Antagonistic effects of actin-specific toxins on Salmonella Typhimurium invasion into mammalian cells.

Competition between bacterial species is a major factor shaping microbial communities. In this work, we explored the hypothesis that competition between bacterial pathogens can be mediated through antagonistic effects of bacterial effector proteins on host systems, particularly the actin cytoskeleton. Using Salmonella Typhimurium invasion into cells as a model, we demonstrate that invasion is inhibited if the host actin cytoskeleton is disturbed by any of the four tested actin-specific toxins: Vibrio cholerae MARTX actin crosslinking and Rho GTPase inactivation domains (ACD and RID, respectively), TccC3 from Photorhabdus luminescens, and Salmonella's own SpvB. We noticed that ACD, being an effective inhibitor of tandem G-actin binding assembly factors, is likely to inhibit the activity of another Vibrio effector, VopF. In reconstituted actin polymerization assays confirmed by live-cell microscopy, we confirmed that ACD potently halted the actin nucleation and pointed-end elongation activities of VopF, revealing competition between these two V. cholerae effectors. Together, the results suggest bacterial effectors from different species that target the same host machinery or proteins may represent an effective but largely overlooked mechanism of indirect bacterial competition in host-associated microbial communities. Whether the proposed inhibition mechanism involves the actin cytoskeleton or other host cell compartments, such inhibition deserves investigation and may contribute to a documented scarcity of human enteric co-infections by different pathogenic bacteria.

The Puerto Rican Infant Metagenomic and Epidemiologic Study of Respiratory Outcomes (PRIMERO): Design and Baseline Characteristics for a Birth Cohort Study of Early-life Viral Respiratory Illnesses and Airway Dysfunction in Puerto Rican Children.

Epidemiologic studies demonstrate an association between early-life respiratory illnesses (RIs) and the development of childhood asthma. However, it remains uncertain whether these children are predisposed to both conditions or if early-life RIs induce alterations in airway function, immune responses, or other human biology that contribute to the development of asthma. Puerto Rican children experience a disproportionate burden of early-life RIs and asthma, making them an important population for investigating this complex interplay. PRIMERO, the Puerto Rican Infant Metagenomics and Epidemiologic Study of Respiratory Outcomes , recruited pregnant women and their newborns to investigate how the airways develop in early life among infants exposed to different viral RIs, and will thus provide a critical understanding of childhood asthma development. As the first asthma birth cohort in Puerto Rico, PRIMERO will prospectively follow 2,100 term healthy infants. Collected samples include post-term maternal peripheral blood, infant cord blood, the child's peripheral blood at the year two visit, and the child's nasal airway epithelium, collected using minimally invasive nasal swabs, at birth, during RIs over the first two years of life, and at annual healthy visits until age five. Herein, we describe the study's design, population, recruitment strategy, study visits and procedures, and primary outcomes.

A mechanistically novel peptide agonist of the IL-7 receptor that addresses limitations of IL-7 cytokine therapy.

Interleukin (IL)-7 is broadly active on T-cell populations, and modified versions have been clinically evaluated for a variety of therapeutic applications, including cancer, lymphopenia, and infectious diseases; and found to be relatively well-tolerated and biologically active. Here we describe novel IL-7R agonists that are unrelated in structure to IL-7, bind to the receptor subunits differently from IL-7, but closely emulate IL-7 biology. The small size, low structural complexity, and the natural amino acid composition of the pharmacologically active peptide MDK1472 allows facile incorporation into protein structures, such as the IgG2-Fc fusion MDK-703. This molecule possesses properties potentially better suited to therapeutic applications than native IL-7 or its derivatives. We compared these compounds with IL-7 for immune cell selectivity, induction of IL-7R signaling, receptor-mediated internalization, proliferation, and generation of immune cell phenotypes in human and non-human primate (NHP) peripheral blood cells in vitro; and found them to be similar in biological activity to IL-7. In cynomolgus macaques, MDK-703 exhibits a circulating half-life of 46 hr and produces sustained T-cell expansion characteristic of IL-7 treatment. In the huCD34+-engrafted NSG mouse model of the human immune system, MDK-703 induces an immune cell profile very similar to that generated by IL-7-derived compounds; including the pronounced expansion of memory T-cells, particularly the population of stem-like memory T-cells (Tscm) which may be important for anti-tumor activities reported with IL-7 treatment. Clinical administration of IL-7 and modified variants has been reported to induce anti-drug antibodies (ADAs), including IL-7 neutralizing antibodies. The novel peptide agonist reported here scores very low in predicted immunogenicity, and because the peptide lacks sequence similarity with IL-7, the problematic immunogenic neutralization of endogenous cytokine should not occur. The properties we report here implicate MDK-703 as a candidate for clinical evaluation in oncology, anti-viral and other infectious disease, vaccine enhancement, and treatment of lymphopenia.

Route and antigen shape immunity to dmLT-adjuvanted vaccines to a greater extent than biochemical stress or formulation excipients.

A key aspect to vaccine efficacy is formulation stability. Biochemical evaluations provide information on optimal compositions or thermal stability but are routinely validated by ex vivo analysis and not efficacy in animal models. Here we assessed formulations identified to improve or reduce stability of the mucosal adjuvant dmLT being investigated in polio and enterotoxigenic E. coli (ETEC) clinical vaccines. We observed biochemical changes to dmLT protein with formulation or thermal stress, including aggregation or subunit dissociation or alternatively resistance against these changes with specific buffer compositions. However, upon injection or mucosal vaccination with ETEC fimbriae adhesin proteins or inactivated polio virus, experimental findings indicated immunization route and co-administered antigen impacted vaccine immunogenicity more so than dmLT formulation stability (or instability). These results indicate the importance of both biochemical and vaccine-derived immunity assessment in formulation optimization. In addition, these studies have implications for use of dmLT in clinical settings and for delivery in resource poor settings.

Immunity-induced criticality of the genotype network of influenza A (H3N2) hemagglutinin.

Seasonal influenza kills hundreds of thousands every year, with multiple constantly changing strains in circulation at any given time. A high mutation rate enables the influenza virus to evade recognition by the human immune system, including immunity acquired through past infection and vaccination. Here, we capture the genetic similarity of influenza strains and their evolutionary dynamics with genotype networks. We show that the genotype networks of influenza A (H3N2) hemagglutinin are characterized by heavy-tailed distributions of module sizes and connectivity indicative of critical behavior. We argue that (i) genotype networks are driven by mutation and host immunity to explore a subspace of networks predictable in structure and (ii) genotype networks provide an underlying structure necessary to capture the rich dynamics of multistrain epidemic models. In particular, inclusion of strain-transcending immunity in epidemic models is dependent upon the structure of an underlying genotype network. This interplay is consistent with self-organized criticality where the epidemic dynamics of influenza locates critical regions of its genotype network. We conclude that this interplay between disease dynamics and network structure might be key for future network analysis of pathogen evolution and realistic multistrain epidemic models.

Risk factors for SARS-CoV-2 infection and transmission in households with children with asthma and allergy: A prospective surveillance study.

BACKGROUND: Whether children and people with asthma and allergic diseases are at increased risk for severe acute respiratory syndrome virus 2 (SARS-CoV-2) infection is unknown. OBJECTIVE: Our aims were to determine the incidence of SARS-CoV-2 infection in households with children and to also determine whether self-reported asthma and/or other allergic diseases are associated with infection and household transmission. METHODS: For 6 months, biweekly nasal swabs and weekly surveys were conducted within 1394 households (N = 4142 participants) to identify incident SARS-CoV-2 infections from May 2020 to February 2021, which was the pandemic period largely before a vaccine and before the emergence of SARS-CoV-2 variants. Participant and household infection and household transmission probabilities were calculated by using time-to-event analyses, and factors associated with infection and transmission risk were determined by using regression analyses. RESULTS: In all, 147 households (261 participants) tested positive for SARS-CoV-2. The household SARS-CoV-2 infection probability was 25.8%; the participant infection probability was similar for children (14.0% [95% CI = 8.0%-19.6%]), teenagers (12.1% [95% CI = 8.2%-15.9%]), and adults (14.0% [95% CI = 9.5%-18.4%]). Infections were symptomatic in 24.5% of children, 41.2% of teenagers, and 62.5% of adults. Self-reported doctor-diagnosed asthma was not a risk factor for infection (adjusted hazard ratio [aHR] = 1.04 [95% CI = 0.73-1.46]), nor was upper respiratory allergy or eczema. Self-reported doctor-diagnosed food allergy was associated with lower infection risk (aHR = 0.50 [95% CI = 0.32-0.81]); higher body mass index was associated with increased infection risk (aHR per 10-point increase = 1.09 [95% CI = 1.03-1.15]). The household secondary attack rate was 57.7%. Asthma was not associated with household transmission, but transmission was lower in households with food allergy (adjusted odds ratio = 0.43 [95% CI = 0.19-0.96]; P = .04). CONCLUSION: Asthma does not increase the risk of SARS-CoV-2 infection. Food allergy is associated with lower infection risk, whereas body mass index is associated with increased infection risk. Understanding how these factors modify infection risk may offer new avenues for preventing infection.

Recurrence of Pyoderma Gangrenosum Potentially Triggered by COVID-19 Vaccination.

Pyoderma gangrenosum (PG) is a rare inflammatory skin disease of unknown origin. As with other vaccines, COVID-19 vaccines have been associated with many cutaneous reactions. Although COVID-19 vaccination is crucial, it is important for dermatologists and other physicians to be aware of the possible cutaneous reactions that can occur following COVID-19 vaccination. In this report, we describe a 73-year-old woman with a personal history of PG who experienced a recurrence after receiving her second dose of the tozinameran vaccine. Although extremely rare, flares of other inflammatory dermatoses, including lichen planus, have been reported following COVID-19 vaccination. Here we discuss the overlap in pathogenesis of PG and COVID-19, proposing possible mechanisms behind this rare phenomenon.

Refractive and keratometric outcomes of supervised novice surgeon-performed limbal relaxing incisions: 1-year results.

PURPOSE: To report refractive and keratometric astigmatism outcomes of resident-performed limbal relaxing incisions (LRIs) during cataract surgery. SETTING: Tertiary care academic teaching hospital. DESIGN: Retrospective case series. METHODS: The length, location, and number of LRIs were determined preoperatively using an online calculator. Variables studied were preoperative keratometry and postoperative uncorrected and corrected distance visual acuity, refraction, and keratometry at 1-month, 3-month, and 12-month visits (POM1, POM3, and POM12, respectively). Subgroup analysis was performed on amount and type of astigmatism. The astigmatism double-angle plot tool and analysis of with-the-wound (WtW) and against-the-wound (AtW) changes were used to assess the effect of astigmatism correction at POM1, POM3, and POM12 visits. RESULTS: In 118 eyes, a higher percentage of eyes demonstrated refractive astigmatism 0.25 diopter (D) or less, 0.50 D or less, 0.75D or less, and 1.0 D or less at POM1 and POM12 (all P < .05) compared with preoperative keratometric astigmatism. Subgroup analysis showed improvement in all groups and types of astigmatism (P < .01). Patients achieved a statistically significant reduction of keratometric astigmatism at POM1, POM3, and POM12 (all P ≤ .0001) relative to baseline, and changes differed significantly based on the preoperative amount of astigmatism (all P ≤ .0001, with greater reductions associated with higher baseline astigmatism) but not by location of the steep meridian. There were significant WtW-AtW changes at POM1, POM3, and POM12. Regression of effect after 1 month was approximately 0.11 D. CONCLUSIONS: Resident-performed LRIs achieved effective and sustained reduction of both refractive and keratometric astigmatism regardless of meridian or magnitude of astigmatism for at least 1 year postoperatively.

Localization, epidemic transitions, and unpredictability of multistrain epidemics with an underlying genotype network.

Mathematical disease modelling has long operated under the assumption that any one infectious disease is caused by one transmissible pathogen spreading among a population. This paradigm has been useful in simplifying the biological reality of epidemics and has allowed the modelling community to focus on the complexity of other factors such as population structure and interventions. However, there is an increasing amount of evidence that the strain diversity of pathogens, and their interplay with the host immune system, can play a large role in shaping the dynamics of epidemics. Here, we introduce a disease model with an underlying genotype network to account for two important mechanisms. One, the disease can mutate along network pathways as it spreads in a host population. Two, the genotype network allows us to define a genetic distance between strains and therefore to model the transcendence of immunity often observed in real world pathogens. We study the emergence of epidemics in this model, through its epidemic phase transitions, and highlight the role of the genotype network in driving cyclicity of diseases, large scale fluctuations, sequential epidemic transitions, as well as localization around specific strains of the associated pathogen. More generally, our model illustrates the richness of behaviours that are possible even in well-mixed host populations once we consider strain diversity and go beyond the "one disease equals one pathogen" paradigm.

Atypical Keratitis Caused by Rothia Dentocariosa.

Purpose: To report a case of atypical keratitis caused by Rothia dentocariosa.Methods: Retrospective case review.Results: A 49 year-old woman of South Asian descent presented with a non-discrete corneal ulcer with a small overlying epithelial defect in the right eye. Cultures were obtained, a topical fluoroquinolone was continued, and a topical steroid was added. The following day, the infiltrate was noted to have worsened and developed a branching appearance. Antifungals were initiated. The culture grew Rothia dentocariosa. A series of intrastromal cefuroxime injections, followed by topical penicillin G drops, led to complete resolution within 8 weeks. A review of the literature revealed only one previously reported case of Rothia dentocariosa keratitis.Conclusions: Rothia dentocariosa may cause an atypical keratitis requiring a prolonged treatment course for resolution. In our case, a combination of cefuroxime and penicillin was effective.

Proliferative Anterior Optic Membranes in Hydrophobic Acrylic Intraocular Lenses.

PURPOSE: To describe the observed stages of development and management of proliferative anterior optic membranes (AOM) seen on a popularly implanted single-piece hydrophobic intraocular lens (IOL). METHODS: This is an observational series of patients with AOMs managed by three surgeons in different geographical areas in the USA. RESULTS: AOMs may clinically present in one of the four distinct stages. Most patients were visually asymptomatic, but those with decreased visual acuity (Stage 4) required Nd:YAG laser treatment to remove the AOM. These patients had improved visual acuity without recurrence of the AOM during the one-year follow-up period. CONCLUSION: AOMs can present and progress in four distinct stages with variable visual significance. AOMs may be safely and effectively treated with Nd:Yag laser. Occurrence of the membrane may be related to many factors, including high fibronectin content of this IOL. Additional studies are needed to identify incidence, etiology, and best management strategies.

Surgical Curriculum for Presbyopia-Correcting Intraocular Lenses: Resident Experiences and Surgical Outcomes.

OBJECTIVE: To describe a stepwise surgical curriculum that was implemented to teach novice surgeons about currently available advanced technology intraocular lenses (ATIOLs) for correction of presbyopia and to report the experiences and surgical results of ATIOL surgery performed by residents who engaged in the curriculum. DESIGN SETTING AND PARTICIPANTS: Third-year ophthalmology residents participated in a curriculum incorporating didactic lectures (with objective assessment and wet-lab practice) and observation of attending-performed ATIOL surgeries prior to performing ATIOL surgery as primary surgeon under direct supervision. Post-operative outcomes studied were best corrected distance visual acuity (BCDVA) and uncorrected distance (UDVA), intermediate (UIVA) near (UNVA) visual acuity and correction of astigmatism with at least 3 months of follow-up (POM3+). Residents were also given a survey to assess experiences with the surgical curriculum, preparedness for use of ATIOLs post-residency, and ATIOL practice pattern post-residency. RESULTS: A total of 12 residents from four consecutive classes completed the curriculum. Residents overall had a favorable opinion of the curriculum and felt well prepared to use ATIOLs after training. Graduates who currently perform cataract surgery felt comfortable using all available ATIOLs. A total of 100 eyes from 72 patients met the inclusion criteria for analysis in the study. At the POM3+ timepoint, 88% of eyes had UDVA of 20/30 or better, 93% had UIVA of 20/30 or better, and 71.2% had UNVA of 20/30 (J2) or better. Among eyes that received an astigmatism-correcting ATIOL, 91% had <1 diopter of astigmatism after surgery. CONCLUSION: Resident surgeons learned to perform ATIOL surgery (medical knowledge) and achieve strong surgical outcomes (patient care) with all currently available ATIOLs after completion of a stepwise curriculum. Educators may be encouraged to incorporate an ATIOL curriculum based on the results of this study. The curriculum presented is a prototype and may be further improved with future experiences and studies.

Adding Epidemiologically Important Meteorological Data to Peanut Rx, the Risk Assessment Framework for Spotted Wilt of Peanut.

Management of disease affecting peanut in the southeastern United States has benefited from extensive field research identifying disease-associated risk factors since the 1990s. An assessment of risk factors associated with tomato spotted wilt (TSW), caused by tomato spotted wilt virus and spread exclusively by thrips, is available to growers through Peanut Rx, a tool developed to inform peanut management decisions. Peanut Rx provides an assessment of relative TSW risk as an index. The assessment provides information about the relative degree to which a field characterized by a specified suite of practices is at risk of crop loss caused by TSW. Loss results when infection occurs, and infection rates are determined, in part, by factors outside a grower's control, primarily the abundance of dispersing, viruliferous thrips. In this study, we incorporated meteorological variables useful for predicting thrips dispersal, increasing the robustness of the Peanut Rx framework in relation to variation in the weather. We used data from field experiments and a large grower survey to estimate the relationships between weather and TSW risk mediated by thrips vectors, and developed an addition to Peanut Rx that proved informative and easy to implement. The expected temporal occurrence of major thrips flights, as a function of heat and precipitation, was translated into the existing risk-point system of Peanut Rx. Results from the grower survey further demonstrated the validity of Peanut Rx for guiding growers' decisions to minimize risk of TSW.

Rapid pathogen identification and antimicrobial susceptibility testing in in vitro endophthalmitis with matrix assisted laser desorption-ionization Time-of-Flight Mass Spectrometry and VITEK 2 without prior culture.

PURPOSE: Prompt clinical diagnosis and initiation of treatment are critical in the management of infectious endophthalmitis. Current methods used to identify causative agents of infectious endophthalmitis are mostly inefficient, owing to suboptimal sensitivity, length, and cost. Matrix Assisted Laser Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) can be used to rapidly identity pathogens without a need for culture. Similarly, automated antimicrobial susceptibility test systems (AST, VITEK 2) provide accurate antimicrobial susceptibility profiles. In this proof-of-concept study, we apply these technologies for the direct identification and characterization of pathogens in vitreous samples, without culture, as an in vitro model of infectious endophthalmitis. METHODS: Vitreous humor aspirated from freshly enucleated porcine eyes was inoculated with different inocula of Staphylococcus aureus (S. aureus) and incubated at 37°C. Vitreous endophthalmitis samples were centrifuged and pellets were directly analyzed with MALDI-TOF MS and VITEK 2 without prior culture. S. aureus colonies that were conventionally grown on culture medium were used as control samples. Time-to-identification, minimum concentration of bacteria required for identification, and accuracy of results compared to standard methods were determined. RESULTS: MALDI-TOF MS achieved accurate pathogen identification from direct analysis of intraocular samples with confidence values of up to 99.9%. Time from sample processing to pathogen identification was <30 minutes. The minimum number of bacteria needed for positive identification was 7.889x106 colony forming units (cfu/µl). Direct analysis of intraocular samples with VITEK 2 gave AST profiles that were up to 94.4% identical to the positive control S. aureus analyzed per standard protocol. CONCLUSION: Our findings demonstrate that the direct analysis of vitreous samples with MALDI-TOF MS and VITEK 2 without prior culture could serve as new, improved methods for rapid, accurate pathogen identification and targeted treatment design in infectious endophthalmitis. In vivo models and standardized comparisons against other microbiological methods are needed to determine the value of direct analysis of intraocular samples from infectious endophthalmitis with MALDI-TOF MS and VITEK 2.

Contralateral amaurosis after a retrobulbar block.

PURPOSE: To describe and discuss the workup and management of a rare complication of retrobulbar anesthesia, as well as CT and MRI findings of this complication. OBSERVATIONS: The patient underwent uncomplicated pars plana vitrectomy with membrane peel for epiretinal membrane removal. Shortly after completion of surgery, the patient noted that he could not see out of his non-operated eye. Visual acuity was bare hand motion, and physical exam revealed a dilated, non-reactive pupil with normal, well-perfused retina. Imaging revealed an air bubble in the optic nerve of the operated eye, suggesting penetration of the optic nerve sheath during retrobulbar block with posterior spread of anesthetic to the contralateral optic nerve. CONCLUSIONS AND IMPORTANCE: After imaging ruled out acute intracranial pathology and confirmed the correct diagnosis, the patient was monitored until vision in the non-operated eye returned to baseline. Excellent visual acuity was attained in the operated eye. Central spread of anesthetic after retrobulbar anesthesia is a rare but potentially life-threatening complication that must be promptly diagnosed and addressed.

Identification of myosin II as a cripto binding protein and regulator of cripto function in stem cells and tissue regeneration.

Cripto regulates stem cell function in normal and disease contexts via TGFbeta/activin/nodal, PI3K/Akt, MAPK and Wnt signaling. Still, the molecular mechanisms that govern these pleiotropic functions of Cripto remain poorly understood. We performed an unbiased screen for novel Cripto binding proteins using proteomics-based methods, and identified novel proteins including members of myosin II complexes, the actin cytoskeleton, the cellular stress response, and extracellular exosomes. We report that myosin II, and upstream ROCK1/2 activities are required for localization of Cripto to cytoplasm/membrane domains and its subsequent release into the conditioned media fraction of cultured cells. Functionally, we demonstrate that soluble Cripto (one-eyed pinhead in zebrafish) promotes proliferation in mesenchymal stem cells (MSCs) and stem cell-mediated wound healing in the zebrafish caudal fin model of regeneration. Notably, we demonstrate that both Cripto and myosin II inhibitors attenuated regeneration to a similar degree and in a non-additive manner. Taken together, our data present a novel role for myosin II function in regulating subcellular Cripto localization and function in stem cells and an important regulatory mechanism of tissue regeneration. Importantly, these insights may further the development of context-dependent Cripto agonists and antagonists for therapeutic benefit.

Actin Cross-Linking Toxin Is a Universal Inhibitor of Tandem-Organized and Oligomeric G-Actin Binding Proteins.

Delivery of bacterial toxins to host cells is hindered by host protective barriers. This obstruction dictates a remarkable efficiency of toxins, a single copy of which may kill a host cell. Efficiency of actin-targeting toxins is further hampered by an overwhelming abundance of their target. The actin cross-linking domain (ACD) toxins of Vibrio species and related bacterial genera catalyze the formation of covalently cross-linked actin oligomers. Recently, we reported that the ACD toxicity can be amplified via a multivalent inhibitory association of actin oligomers with actin assembly factors formins, suggesting that the oligomers may act as secondary toxins. Importantly, many proteins involved in nucleation, elongation, severing, branching, and bundling of actin filaments contain G-actin-binding Wiskott-Aldrich syndrome protein (WASP)-homology motifs 2 (WH2) organized in tandem and therefore may act as a multivalent platform for high-affinity interaction with the ACD-cross-linked actin oligomers. Using live-cell single-molecule speckle (SiMS) microscopy, total internal reflection fluorescence (TIRF) microscopy, and actin polymerization assays, we show that, in addition to formins, the oligomers bind with high affinity and potently inhibit several families of actin assembly factors: Ena/vasodilator-stimulated phosphorprotein (VASP); Spire; and the Arp2/3 complex, both in vitro and in live cells. As a result, ACD blocks the actin retrograde flow and membrane dynamics and disrupts association of Ena/VASP with adhesion complexes. This study defines ACD as a universal inhibitor of tandem-organized G-actin binding proteins that overcomes the abundance of actin by redirecting the toxicity cascade toward less abundant targets and thus leading to profound disorganization of the actin cytoskeleton and disruption of actin-dependent cellular functions.

Long-term effect of anti-vascular endothelial growth factor injections on intraocular pressure.

OBJECTIVE: There is a substantial debate in the ophthalmology community about whether anti-vascular endothelial growth factor (VEGF) injections result in a long-term increase in intraocular pressure (IOP). DESIGN: We performed a retrospective study to investigate how the number and timing of intravitreal injections in patients with age-related macular degeneration (AMD) and diabetic macular edema (DME) affect IOP over time. METHODS: We collected long-term IOP data on patients receiving anti-VEGF injections at our institution. Patients over the age of 40 years who received injections for AMD (n = 76) or DME (n = 55) were included. Patients were grouped according to indication as well as number of injections received (1-3, 4-6, 7-9, or 10+ injections). IOP measurements were then placed into time points (0-6, 6-12, 12-18, 18-24, or 24+ months) and compared to the preinjection average IOP. RESULTS: For patients with DME, average preinjection IOP was 15.7 mmHg. At 24+ months after injection, the average IOP was 15.2 (P = 0.68) for patients receiving 1-3 injections, 16.8 (P = 0.23) for 4-6 injections, and 14.4 (P = 0.66) for 7-9 injections. For patients with AMD, average initial IOP was 15.6 mmHg. At 24+ months after injection, the average IOP was 12.6 (P = 0.97) for 1-3 injections, 14.9 (P = 0.96) for 4-6 injections, 14.8 (P = 0.84) for 7-9 injections, and 15.7 (P = 0.56) for 10+ injections. CONCLUSIONS: There was no increase in IOP over time for AMD or DME patients, regardless of how many injections they received. For patients receiving unilateral injections, there was no increase in IOP in the injected eye when compared to the noninjected eye.

Social Crowding during Development Causes Changes in GnRH1 DNA Methylation.

Gestational and developmental cues have important consequences for long-term health, behavior and adaptation to the environment. In addition, social stressors cause plastic molecular changes in the brain that underlie unique behavioral phenotypes that also modulate fitness. In the adult African cichlid, Astatotilapia burtoni, growth and social status of males are both directly regulated by social interactions in a dynamic social environment, which causes a suite of plastic changes in circuits, cells and gene transcription in the brain. We hypothesized that a possible mechanism underlying some molecular changes might be DNA methylation, a reversible modification made to cytosine nucleotides that is known to regulate gene function. Here we asked whether changes in DNA methylation of the GnRH1 gene, the central regulator of the reproductive axis, were altered during development of A. burtoni. We measured changes in methylation state of the GnRH1 gene during normal development and following the gestational and developmental stress of social crowding. We found differential DNA methylation within developing juveniles between 14-, 28- and 42-day-old. Following gestational crowding of mouth brooding mothers, we saw differential methylation and transcription of GnRH1 in their offspring. Taken together, our data provides evidence for social control of GnRH1 developmental responses to gestational cues through DNA methylation.