrolein toxicity in the cornea. Primary human corneal stromal fibroblasts cultures (hCSFs) from human donor cornea were cultured and exposed to acrolein toxicity with -/+ N-acetylcysteine (NAC) to study the mode of action in the presence of Buthionine sulphoximine (BSO). PrestoBlue and MTT assays were used to optimize acrolein, NAC, and BSO doses for hCSFs. Cell-based assays and qRT-PCR analyses were performed to understand the acrolein toxicity and mechanisms. Acrolein exposure leads to an increased reactive oxygen species (ROS), compromised glutathione (GSH) levels, and mitochondrial dysfunction. The TUNEL and caspase assays showed that acrolein caused cell death in hCSFs. These deleterious effects can be mitigated using NAC in hCSFs, suggesting that GSH can be a potential target for acrolein toxicity in the cornea.

An Ancillary Central Catheter Emergency Support Service Team Staffed by Surgical Personnel Improves Workflow During the Coronavirus Disease 2019 Crisis.

Background. The SARS-CoV-2 novel coronavirus disease 2019 (COVID-19) pandemic has posed significant challenges to urban health centers across the United States. Many hospitals are reallocating resources to best handle the influx of critical patients. Methods. At our New York City hospital, we developed the ancillary central catheter emergency support service (ACCESS), a team for dedicated central access staffed by surgical residents to assist in the care of critical COVID-19 patients. We conducted a retrospective review of all patients for whom the team was activated. Furthermore, we distributed a survey to the critical care department to assess their perceived time saved per patient. Results. The ACCESS team placed 104 invasive catheters over 10 days with a low complication rate of .96%. All critical care providers surveyed found the service useful and felt it saved at least 30 minutes of procedural time per patient, as patient to critical care provider ratios were increased from 12 patients to one provider to 44 patients to one provider. Conclusions. The ACCESS team has helped to effectively redistribute surgical staff, provide a learning experience for residents, and improve efficiency for the critical care team during this pandemic.

Role of inhibitor of differentiation 3 gene in cellular differentiation of human corneal stromal fibroblasts.

Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGFß)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGFß-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation. Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGFß1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGFß-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (α-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis. Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGFß1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in α-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGFß1 showed the expected low expression of α-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGFß-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGFß-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein. Conclusions: Id3 regulates TGFß-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.

A Novel Topical Ophthalmic Formulation to Mitigate Acute Mustard Gas Keratopathy In Vivo: A Pilot Study.

Purpose: This pilot study investigated the in vivo therapeutic potential and tolerability of a multimodal ophthalmic formulation, topical eye drops (TED), for acute mustard gas keratopathy (MGK) using a rabbit model. Methods: Twenty New Zealand White rabbits were used. Only right eyes of 18 rabbits (oculus dexter [OD]) received single sulfur mustard gas (SM) vapor injury, whereas contralateral eyes were left untreated or received TED for tolerabilty evaluation. Two rabbit eyes received no treatment and served as age-matched naive control. The four groups were: Naive (oculus sinister [OS] untreated eyes; n = 9); TED (OS treated only with TED BID for 3 days; n = 9); SM (OD exposed to SM vapor; n = 9); and SM+TED (OD exposed to SM+TED BID for 3 days; n = 9). Ocular examination in live rabbits were performed utilizing slit-lamp biomicroscopy, Fantes grading system, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry. Cellular and molecular changes in rabbit corneas were assessed after humane euthanasia on day-3 and day-7 with histopathological and real-time polymerase chain reaction PCR techniques. Results: TED to rabbit eyes was found tolerable in vivo. SM-exposed eyes showed significant increase in Fantes scores, central corneal thickness (CCT), Schirmer's test, epithelium-stroma separation, and corneal edema. TED mitigated clinical symptoms by reducing corneal edema, Fantes scores, CCT, and Schirmer's test. Further, TED decreased SM-induced corneal haze, inflammatory and profibrotic markers, transforming growth factor-TGF-ß1 and cyclooxygenase-2COX-2, and damage to corneal structure, including epithelial-stromal integrity. Conclusions: The developed multimodal eyedrop formulation, TED, has potential to mitigate acute MGK effectively in vivo. Translational Relevance: TED is effective against MGK.

A rabbit model for evaluating ocular damage from acrolein toxicity in vivo.

Acrolein is a highly reactive and volatile unsaturated aldehyde commonly used for producing scores of commercial products. It has been recognized as a chemical weapon since its use during World War I, and more recently, in Syria. Acrolein exposure causes severe eye, skin, and lung damage in addition to many casualties. In the eye, it causes severe pain, eyelid swelling, corneal burns, and vision impairment. Very little information is available about how acrolein damages the cornea and causes vision loss. At present, the lack of clinically relevant animal models limits evaluation of acrolein toxicity and mechanisms specific to the eye. We aim to standardize the mode of delivery and exposure duration of acrolein, damaging the rabbit eye in vivo as an ocular injury model for studying the toxicity of acrolein and developing medical countermeasures. Rabbit eyes were exposed to two modes of delivery (topical and vapor) for different durations (1-5 minutes). Clinical ophthalmic examinations with a slit lamp, stereomicroscope, fluorescein dye, pachymeter, tonometer, and tearing examinations in live rabbits were performed at various times up to 4 weeks. Corneas were histologically diagnosed for transparency, fibrosis, collagens, and neovascularization. Our study successfully established an in vivo rabbit model for evaluating acrolein toxicity to the eye, accounting for different modes and durations of exposure.

Enhanced recovery program implementation: an evidence-based review of the art and the science.

BACKGROUND: The benefits of enhanced recovery program (ERP) implementation include patient engagement, improved patient outcomes and satisfaction, better team relationships, lower per episode costs of care, lower public consumption of narcotic prescription pills, and the promise of greater access to quality surgical care. Despite these positive attributes, vast numbers of surgical patients are not treated on ERPs, and many of those considered "on pathway" are unlikely to be exposed to a majority of recommended ERP elements. METHODS: To explain the gap between ERP knowledge and action, this manuscript reviewed formal implementation strategies, proposed a novel change adoption model and focused on common barriers (and corollary solutions) that are encountered during the journey to a fully implemented and successful ERP. Given the nature of this review, IRB approval was not required/obtained. RESULTS: The information reviewed indicates that implementation of best practice is both a science and an art. What many surgeons have learned is that the "soft" skills of emotional intelligence, leadership, team dynamics, culture, buy-in, motivation, and sustainability are central to a successful ERP implementation. CONCLUSIONS: To lead teams toward achievement of pervasive and sustained adherence to best practices, surgeons need to learn new strategies, techniques, and skills.

Meta-analysis of genomic variants and gene expression data in schizophrenia suggests the potential need for adjunctive therapeutic interventions for neuropsychiatric disorders.

Schizophrenia (SZ) is a debilitating mental illness with a multigenic aetiology and significant heritability. Despite extensive genetic studies, the molecular aetiology has remained enigmatic. A recent systems biology study suggested a protein-protein interaction network for SZ with 504 novel interactions. The onset of psychiatric disorders is predominant during adolescence, often accompanied by subtle structural abnormalities in multiple regions of the brain. The availability of BrainSpan Atlas data allowed us to re-examine the genes present in the SZ interactome as a function of space and time. The availability of genomes of healthy centenarians and nonpsychiatric Exome Aggregation Consortium database allowed us to identify the variants of criticality. The expression of the SZ candidate genes responsible for cognition and disease onset was studied in different brain regions during particular developmental stages. A subset of novel interactors detected in the network was further validated using gene expression data of post-mortem brains of patients with psychiatric illness. We have narrowed down the list of drug targets proposed by theprevious interactome study to 10 proteins. These proteins belonging to 81 biological pathways are targeted by 34 known Food and Drug Administration-approved drugs that have distinct potential for the treatment of neuropsychiatric disorders. We also report the possibility of targeting key genes belonging to celecoxib pharmacodynamics, Gα signalling and cGMP-PKG signalling pathwaysthat are not known to be specific to SZ aetiology.