Ocular surface staining: Current concepts and techniques.

The health of the ocular surface is vital for clear vision and comfort. Various factors can adversely influence the ocular surface and tear film homeostasis, and these include procedures like cataract and corneal refractive surgery. It is, therefore, important to assess the integrity of the ocular surface in a rapid, predictable, and consistent manner in the clinic. Various tests and devices have been described, and while these are useful, this article highlights the importance of using fluorescein staining of the ocular surface in detecting changes. This is a simple, inexpensive, rapidly performed test that is available in most eye clinics. However, a proper technique of dye instillation and assessment is important to recognize the changes that can occur. Once detected, these changes can be quantified, and the location and patterns can be used to diagnose the diseases that are present; these changes can also be used to monitor treatment outcomes and disease progression. The article discusses the technique, assessment, and interpretation of fluorescein staining of the ocular surface, along with the role of the two other vital dyes - rose bengal and lissamine green.

An orally available small molecule that targets soluble TNF to deliver anti-TNF biologic-like efficacy in rheumatoid arthritis.

Tumor necrosis factor (TNF) is a pleiotropic cytokine belonging to a family of trimeric proteins with both proinflammatory and immunoregulatory functions. TNF is a key mediator in autoimmune diseases and during the last couple of decades several biologic drugs have delivered new therapeutic options for patients suffering from chronic autoimmune diseases such as rheumatoid arthritis and chronic inflammatory bowel disease. Attempts to design small molecule therapies directed to this cytokine have not led to approved products yet. Here we report the discovery and development of a potent small molecule inhibitor of TNF that was recently moved into phase 1 clinical trials. The molecule, SAR441566, stabilizes an asymmetrical form of the soluble TNF trimer, compromises downstream signaling and inhibits the functions of TNF in vitro and in vivo. With SAR441566 being studied in healthy volunteers we hope to deliver a more convenient orally bioavailable and effective treatment option for patients suffering with chronic autoimmune diseases compared to established biologic drugs targeting TNF.

An inducible potato (E,E)-farnesol synthase confers tolerance against bacterial pathogens in potato and tobacco.

Terpene synthases (TPSs) have diverse biological functions in plants. Though the roles of TPSs in herbivore defense are well established in many plant species, their role in bacterial defense has been scarce and is emerging. Through functional genomics, here we report the in planta role of potato (Solanum tuberosum) terpene synthase (StTPS18) in bacterial defense. Expression of StTPS18 was highest in leaves and was induced in response to Pseudomonas syringae and methyl jasmonate treatments. The recombinant StTPS18 exhibited bona fide (E,E)-farnesol synthase activity forming a sesquiterpenoid, (E,E)-farnesol as the sole product, utilising (E,E)-farnesyl diphosphate (FPP). Subcellular localization of GFP fusion protein revealed that StTPS18 is localized to the cytosol. Silencing and overexpression of StTPS18 in potato resulted in reduced and enhanced tolerance, respectively, to bacterial pathogens P. syringae and Ralstonia solanacearum. Bacterial growth assay using medium containing (E,E)-farnesol significantly inhibited P. syringae growth. Moreover, StTPS18 overexpressing transgenic potato and Nicotiana tabacum leaves, and (E,E)-farnesol and P. syringae infiltrated potato leaves exhibited elevated expression of sterol pathway and members of pathogenesis-related genes with enhanced phytosterol accumulation. Interestingly, enhanced phytosterols in 13 C3 -(E,E)-farnesol infiltrated potato leaves were devoid of any noticeable 13 C labeling, indicating no direct utilization of (E,E)-farnesol in phytosterols formation. Furthermore, leaves of StTPS18 overexpressing transgenic lines had no detectable (E,E)-farnesol similar to the control plant, and emitted lower levels of sesquiterpenes than the control. These findings point towards an indirect involvement of StTPS18 and its product (E,E)-farnesol in bacterial defense through upregulation of phytosterol biosynthesis and defense genes.

Evaluation of ocular surface and tear function - A review of current approaches for dry eye.

An increasing prevalence of dry eye disease in the past decade has resulted in a greater focus on diagnostic methods for this condition. There has been a proliferation of technologies that attempt to quantify various aspects of tear function and ocular surface health. However, a cost-effective, simple, and efficient method remains elusive. In the Indian context, the majority of these patients present to the general ophthalmologist, and a clinical approach that is quick and easy to perform would allow widespread usage for accurate diagnosis. This article reviews currently available methods and their relevance to the general ophthalmologist.

Inflammation and dry eye disease-where are we?

The presence of inflammation in dry eye disease (DED) results in increased patient symptomatology, ocular surface damage and worsening tear dysfunction. It also affects the health of meibomian glands and their secretions which further aggravates ocular surface disease. This article reviews current knowledge regarding ocular surface inflammation in DED and explores the relationships between the vicious cycles of DED, inflammation and meibomian gland dysfunction (MGD). The clinical evaluation of eyes with such changes, markers that identify the presence of inflammation on the ocular surface and current treatment options are discussed.

Safety and target engagement of an oral small-molecule sequestrant in adolescents with autism spectrum disorder: an open-label phase 1b/2a trial.

Autism spectrum disorder (ASD) is defined by hallmark behaviors involving reduced communication and social interaction as well as repetitive activities and restricted interests. ASD represents a broad spectrum, from minimally affected individuals to those requiring intense support, with additional manifestations often including anxiety, irritability/aggression and altered sensory processing. Gastrointestinal (GI) issues are also common in ASD, and studies have identified changes in the gut microbiome of individuals with ASD compared to control populations, complementing recent findings of differences in gut-derived metabolites in feces and circulation. However, a role for the GI tract or microbiome in ASD remains controversial. Here we report that an oral GI-restricted adsorbent (AB-2004) that has affinity for small aromatic or phenolic molecules relieves anxiety-like behaviors that are driven by a gut microbial metabolite in mice. Accordingly, a pilot human study was designed and completed to evaluate the safety of AB-2004 in an open-label, single-cohort, multiple-ascending-dose clinical trial that enrolled 30 adolescents with ASD and GI symptoms in New Zealand and Australia. AB-2004 was shown to have good safety and tolerability across all dose levels, and no drug-related serious adverse events were identified. Significant reductions in specific urinary and plasma levels of gut bacterial metabolites were observed between baseline and end of AB-2004 treatment, demonstrating likely target engagement. Furthermore, we observed improvements in multiple exploratory behavioral endpoints, most significantly in post hoc analysis of anxiety and irritability, as well as GI health, after 8 weeks of treatment. These results from an open-label study (trial registration no. ACTRN12618001956291) suggest that targeting gut-derived metabolites with an oral adsorbent is a safe and well-tolerated approach to improving symptoms associated with ASD, thereby emboldening larger placebo-controlled trials.

Comparative analysis of stress distribution around CFR­PEEK implants and titanium implants with different prosthetic crowns: A finite element analysis.

BACKGROUND: Polyetheretherketone (PEEK) is a new material that was introduced for the fabrication of implants and their superstructure along with other available materials. It is not yet known whether the carbon fiber-reinforced polyetheretherketone (CFR­PEEK) material can be used as an implant and its superstructure in place of titanium (Ti). OBJECTIVES: The study evaluated stress distribution around CFR­PEEK implants and Ti implants with 5 different prosthetic crowns. MATERIAL AND METHODS: A three-dimensional (3D) model of a bone block was created to represent the right maxillary premolar area with a bone-level implant system with 100% osseointegration, using the Ansys Workbench software, v. 15.0. In total, 10 3D finite element analysis (FEA) models were created. The models were divided into 2 groups according to the type of implant: the CFR­PEEK group (n = 5); and the Ti group (n = 5). Each group was subdivided to imitate 5 different restorative crown materials (PEEK, zirconia, porcelain fused to metal (PFM), metal, and acrylic resin). Each implant model was loaded vertically (200 N) and obliquely (100 N). Stress distribution in the implants, the abutments, the cement layers, and the crowns was evaluated using the von Mises stress analysis. Maximum and minimum principal stress analyses were used to determine the stress generated in the bone. RESULTS: The CFR­PEEK implants bore more stress in vertical and oblique loading as compared to the Ti implants. The stress generated in the bone with the CFR­PEEK implants was similar to that generated with the Ti implants under vertical loading. Under oblique loading, less stress was transferred to the bone with the CFR­PEEK implants as compared to the Ti implants, showing better adaptation of the CFR­PEEK implants to lateral stress. CONCLUSIONS: In this FEA study, the amount of stress generated within the bone in the case of the CFR­PEEK implants with different restorative crowns was smaller in comparison with the Ti implants in oblique loading. This could help reduce lateral stress on implants as well as crestal bone loss.

Rehabilitation of Severe Atrophic Ridges with Zygomatic Implant and/or All-on-4 Treatment Concepts-A Case Series.

Rehabilitation of completely edentulous patients with implant-supported prosthesis (ISP) is the need of the day, but in many patients, the routine ISP is not possible due to the severe atrophic residual ridges. The present case series describes three cases with atrophic ridges rehabilitated using zygomatic implants and/or All-on-4 treatment concepts. In case 1 and case 3, in maxillary arch, there was not enough bone in Zone 3 to rehabilitate the patient with routine protocol followed for the All-on-4 treatment concept, so in these two cases, zygomatic implants were placed. Case 1 and case 2 were rehabilitated with Malo Bridge and case 3 with acrylic teeth-hybrid prosthesis using a computerized milling procedure to obtain improved fit, function, esthetics, and ease of retrievability whenever required.

CRISPR/Cas9 generated knockout mice lacking phenylalanine hydroxylase protein as a novel preclinical model for human phenylketonuria.

Phenylketonuria (PKU) is an autosomal recessive inborn error of L-phenylalanine (Phe) metabolism. It is caused by a partial or complete deficiency of the enzyme phenylalanine hydroxylase (PAH), which is necessary for conversion of Phe to tyrosine (Tyr). This metabolic error results in buildup of Phe and reduction of Tyr concentration in blood and in the brain, leading to neurological disease and intellectual deficits. Patients exhibit retarded body growth, hypopigmentation, hypocholesterolemia and low levels of neurotransmitters. Here we report first attempt at creating a homozygous Pah knock-out (KO) (Hom) mouse model, which was developed in the C57BL/6 J strain using CRISPR/Cas9 where codon 7 (GAG) in Pah gene was changed to a stop codon TAG. We investigated 2 to 6-month-old, male, Hom mice using comprehensive behavioral and biochemical assays, MRI and histopathology. Age and sex-matched heterozygous Pah-KO (Het) mice were used as control mice, as they exhibit enough PAH enzyme activity to provide Phe and Tyr levels comparable to the wild-type mice. Overall, our findings demonstrate that 6-month-old, male Hom mice completely lack PAH enzyme, exhibit significantly higher blood and brain Phe levels, lower levels of brain Tyr and neurotransmitters along with lower myelin content and have significant behavioral deficit. These mice exhibit phenotypes that closely resemble PKU patients such as retarded body growth, cutaneous hypopigmentation, and hypocholesterolemia when compared to the age- and sex-matched Het mice. Altogether, biochemical, behavioral, and pathologic features of this novel mouse model suggest that it can be used as a reliable translational tool for PKU preclinical research and drug development.

A 3D Polymer Scaffold Platform for Enhanced in vitro Culture of Human & Rabbit Buccal Epithelial Cells for Cell Therapies.

BACKGROUND: Buccal mucosal epithelial cells show promising application for various regenerative medicine approaches. In this study, we examined the feasibility of culturing rabbit and human buccal mucosal epithelial cells in a novel thermoreversible gelation polymer (TGP) scaffold, without feeder layers or other foreign proteins. METHODS & RESULTS: The results of this 28-day in vitro culture, u sing the conventional technique (2D) and TGP (3D) showed that the epithelial cell morphology could be maintained only in the TGP group while cells in the 2D group de-differentiated to fibroblast morphology in both human and rabbit samples. CK3 expression, a marker for epithelial differentiation was higher in 3D-TGP cultured cells than 2D. CONCLUSION: TGP based in vitro cell culture is a prospective methodology to culture buccal mucosal epithelial cells efficiently without using foreign biological components for tissue engineering applications.

Functional characterization of a defense-responsive bulnesol/elemol synthase from potato.

Terpene synthases (TPSs) produce a variety of terpenoids that play numerous functional roles in primary and secondary metabolism, as well as in ecological interactions. Here, we report the functional characterization of an inducible potato TPS gene encoding bulnesol/elemol synthase (StBUS/ELS). The expression of StBUS/ELS in potato leaves was significantly induced in response to both bacterial (Pseudomonas syringae) and fungal (Alternaria solani) infection as well as methyl jasmonate treatment, indicating its role in defense. The leaves exhibited the highest StBUS/ELS expression followed by the stem with least and similar expression in tuber, sprout and root. Recombinant StBUS/ELS catalyzed the formation of different sesquiterpenes by utilizing farnesyl diphosphate as substrate, and the monoterpene geraniol from geranyl diphosphate. Among the sesquiterpenes formed by StBUS/ELS, elemol was the predominant product followed by α-bulnesene, bulnesol and ß-elemene. Further gas chromatography-mass spectrometry (GC-MS) analysis of StBUS/ELS assay products at different injection temperatures revealed elemol and bulnesol as the major products at 275 and 200/150°C, respectively, without much change in the levels of minor products. This indicated thermal rearrangement of bulnesol into elemol at higher temperatures. Transient overexpression of StBUS/ELS in potato leaves conferred tolerance against the growth of bacteria P. syringae and Ralstonia solanacearum, and the fungus A. solani. Further, expression analysis of pathogenesis-related (PR) genes in StBUS/ELS overexpressing leaves showed no significant change in comparison to control, indicating a direct involvement of StBUS/ELS enzymatic products against pathogens. Overall, our study suggested that StBUS/ELS is a pathogen-inducible TPS encoding bulnesol/elemol synthase and could provide a direct role in defense against biotic stress in potato.

ACTIVating Resources for the COVID-19 Pandemic: In Vivo Models for Vaccines and Therapeutics.

The Preclinical Working Group of Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV), a public-private partnership spearheaded by the National Institutes of Health, has been charged with identifying, prioritizing, and communicating SARS-CoV-2 preclinical resources. Reviewing SARS-CoV-2 animal model data facilitates standardization and harmonization and informs knowledge gaps and prioritization of limited resources. To date, mouse, hamster, ferret, guinea pig, and non-human primates have been investigated. Several species are permissive for SARS-CoV-2 replication, often exhibiting mild disease with resolution, reflecting most human COVID-19 cases. More severe disease develops in a few models, some associated with advanced age, a risk factor for human disease. This review provides a snapshot that recommends the suitability of models for testing vaccines and therapeutics, which may evolve as our understanding of COVID-19 disease biology improves. COVID-19 is a complex disease, and individual models recapitulate certain aspects of disease; therefore, the coordination and assessment of animal models is imperative.

Prospects for a safe COVID-19 vaccine.

Rapid development of an efficacious vaccine against the viral pathogen severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of the coronavirus disease 2019 (COVID-19) pandemic, is essential, but rigorous studies are required to determine the safety of candidate vaccines. Here, on behalf of the Accelerating COVID-19 Therapeutic Interventions and Vaccines (ACTIV) Working Group, we evaluate research on the potential risk of immune enhancement of disease by vaccines and viral infections, including coronavirus infections, together with emerging data about COVID-19 disease. Vaccine-associated enhanced disease has been rarely encountered with existing vaccines or viral infections. Although animal models of SARS-CoV-2 infection may elucidate mechanisms of immune protection, we need observations of enhanced disease in people receiving candidate COVID-19 vaccines to understand the risk of immune enhancement of disease. Neither principles of immunity nor preclinical studies provide a basis for prioritizing among the COVID-19 vaccine candidates with respect to safety at this time. Rigorous clinical trial design and postlicensure surveillance should provide a reliable strategy to identify adverse events, including the potential for enhanced severity of COVID-19 disease, after vaccination.

Translational strategy using multiple nuclear imaging biomarkers to evaluate target engagement and early therapeutic efficacy of SAR439859, a novel selective estrogen receptor degrader.

PURPOSE: Preclinical in vivo nuclear imaging of mice offers an enabling perspective to evaluate drug efficacy at optimal dose and schedule. In this study, we interrogated sufficient estrogen receptor occupancy and degradation for the selective estrogen receptor degrader (SERD) compound SAR439859 using molecular imaging and histological techniques. MATERIAL AND METHODS: [18F]FluoroEstradiol positron emission tomography (FES-PET), [18F]FluoroDeoxyGlucose (FDG) PET, and [18F]FluoroThymidine (FLT) PET were investigated as early pharmacodynamic, tumor metabolism, and tumor proliferation imaging biomarkers, respectively, in mice bearing subcutaneous MCF7-Y537S mutant ERα+ breast cancer model treated with the SERD agent SAR439859. ER expression and proliferation index Ki-67 were assessed by immunohistochemistry (IHC). The combination of palbociclib CDK 4/6 inhibitor with SAR439859 was tested for its potential synergistic effect on anti-tumor activity. RESULTS: After repeated SAR439859 oral administration over 4 days, FES tumoral uptake (SUVmean) decreases compared to baseline by 35, 57, and 55% for the 25 mg/kg qd, 12.5 mg/kg bid and 5 mg/kg bid treatment groups, respectively. FES tumor uptake following SAR439859 treatment at different doses correlates with immunohistochemical scoring for ERα expression. No significant difference in FDG uptake is observed after SAR439859 treatments over 3 days. FLT accumulation in tumor is significantly decreased when palbociclib is combined to SAR439859 (- 64%) but not different from the group dosed with palbociclib alone (- 46%). The impact on proliferation is corroborated by Ki-67 IHC data for both groups of treatment. CONCLUSIONS: In our preclinical studies, dose-dependent inhibition of FES tumoral uptake confirmed target engagement of SAR439859 to ERα. FES-PET thus appears as a relevant imaging biomarker for measuring non-invasively the impact of SAR439859 on tumor estrogen receptor occupancy. This study further validates the use of FLT-PET to directly visualize the anti-proliferative tumor effect of the palbociclib CDK 4/6 inhibitor alone and in combination with SAR439859.

Predicting Forage Quality of Warm-Season Legumes by Near Infrared Spectroscopy Coupled with Machine Learning Techniques.

Warm-season legumes have been receiving increased attention as forage resources in the southern United States and other countries. However, the near infrared spectroscopy (NIRS) technique has not been widely explored for predicting the forage quality of many of these legumes. The objective of this research was to assess the performance of NIRS in predicting the forage quality parameters of five warm-season legumes-guar (Cyamopsis tetragonoloba), tepary bean (Phaseolus acutifolius), pigeon pea (Cajanus cajan), soybean (Glycine max), and mothbean (Vigna aconitifolia)-using three machine learning techniques: partial least square (PLS), support vector machine (SVM), and Gaussian processes (GP). Additionally, the efficacy of global models in predicting forage quality was investigated. A set of 70 forage samples was used to develop species-based models for concentrations of crude protein (CP), acid detergent fiber (ADF), neutral detergent fiber (NDF), and in vitro true digestibility (IVTD) of guar and tepary bean forages, and CP and IVTD in pigeon pea and soybean. All species-based models were tested through 10-fold cross-validations, followed by external validations using 20 samples of each species. The global models for CP and IVTD of warm-season legumes were developed using a set of 150 random samples, including 30 samples for each of the five species. The global models were tested through 10-fold cross-validation, and external validation using five individual sets of 20 samples each for different legume species. Among techniques, PLS consistently performed best at calibrating (R2c = 0.94-0.98) all forage quality parameters in both species-based and global models. The SVM provided the most accurate predictions for guar and soybean crops, and global models, and both SVM and PLS performed better for tepary bean and pigeon pea forages. The global modeling approach that developed a single model for all five crops yielded sufficient accuracy (R2cv/R2v = 0.92-0.99) in predicting CP of the different legumes. However, the accuracy of predictions of in vitro true digestibility (IVTD) for the different legumes was variable (R2cv/R2v = 0.42-0.98). Machine learning algorithms like SVM could help develop robust NIRS-based models for predicting forage quality with a relatively small number of samples, and thus needs further attention in different NIRS based applications.

Trispecific antibodies enhance the therapeutic efficacy of tumor-directed T cells through T cell receptor co-stimulation.

Despite the significant therapeutic advances provided by immune-checkpoint blockade and chimeric antigen receptor T cell treatments, many malignancies remain unresponsive to immunotherapy. Bispecific antibodies targeting tumor antigens and activating T cell receptor signaling have shown some clinical efficacy; however, providing co-stimulatory signals may improve T cell responses against tumors. Here, we developed a trispecific antibody that interacts with CD38, CD3 and CD28 to enhance both T cell activation and tumor targeting. The engagement of both CD3 and CD28 affords efficient T cell stimulation, whereas the anti-CD38 domain directs T cells to myeloma cells, as well as to certain lymphomas and leukemias. In vivo administration of this antibody suppressed myeloma growth in a humanized mouse model and also stimulated memory/effector T cell proliferation and reduced regulatory T cells in non-human primates at well-tolerated doses. Collectively, trispecific antibodies represent a promising platform for cancer immunotherapy.

Usefulness of ambulatory blood pressure measurement for hypertension management in India: the India ABPM study.

The present paper reports differences between office blood pressure (BP) measurement (OBPM) and ambulatory blood pressure measurement (ABPM) in a large multi-centre Indian all comers' population visiting primary care physicians. ABPM and OBPM data from 27,472 subjects (aged 51 ± 14 years, males 68.2%, treated 45.5%) were analysed and compared. Patients were classified based on the following hypertension thresholds: systolic BP (SBP) ≥ 140 and/or diastolic BP (DBP) ≥90 mmHg for OBPM, and SBP ≥ 130 and/or DBP ≥ 80 mmHg for 24-h ABPM, and SBP ≥ 120 and/or DBP ≥ 70 mmHg for night-time ABPM and SBP ≥ 135 and/or DBP ≥ 85 mmHg for daytime ABPM, all together. White coat hypertension (WCH) was seen in 12.0% (n = 3304), masked hypertension (MH) in 19.3% (n = 5293) and 55.5% (n = 15,246) had sustained hypertension. Isolated night-time hypertension (INH) was diagnosed in 11.9% (n = 3256). Untreated subjects had MH relatively more often than treated subjects (23.0% vs. 14.8%, p < 0.0001; respectively). Females had higher relative risk (RR) of having WCH than males (RR 1.16 [CI 95, 1.07-1.25], p < 0.0001). Whereas, males had higher RR of MH than females (RR 1.09 [CI 95, 1.02-1.17] p < 0.01). INH subjects had lower average systolic and diastolic dipping percentages (0.7 ± 6.6/ 2.2 ± 7.9 vs. 9.0 ± 7.3/11.9 ± 8.5, p < 0.001) than those without INH. In conclusion, for diagnosis of hypertension there was a contradiction between OBPM and ABPM in approximately one-third of all patients, and a substantial number of patients had INH. Using ABPM in routine hypertension management can lead to a reduction in burden and associated costs for Indian healthcare.

Blood pressure related to age: The India ABPM study.

The present paper reports trends in office blood pressure (BP) measurement (OBPM) and ambulatory blood pressure measurement (ABPM) with age in a large multi-center Indian all comers' population visiting primary care physicians. ABPM and OBPM data from 27 472 subjects (aged 51 ± 14 years, males 68.2%, treated 45.5%) were analyzed and compared. Individual differences between OBPM and ABPM patterns were compared for patients according to 10-year age categories. Results showed that systolic (S) BP values started to increase with age from the age of 40, BP variability (SD) increased from the age of 30 years. Diastolic (D) BP values started to decrease from the age of 50 years. Mean OBPM values were higher than daytime ABPM values (all P < .001) in all age-groups. The prevalence of white coat hypertension (WCH) and masked hypertension (MH) was based on OBPM and daytime, 24-hour, and nighttime average BPs together. WCH decreased with age from 15.1% and 12.4% in treated and untreated subjects at the youngest age to 7.2% and 6.9% in the oldest age, respectively. MH prevalence was higher for untreated than for treated subjects but remained similar for all age-groups (range of 18.6%-21.3%). The prevalence of reverse dippers increased with age from the youngest to oldest group with 7.3%-34.2% (P < .001 for trend). Dippers prevalence decreased from 42.5% to 17.9% from the youngest to oldest age-groups, respectively (P < .001 for trend). These findings confirm that BP patterns show clear differences in trends with age, particularly regarding nighttime BP.