A hydrolyzed N-propionylthiosuccinimide linker is cleaved by metastable fragmentation, increasing reliability of conjugation site identification in conjugate vaccines.

RATIONALE: Conjugation sites are a quality attribute of conjugate vaccines. Proteolysis of bioconjugates synthesized by maleimide-thiol chemistry generates type 2 peptides with a hydrolyzed thiosuccinimide linker containing information on the conjugation sites. A mass spectrometry (MS)-cleavable linker could make the identification of conjugation sites by MS more reliable. METHODS: Four synthetic type 2 peptides with a hydrolyzed thiosuccinimide linker were analyzed by matrix-assisted laser desorption ionization (MALDI) MS/MS with and without collision gas. These peptides were also partially labeled with 18O in the linker to confirm the proposed fragmentation mechanism. A conjugate vaccine with the hydrolyzed thiosuccinimide linker was reduced and S-alkylated, digested with trypsin and analyzed by liquid chromatography-MS/MS using collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD) fragmentation methods at a normalized collision energy of 30. RESULTS: A metastable fragmentation preferentially cleaves the newly formed pseudopeptide bond within the hydrolyzed thiosuccinimide linker of type 2 peptides to yield P + 71 and C + 98 ions. These ions make the assignment of conjugation sites more reliable. Partial 18O-labeling and MS/MS analysis confirmed the proposed structures. CID produces these ions as the two most intense signals more favorably than HCD. The latter also yields these ions, guarantees better sequence coverage and promotes other fragmentations in the linker. CONCLUSIONS: Hydrolyzed thiosuccinimide linker is cleavable in MALDI and electrospray ionization MS/MS analysis by a gas-phase metastable fragmentation. The resulting fragment ions (P + 71 and C + 98) make the identification of conjugation sites more reliable. These results could be extended to self-hydrolyzing maleimides, which efficiently stabilize the thiosuccinimide linker upon hydrolysis, in antibody-drug conjugates.

Screening of Exosome-Derived Proteins and Their Potential as Biomarkers in Diagnostic and Prognostic for Pancreatic Cancer.

In the oncological area, pancreatic cancer is one of the most lethal diseases, with 5-year survival rising just 10% in high-development countries. This disease is genetically characterized by KRAS as a driven mutation followed by SMAD4, CDKN2, and TP53-associated mutations. In clinical aspects, pancreatic cancer presents unspecific clinical symptoms with the absence of screening and early plasmatic biomarker, being that CA19-9 is the unique plasmatic biomarker having specificity and sensitivity limitations. We analyzed the plasmatic exosome proteomic profile of 23 patients with pancreatic cancer and 10 healthy controls by using Nanoscale liquid chromatography coupled to tandem mass spectrometry (NanoLC-MS/MS). The pancreatic cancer patients were subdivided into IPMN and PDAC. Our findings show 33, 34, and 7 differentially expressed proteins when comparing the IPMN vs. control, PDAC-No treatment vs. control, and PDAC-No treatment vs. IPMN groups, highlighting proteins of the complement system and coagulation, such as C3, APOB, and SERPINA. Additionally, PDAC with no treatment showed 11 differentially expressed proteins when compared to Folfirinox neoadjuvant therapy or Gemcitabine adjuvant therapy. So here, we found plasmatic exosome-derived differentially expressed proteins among cancer patients (IPMN, PDAC) when comparing with healthy controls, which could represent alternative biomarkers for diagnostic and prognostic evaluation, supporting further scientific and clinical studies on pancreatic cancer.

Novel probabilistic model of core vitreous traction using microsurgical vitrectomy tools.

PURPOSE: Traction exerted on the vitreous base during vitrectomy poses a risk for retinal tears. We aimed to quantify core vitreous traction during vitrectomy using spring return and pneumatic cutters. METHODS: Juvenile porcine vitreous was vacuum held in a vitreous bath while traction was measured using precision force gauge during vitrectomy. The parameters included were aspiration rate, cut-rate, cutter size, and machine types. RESULTS: An empirical probabilistic model was developed. The traction was proportional to the aspiration rate but insignificantly dependent on the cut-rate. The traction probability was inversely proportional to the exponential function of the traction (p < 0.05). The traction was < 0.003 N for 99% of the time using either 23- or 25-gauge cutters. CONCLUSION: The tractions measured were considered similar to the causative forces of an iatrogenic retinal tear during a pars plana vitrectomy. The results provide a safety reference matrix of instrumental parameters during vitrectomy.

Intraocular tumour necrosis factor ligand related molecule 1 A links disease progression of proliferative diabetic retinopathy after primary vitrectomy.

Tumour necrosis factor ligand related molecule 1 A (TL1A), a member of tumour necrosis factor superfamily, has been identified as a crucial regulator for vascular homeostasis and inflammation. However, the function of TL1A in diabetic retinopathy (DR) is largely unknown. This study aims to examine levels of TL1A in serum and intraocular fluid in patients with proliferative diabetic retinopathy (PDR), and to explore the correlation of intraocular TL1A with the prognosis of PDR progression after primary vitrectomy. Seventy-five patients (75 eyes) with PDR who underwent pars plana vitrectomy (PPV) and 19 patients (19 eyes) who received vitrectomy for idiopathic macular holes (IMH) as non-diabetic control group were enrolled in this prospective study. Serum, aqueous and vitreous fluid samples were collected during cataract and PPV surgery. Protein expressions of TL1A as well as other angiogenic and inflammatory cytokines in serum and intraocular fluid were measured. Correlations of intraocular TL1A concentrations with inflammatory cytokines were analyzed. We found both aqueous and vitreous TL1A levels were significantly higher in the PDR group than in control group (Paqueous  = 0.026; Pvitreous <0.001). Angiogenic and inflammatory cytokines such as VEGF, IL-6, IL-8, MCP-1, MIP-1α, and MIP-1ß were significantly higher in intraocular fluid in PDR group than in controls, which MCP-1 and MIP-1α showed positive correlation with intraocular TL1A levels. There is no significant difference in the levels of serum TL1A as well as other inflammatory cytokines between PDR patients and controls. Intraocular levels of TL1A were significantly lower in PDR progression group than in the stable group (Paqueous <0.001; Pvitreous <0.001). Multivariate logistic regression analyses revealed that lower levels of intraocular TL1A was an important risk factor for predicting PDR progression after primary PPV (ORaqueous  = 0.717, Paqueous  = 0.001; ORvitreous  = 0.684; Pvitreous  = 0.002). In conclusion, TL1A and multiple inflammatory cytokines were highly enriched in the intraocular fluid of PDR patients compared with the controls. Lower levels of intraocular TL1A were associated with development of PDR complications after primary PPV and might be used as prognostic factor in predicting the vitrectomy outcome in PDR patients.

Characteristics and outcomes of vitrectomy for proliferative diabetic retinopathy in young versus senior patients.

BACKGROUND: Proliferative diabetic retinopathy (PDR) is one of the most common cause of vision loss in diabetic patients, and the incidence age of PDR patients gradually gets younger. This study aims to compare the characteristics of PDR and outcomes following vitrectomy in young and senior patients. METHODS: This is a retrospective case series study. Data of 116 eyes of 92 patients who underwent vitrectomy for PDR from February 2012 to February 2017 were reviewed, which were divided into young and senior patient groups. All patients were followed up for 24 months at least. RESULTS: There were 62.1% of eyes with tractional retinal detachment secondary to PDR in the young patient group, while only 12.1% of eyes in the senior patient group with this surgery indication. (P < 0.001) The best corrected visual acuity increased in 41 eyes (70.7%), stable in 9 eyes (15.5%), and decreased in 8 eyes (13.8%) in young patients at the final follow-up. And it increased in 47 eyes (81.0%), stable in 2 eyes (3.4%), and decreased in 9 eyes (15.5%) in senior patients.(P = 0.085) Postoperative complications mainly included recurrent vitreous hemorrhage (24.1%), retinal detachment (3.4%), neovascular glaucoma (NVG) (27.6%) and nuclear sclerosis (53.4%) in young patients, and it was 19.0, 0.0, 1.7 and 3.4% in senior patients respectively. CONCLUSION: PDR of young patients is more severe than that of senior patients, and vitrectomy is an effective and safe method for PDR treatment. NVG is a main and severe complication besides nuclear sclerosis in young patients, and the incidence of NVG is higher compared to that in senior patients.

Is it necessary to use tobramycin-dexamethasone eye ointment prophylactically in eyes at the end of intraocular surgery?

BACKGROUND: There are no data available regarding the complications associated with using antibiotic ointment at the end of intraocular surgery. This study aimed to explore the necessity of using ocular tobramycin-dexamethasone prophylactically at the end of intraocular surgery. METHODS: This was a retrospective cohort study of patients who received intraocular surgery at Tianjin Medical University General Hospital from January 2015 to December 2017. The patients were grouped according to whether they received tobramycin-dexamethasone eye ointment or not after surgery. The Tobramycin dexamethasone eye ointment was sampled to observe bacterial contamination pathogens at 0.5, 1, 1.5, 2, 2.5, 3, 6, 8, 24, 36, 48, 72, and 168 h after being opened. RESULTS: A total of 3811 eyes in 3811 patients (mean age of 63 ± 12 years) were included: 2397 eyes that received prophylactic tobramycin-dexamethasone eye ointment and 1414 eyes that did not. The overall rate of endophthalmitis was 0.08% (3/3811) in our study, all in the eye ointment group (0.12%, 3/2397); no patients developed endophthalmitis in the non-ointment group (0%, 0/1414)(P = 0.184). The anterior chamber reactions 1 day after surgery were more serious in the eye ointment group compared with the non-ointment group (all P < 0.05), but there were no statistically significant differences at 1 month postoperatively (all P > 0.05). The contamination rate was 0% at all time points over 7 days. CONCLUSION: We did not observe a statistically significant difference in the incidence of endophthalmitis in patients with or without prophylactic tobramycin-dexamethasone eye ointment. And tobramycin-dexamethasone eye ointment seemed to increase some side effects such as eye secretions increasing and foreign body feeling.

Characteristics and treatments of ocular blast injury in Tianjin explosion in China.

BACKGROUND: To document characteristics and treatments of ocular blast injury from a fire and explosion. METHOD: Authors retrospectively evaluated 116 patients with 166 eye injuries from six hospitals. Terminology of ocular injury referred to Birmingham Eye Trauma Terminology, and best-corrected visual acuity (BCVA) was categorized with the ocular trauma score (OTS) grading system. Incidence, preoperational and follow-up BCVA, treatment of severe ocular blast injuries were surveyed. RESULTS: Oculoplastic injuries accounted for the majority of eye injuries, while globe injuries were presented in 52 eyes with median baseline OTS 70 ranging from 26 to 100. No endophthalmitis occurred. The mean timing of the first-stage operations was 9.4 ± 6.4 h after blast, while second-stage operations were performed on average 14.7 ± 0.9 days post blast. Final BCVA of 68.8% of eyes achieved 20/200 or better as followed, 7 open globe injuries had a BCVA of no light perception. Additionally, eyes presenting rupture, retinal detachment, vitreous hemorrhage, choroidal injury and initial BCVA less than 20/200 had worse final visual outcomes, while globe penetration was not associated with poor visual acuity. CONCLUSION: Various ocular injuries were commonly in the casualties of blast, in which open-globe injuries have worst visual prognosis. OTS is a valid approach for evaluation of prognosis and optimizing the therapeutic strategies subsequently in the massive casualty. Intense rescue and careful examination, proper surgery should be performed correctly to rescue patients.

Subretinal Implantation of a Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium Monolayer in a Porcine Model.

The goal of this study was to quantitatively assess retinal thickness using spectral domain optical coherence tomography (SD-OCT) after subretinal implantation of human embryonic stem cell-derived retinal pigment epithelium in a porcine model. The implant is called CPCB-RPE1 for the California Project to Cure Blindness-Retinal Pigment Epithelium 1. Data were derived from previous experiments on 14 minipigs that received either subretinal implantation of CPCB-RPE1 (n = 11) or subretinal bleb formation alone (sham; n = 3) using previously described methods and procedures (Brant Fernandes et al. Ophthalmic Surg Lasers Imaging Retina 47:342-51, 2016; Martynova et al. (2016) Koss et al. Graefes Arch Clin Exp Ophthalmol 254:1553-65, 2016; Hu et al. Ophthalmic Res 48:186-91, 2016; Martynova et al. ARVO Abstract 2016. SD-OCT retinal thickness (RT) and sublayer thickness over the implant were compared with topographically similar preimplantation regions as described previously Martynova et al. ARVO Abstract 2016. Imaging results were compared to postmortem histology using hematoxylin-eosin staining. RT overlying the CPCB-RPE1 postimplantation was not significantly different from preimplantation (308 ± 72 µm vs 292 ± 41 µm; p = 0.44). RT was not significantly different before and after implantation in any retinal sublayer at 1 month. Histology demonstrated grossly normal retinal anatomy as well as photoreceptor interdigitation with RPE.

Parylene scaffold for cartilage lesion.

Evaluate parylene scaffold feasibility in cartilage lesion treatment, introducing a novel paradigm combining a reparative and superficial reconstructive procedure. Fifteen rabbits were used. All animals had both knees operated and the same osteochondral lesion model was created bilaterally. The parylene scaffold was implanted in the right knee, and the left knee of the same animal was used as control. The animals were euthanized at different time points after surgery: four animals at three weeks, three animals at six weeks, four animals at nine weeks, and four animals at 12 weeks. Specimens were analyzed by International Cartilage Repair Society (ICRS) macroscopic evaluation, modified Pineda histologic evaluation of cartilage repair, and collagen II immunostaining. Parylene knees were compared to its matched contra-lateral control knees of the same animal using the Wilcoxon matched-pairs signed rank. ICRS mean ± SD values for parylene versus control, three, six, nine and twelve weeks, respectively: 7.83 ± 1.85 versus 4.42 ± 1.08, p = 0.0005; 10.17 ± 1.17 versus 6.83 ± 1.17, p = 0.03; 10.89 ± 0.60 versus 7.33 ± 2.18, p = 0.007; 10.67 ± 0.78 versus 7.83 ± 3.40, p = 0.03. Modified Pineda mean ± SD values for parylene versus control, six, nine and twelve weeks, respectively: 3.37 ± 0.87 versus 6.94 ± 1.7, p < 0.0001; 5.73 ± 2.05 versus 6.41 ± 1.7, p = 0.007; 3.06 ± 1.61 versus 6.52 ± 1.51, p < 0.0001. No inflammation was seen. Parylene implanted knees demonstrated higher collagen II expression via immunostaining in comparison to the control knees. Parylene scaffolds are a feasible option for cartilage lesion treatment and the combination of a reparative to a superficial reconstructive procedure using parylene scaffolds led to better results than the reparative procedure alone.

Subretinal implantation of a monolayer of human embryonic stem cell-derived retinal pigment epithelium: a feasibility and safety study in Yucatán minipigs.

PURPOSE: A subretinal implant termed CPCB-RPE1 is currently being developed to surgically replace dystrophic RPE in patients with dry age-related macular degeneration (AMD) and severe vision loss. CPCB-RPE1 is composed of a terminally differentiated, polarized human embryonic stem cell-derived RPE (hESC-RPE) monolayer pre-grown on a biocompatible, mesh-supported submicron parylene C membrane. The objective of the present delivery study was to assess the feasibility and 1-month safety of CPCB-RPE1 implantation in Yucatán minipigs, whose eyes are similar to human eyes in size and gross retinal anatomy. METHODS: This was a prospective, partially blinded, randomized study in 14 normal-sighted female Yucatán minipigs (aged 2 months, weighing 24-35 kg). Surgeons were blinded to the randomization codes and postoperative and post-mortem assessments were performed in a blinded manner. Eleven minipigs received CPCB-RPE1 while three control minipigs underwent sham surgery that generated subretinal blebs. All animals except two sham controls received combined local (Ozurdex™ dexamethasone intravitreal implant) and systemic (tacrolimus) immunosuppression or local immunosuppression alone. Correct placement of the CPCB-RPE1 implant was assessed by in vivo optical coherence tomography and post-mortem histology. hESC-RPE cells were identified using immunohistochemistry staining for TRA-1-85 (a human marker) and RPE65 (an RPE marker). As the study results of primary interest were nonnumerical no statistical analysis or tests were conducted. RESULTS: CPCB-RPE1 implants were reliably placed, without implant breakage, in the subretinal space of the minipig eye using surgical techniques similar to those that would be used in humans. Histologically, hESC-RPE cells were found to survive as an intact monolayer for 1 month based on immunohistochemistry staining for TRA-1-85 and RPE65. CONCLUSIONS: Although inconclusive regarding the necessity or benefit of systemic or local immunosuppression, our study demonstrates the feasibility and safety of CPCB-RPE1 subretinal implantation in a comparable animal model and provides an encouraging starting point for human studies.

Image quality comparison of AirDoc portable retina camera versus eyer in a diabetic retinopathy screening program.

BACKGROUND: Diabetic retinopathy (DR) stands as the foremost cause of preventable blindness in adults. Despite efforts to expand DR screening coverage in the Brazilian public healthcare system, challenges persist due to various factors including social, medical, and financial constraints. Our objective was to evaluate the quality of images obtained with the AirDoc, a novel device, compared to Eyer portable camera which has already been clinically validated. METHODS: Images were captured by two portable retinal devices: AirDoc and Eyer. The included patients had their fundus images obtained in a screening program conducted in Blumenau, Santa Catarina. Two retina specialists independently assessed image's quality. A comparison was performed between both devices regarding image quality and the presence of artifacts. RESULTS: The analysis included 129 patients (mean age of 61 years), with 29 (43.28%) male and an average disease duration of 11.1 ± 8 years. In Ardoc, 21 (16.28%) images were classified as poor quality, with 88 (68%) presenting artifacts; in Eyer, 4 (3.1%) images were classified as poor quality, with 94 (72.87%) presenting artifacts. CONCLUSIONS: Although both Eyer and AirDoc devices show potential as screening tools, the AirDoc images displayed higher rates of ungradable and low-quality images, that may directly affect the DR and DME grading. We must acknowledge the limitations of our study, including the relatively small sample size. Therefore, the interpretations of our analyses should be approached with caution, and further investigations with larger patient cohorts are warranted to validate our findings.

A dataset for developing proteomic tools for pathogen detection via differential cell lysis of whole blood samples.

This data descriptor presents a curated dataset for pathogen detection and identification (Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans) directly from whole-blood samples. The dataset was created using differential cell lysis combined with rapid extraction, digestion, and mass spectrometry-based proteomics. Our method offers a rapid diagnostic alternative to traditional culture, enabling timely disease management, such as sepsis. Highlighting our dataset's uniqueness, it features a three-tier structure: Spectral Libraries of Pathogens for identifying peptide peaks for putative biomarkers; Spiked pathogen in blood MS data for biomarker panel optimization through varied concentration samples; and Parallel Reaction Monitoring (PRM) data from sepsis patients for validating our biomarker panel, achieving 83.3% sensitivity within seven hours without microbial enrichment culture. This dataset serves as a comprehensive reference for bioinformatic tool development and biomarker panel proposals, advancing microbial detection, antimicrobial resistance, and epidemiological studies.

Diabetic retinopathy screening and treatment through the Brazilian National Health Insurance.

The current study aimed to investigate diabetic retinopathy (DR) screening and treatment coverages among diabetic patients evaluated through the Brazilian National Health Insurance from 2014 to 2019. The Brazilian Public Health System Information Database was used as the primary data source. DR screening coverage was calculated as the rate of procedures of clinical dilated fundus exam and color fundus photograph over the number of diabetic patients. DR treatment coverage was calculated as the rate of procedures of intravitreal injection, photocoagulation, and panretinal photocoagulation over the number of diabetic patients presumably in need of DR treatment. The overall screening coverage increased from 12.1% in 2014 to 21.2% in 2019 (p < 0.001) with substantial regional discrepancies so that North region was the only one with no changes along the period. The overall treatment coverage increased from 27.7% in 2014 to 44.1% in 2019, with Southeast and Midwest absorbing the demand for service from the North, Northeast and South. Despite an improvement along the past years, both screening and treatment coverages for DR in diabetes patients are ineffective in Brazil. Public health policies should address resources disparities throughout the country aiming to offer same healthcare conditions to patients regardless their geographic location.

Trends in treatment of retinal disorders in the Brazilian Public Health System over a 10-year period.

OBJECTIVE: To investigate trends in terms of number and cost of intravitreal injection, photocoagulation and panphotocoagulation procedures performed by the Brazilian Public Health System, from 2010 to 2019. METHODS: The Brazilian Public Health System Database was used as the primary source of data. Intravitreal injection, photocoagulation and panphotocoagulation procedures performed from 2010 to 2019 were investigated. Procedure prevalence and cost trends were analyzed according to year and region. Annual trends were examined using generalized linear models, with a significance level of 5% (p=0.05). RESULTS: There was a significant increase in the prevalence of intravitreal injections (1,088%), panphotocoagulation (51%) and photocoagulation (37%) procedures from 2010 to 2019. Intravitreal injections accounted for the most significant increase. However, costs were not significantly readjusted over the years. CONCLUSION: Over a 10-year period, there was a significant increase in the number of procedures associated with retinal disorders. Procedure costs saw little readjustments over time. In spite of limitations, inaccuracies and lack of details, the Brazilian Public Health System Database is the primary source of data for the Public Health System related research in Brazil, and can contribute with information on ocular health and costs of ophthalmic procedures.

Effects of the Pars Plana Vitrectomy on the Chronic Total Rhegmatogenous Retinal Detachment in the Young Adults.

Objective: To observe the characteristics and evaluate the efficacy and safety of the chronic total rhegmatogenous retinal detachment (RRD) treatment by the 23-gauge pars plana vitrectomy (PPV) in young adults and to analyze the related factors. Methods: A retrospective chart review was performed for the young adults who underwent the 23-gauge PPV for the chronic total RRD at the Tianjin Medical University General Hospital from 2011 to 2018. A total of 54 eyes of 48 patients were included in this study. The preoperative vision ranged from 2.00 to 1.00. The mean duration of RRD was 9 ± 0.6 months with a range from 4 to 18 months. The proliferative vitreoretinopathy (PVR) grade D1 and grade D2 was diagnosed in 48 eyes and 6 eyes, respectively. About 37 eyes were filled with C3F8 and 17 eyes were filled with silicone oil tamponade. The follow-up ranged from 9 to 78 months with a mean of 23 ± 2.2 months. Results: The postoperative visual acuity increased in all the eyes at the final observation. The retinal attachment was achieved in 49 eyes (90.7%) in the primary PPV. Five eyes (9.3%) with the failed retinal attachment finally achieved the attachment after the second procedure. The postoperative complications mainly included temporary intraocular pressure (IOP) elevation, hyphema, and retinal redetachment. Conclusion: Chronic total RRD can be treated via the 23-gauge PPV with a great anatomical and visual prognosis in the young adult. The successful treatment of the chronic total RRD in young adults is mainly associated with the complete dissection of the severe vitreoretinopathy, especially for the epiretinal membrane at the retinal breaks and degenerations and the subretinal proliferation during surgery.

Epidemiology of Sports-Related Eye Injuries Among Athletes in Tianjin, China.

Purpose: To investigate the incidence, characteristics, and risk factors of sports-related eye injuries among athletes in Tianjin, China. Methods: A cross-sectional study was carried out from March 2018 to October 2018. In this study, the athletes from Tianjin University of Sports, Tianjin Vocational College of Sports, and Tianjin provincial sports teams were selected for general investigation. In total, 1,673 athletes were invited and 1,413 participated in the study (response rate of 84.5%). Results: In total, 1,413 athletes were enrolled; 151 had suffered from sports-related eye injuries, with an incidence of 10.7% (95% CI: 9.1-12.0%). Handball (38.5%) was the sport with the highest incidence of eye injuries, followed by water polo (36.4%) and diving (26.7%). Overall, 42.4% of the athletes were injured by ball and 22.5% of injuries came from teammates. The eye injuries usually occurred during training (64.2%) and competitions (14.6%). Adnexa wound (51.7%) was the most common type of injury. About 11.9% of the athletes with eye injuries had the impaired vision; 66.7% failed to see doctors on time. The athletes <18 years of age had a higher risk of eye injuries (odds ratio [OR] =1.60, 95% CI: 1.06-2.40). The athletes with lower family income (<1,000 RMB) were at risk population for sports-related eye injuries (OR = 3.91, 95% CI: 2.24-6.82). Training >4 h a day increased the risk of eye injuries (OR = 2.21, 95% CI: 1.42-3.43). Conclusion: The incidence of sports-related eye injuries among athletes was 10.7% in Tianjin, China. Handball, water polo, and diving were the most common activities of injury. Age, family income, and training time were the risk factors for sports-related eye injuries.

CREditing: a tool for gene tuning in Trypanosoma cruzi.

The genetic manipulation of Trypanosoma cruzi continues to be a challenge, mainly due to the lack of available and efficient molecular tools. The CRE-lox recombination system is a site-specific recombinase technology, widely used method of achieving conditional targeted deletions, inversions, insertions, gene activation, translocation, and other modifications in chromosomal or episomal DNA. In the present study, the CRE-lox system was adapted to expand the current genetic toolbox for this hard-to-manipulate parasite. For this, evaluations of whether direct protein delivery of CRE recombinase through electroporation could improve CRE-mediated recombination in T. cruzi were performed. CRE recombinase was fused to the C-terminus of T. cruzi histone H2B, which carries the nuclear localization signal and is expressed in the prokaryotic system. The fusion protein was affinity purified and directly introduced into epimastigotes and tissue culture-derived trypomastigotes. This enabled the control of gene expression as demonstrated by turning on a tandem dimer fluorescent protein reporter gene that had been previously transfected into parasites, achieving CRE-mediated recombination in up to 85% of parasites. This system was further tested for its ability to turn off gene expression, remove selectable markers integrated into the genome, and conditionally knock down the nitroreductase gene, which is involved in drug resistance. Additionally, CREditing also enabled the control of gene expression in tissue culture trypomastigotes, which are more difficult to transfect than epimastigotes. The considerable advances in genomic manipulation of T. cruzi shown in this study can be used by others to aid in the greater understanding of this parasite through gain- or loss-of-function approaches.

Disintegrin-Integrin Binding for Attachment of Polymer Substrate to the Retina.

OBJECTIVE: We propose a novel attachment method for retinal tissue that utilizes silicone modified with bioactive molecules. DESIGN: This is an experimental study divided into an in vitro section performed in cadaveric pig eyes and an in vivo section performed in rabbits. SUBJECTS: During in vitro experiments 36 cadaveric pig eyes were used. During in vivo experiments 4 rabbits were used. METHODS: Different types of silicone went through a laser irradiation process to determine if binding sites for disintegrins could be created. Laser treated silicones that showed disintegrin binding were evaluated with in vitro testing for retina-silicone attachment. The best silicone binding in vitro was implanted into a rabbit's eye after a full vitrectomy was performed. Post-operative exams were done every two weeks to evaluate placement, attachment and sterilization method. After three months animals were euthanized and eye was enucleated for histology analysis. MAIN OUTCOME MEASURES: Attachment strength between silicone-disintegrin-retina, and signs of endophthalmitis during in vivo studies for biocompatibility purposes. RESULTS: A technique to successfully lase and produce an active area on the silicone surface was described. Scanning electron microscope (SEM) images were evaluated to assess physical ablation/debris field area on the surface, definition of edges, evenness, and symmetry of the lased area allowing us to select MED 4800 silicone family for further testing. Cell culture experiments showed disintegrin binding to the silicone active area. In vitro experiments with cadaveric eyes were performed to test retina-silicone attachment. MED 4860 showed strongest attachment to the retina and it was used during in vivo experiments. A sterilization protocol was tested and proved to be reliable for bioactive materials. Disintegrin coated silicone showed attachment in 2 of 4 rabbits during the 3-month implant period. The adhesion was persistent until reversed with plasmin. All rabbits were implanted for 3 months regardless of attachment, to test the feasibility of the sterilization method. None of the rabbits developed any type of eye infection during the implant period. CONCLUSION: We successfully lased and produced an active area on the silicone surface to allow disintegrin-silicone binding. Different silicones interact differently with the laser energy, and this is reflected in the strength of the silicone-disintegrin-retina attachment.

Trends in treatment of retinal disorders in the Brazilian Public Health System over a 10-year period / Tendências no tratamento de distúrbios de retina no Sistema Único de Saúde brasileiro em um período de 10 anos

ABSTRACT Objective: To investigate trends in terms of number and cost of intravitreal injection, photocoagulation and panphotocoagulation procedures performed by the Brazilian Public Health System, from 2010 to 2019. Methods: The Brazilian Public Health System Database was used as the primary source of data. Intravitreal injection, photocoagulation and panphotocoagulation procedures performed from 2010 to 2019 were investigated. Procedure prevalence and cost trends were analyzed according to year and region. Annual trends were examined using generalized linear models, with a significance level of 5% (p=0.05). Results: There was a significant increase in the prevalence of intravitreal injections (1,088%), panphotocoagulation (51%) and photocoagulation (37%) procedures from 2010 to 2019. Intravitreal injections accounted for the most significant increase. However, costs were not significantly readjusted over the years. Conclusion: Over a 10-year period, there was a significant increase in the number of procedures associated with retinal disorders. Procedure costs saw little readjustments over time. In spite of limitations, inaccuracies and lack of details, the Brazilian Public Health System Database is the primary source of data for the Public Health System related research in Brazil, and can contribute with information on ocular health and costs of ophthalmic procedures.

RESUMO Objetivo: Investigar as tendências dos números e dos custos dos procedimentos de injeção intravítrea, fotocoagulação e panfotocoagulação realizados pelo Sistema Único de Saúde brasileiro, no período de 2010 a 2019. Métodos: Foram extraídos dados do Departamento de Informática do Sistema Único de Saúde acerca dos atendimentos associados aos procedimentos de injeção intravítrea, fotocoagulação e panfotocoagulação, realizados de 2010 a 2019. A prevalência de procedimentos e os custos foram analisados por ano e por região de atendimento. As tendências ao longo dos anos foram avaliadas por meio de modelos lineares generalizados. Valores de p=0,05 foram considerados estatisticamente significantes. Resultados: Em relação aos procedimentos específicos, houve aumento nas frequências de injeção intravítrea (1.088%), panfotocoagulação (51%) e fotocoagulação (37%), no comparativo de 2010 a 2019. A injeção intravítrea foi o procedimento que apresentou maior crescimento ao longo dos anos, porém não houve reajuste de custo significativo durante o período estudado. Conclusão: Ao longo de 10 anos, houve aumento significativo do número de atendimentos associados aos tratamentos de distúrbios da retina. Os custos relacionados aos procedimentos mostraram pouco reajuste ao longo dos anos. Embora o Departamento de Informática do Sistema Único de Saúde apresente algumas limitações, como imprecisões e falta de detalhamento em alguns procedimentos, esta é a ferramenta de dados disponível no Brasil para o acesso a pesquisa relacionada ao Sistema Único de Saúde e pode contribuir com informações da saúde ocular e os custos dos procedimentos.

Stem cell therapy for retinal diseases.

In this review, we discuss about current knowledge about stem cell (SC) therapy in the treatment of retinal degeneration. Both human embryonic stem cell and induced pluripotent stem cell has been growth in culture for a long time, and started to be explored in the treatment of blinding conditions. The Food and Drug Administration, recently, has granted clinical trials using SC retinal therapy to treat complex disorders, as Stargardt's dystrophy, and patients with geographic atrophy, providing good outcomes. This study's intent is to overview the critical regeneration of the subretinal anatomy through retinal pigment epithelium transplantation, with the goal of reestablish important pathways from the retina to the occipital cortex of the brain, as well as the differentiation from pluripotent quiescent SC to adult retina, and its relationship with a primary retinal injury, different techniques of transplantation, management of immune rejection and tumorigenicity, its potential application in improving patients' vision, and, finally, approaching future directions and challenges for the treatment of several conditions.