Unearthing who and Y at Harewood Cemetery and inference of George Washington's Y-chromosomal haplotype.

An excavation conducted at Harewood Cemetery to identify the unmarked grave of Samuel Washington resulted in the discovery of burials presumably belonging to George Washington's paternal grandnephews and their mother, Lucy Payne. To confirm their identities this study examined Y-chromosomal, mitochondrial, and autosomal DNA from the burials and a living Washington descendant. The burial's Y-STR profile was compared to FamilyTreeDNA's database, which resulted in a one-step difference from the living descendant and an exact match to another Washington. A more complete Y-STR and Y-SNP profile from the descendant was inferred to be the Washington Y profile. Kinship comparisons performed in relation to the descendant, who is a 4th and 5th degree relative of the putative individuals, resulted in >37,000 overlapping autosomal SNPs and strong statistical support with likelihood ratios exceeding one billion. This study highlights the benefits of a multi-marker approach for kinship prediction and DNA-assisted identification of historical remains.

Toward Translational Impact of Low-Glucose Strategies on Red Blood Cell Storage Optimization.

Transfusion of stored red blood cells (RBCs) to patients is a critical component of human healthcare. Following purification from whole blood, RBCs are stored in one of many media known as additive solutions for up to 42 days. However, during the storage period, the RBCs undergo adverse chemical and physical changes that are often collectively known as the RBC storage lesion. Storage of RBCs in additive solutions modified to contain physiological levels of glucose, as opposed to hyperglycemic levels currently used in most cases, reduces certain markers of the storage lesion, although intermittent doses of glucose are required to maintain normoglycemic conditions. Here, we describe an electrically actuated valving system to dispense small volumes of glucose into 100 mL PVC storage bags containing packed RBCs from human donors. The RBCs were stored in a conventional additive solution (AS-1) or a normoglycemic version of AS-1 (AS-1N) and common markers of stored RBC health were measured at multiple time points throughout storage. The automated feeding device delivered precise and predictable volumes of concentrated glucose to maintain physiological glucose levels for up to 37 days. Hemolysis, lactate accumulation, and pH values of RBCs stored in AS-1N were statistically equivalent to values measured in AS-1, while significant reductions in osmotic fragility and intracellular sorbitol levels were measured in AS-1N. The reduction of osmotic fragility and oxidative stress markers in a closed system may lead to improved transfusion outcomes for an important procedure affecting millions of people each year.

Optometrist's perspectives of Artificial Intelligence in eye care.

PURPOSE: The application of artificial intelligence (AI) in diagnosing and managing ocular disease has gained popularity as research highlights the utilization of AI to improve personalized medicine and healthcare outcomes. The objective of this study is to describe current optometric perspectives of AI in eye care. METHODS: Members of the American Academy of Optometry were sent an electronic invitation to complete a 17-item survey. Survey items assessed perceived advantages and concerns regarding AI using a 5-point Likert scale ranging from "strongly agree" to "strongly disagree." RESULTS: A total of 400 optometrists completed the survey. The mean number of years since optometry school completion was 25 ± 15.1. Most respondents reported familiarity with AI (66.8%). Though half of optometrists had concerns about the diagnostic accuracy of AI (53.0%), most believed it would improve the practice of optometry (72.0%). Optometrists reported their willingness to incorporate AI into practice increased from 53.3% before the COVID-19 pandemic to 65.5% after onset of the pandemic (p<0.001). CONCLUSION: In this study, optometrists are optimistic about the use of AI in eye care, and willingness to incorporate AI in clinical practice also increased after the onset of the COVID-19 pandemic.

Retrospective review COVID-19 vaccine induced thrombotic thrombocytopenia and cerebral venous thrombosis-what can we learn from the immune response.

INTRODUCTION: Cerebral Venous Thrombosis (CVT), prior to the COVID pandemic, was rare representing 0.5 of all strokes, with the diagnosis made by MRI or CT venography.1-,3 COVID-19 patients compared to general populations have a 30-60 times greater risk of CVT compared to non-affected populations, and up to a third of severe COVID patients may have thrombotic complications.4-8 Currently, vaccines are the best way to prevent severe COVID-19. In February 2021, reports of CVT and Vaccine-induced immune thrombotic thrombocytopenia (VITT) related to adenovirus viral vector vaccines including the Oxford-AstraZeneca vaccine (AZD1222 (ChAdOx1)) and Johnson & Johnson COVID-19 vaccine (JNJ-78436735 (Ad26.COV2·S)), were noted, with a 1/583,000 incidence from Johnson and Johnson vaccine in the United States.11, 12 This study retrospectively analyzed CVT and cross-sectional venography at an Eastern Medical Center from 2018 to 2021, and presents radiographic examples of CVT and what is learned from the immune response. METHODS: After IRB approval, a retrospective review of cross-sectional CTV and MRVs from January 1st 2018 to April 30th 2021, at a single health system was performed. Indications, vaccine status, patient age, sex, and positive finding incidence were specifically assessed during March and April for each year. A multivariable-adjusted trends analysis using Poisson regression estimated venogram frequencies and multivariable logistic regression compared sex, age, indications and vaccination status. RESULTS AND DISCUSSION: From January 1, 2018 to April 30, 2021, (Fig. 1), a total of n = 2206 in patient and emergency room cross-sectional venograms were obtained, with 322 CTVs and 1884 MRVs. In 2018, 2019, 2020, respective totals of cross-sectional venograms were 568, 657, 660, compared to 321 cross-sectional venograms in the first four months of 2021. CTV in 2018, 2019, 2020, respective totals were 51, 86, 97, MRV totals were 517, 571, 563, compared to the 2021 first four month totals of 88 CTVs and 233 MRVs. March, April 2018, 2019, 2020, CTVs respectively were 6, 17, 11, compared to the 2021 first four months of 59 CTVs, comprising 63% of the total 93 CTVs, respective MRVs were 79, 97, 52, compared to 143 MRVs in the first four months of 2021 for 39% of the total 371 MRVs. In March, April 2020 during the pandemic onset, cross-sectional imaging at the East Coast Medical Center decreased, as priorities were on maintaining patient ventilation, high level of care and limiting spread of disease. In March/April 2021, reports of VITT and CVT likely contributed to increased CTVs and MRVs, of 39.65% [1.20-1.63] increase (P < 0.001) from prior. In March, April 2021 of 202 venograms obtained, 158 (78.2.%) were unvaccinated patients, 16 positive for CVT (10.1%), 44 were on vaccinated patients (21.7%), 8 specifically ordered with vaccination as a clinical indication, 2 positive for CVT (4.5%), (odds ratio = 0.52 [0.12-2.38], p = 0.200). CONCLUSION: CTV prior to the COVID pandemic, was rare, responsible for 0.5 of all strokes, at the onset of the pandemic in the East Coast, overall cross-sectional imaging volumes declined due to maintaining ventilation, high levels of care and limiting disease spread, although COVID-19 patients have a 30-60 times greater risk of CVT compared to the general population, and vaccination is currently the best option to mitigate severe disease. In early 2021, reports of adenoviral vector COVID vaccines causing CTV and VITT, led to at 39.65% increase in cross-sectional venography, however, in this study unvaccinated patients in 2021 had higher incidence of CVT (10.1%), compared to the vaccinated patients (4.5%). Clinicians should be aware that VITT CVT may present with a headache 5-30 days post-vaccination with thrombosis best diagnosed on CTV or MRV. If thrombosis is present with thrombocytopenia, platelets <150 × 109, elevated D-Dimer >4000 FEU, and positive anti-PF4 ELISA assay, the diagnosis is definitive.13 VITT CVT resembles spontaneous autoimmune heparin induced thrombocytopenia (HIT), and is postulated to occur from platelet factor 4 (PF4) binding to vaccine adenoviral vectors forming a novel antigen, anti-PF4 memory B-cells and anti-PF4 (VITT) antibodies.14-17.

A 3D-printed transfusion platform reveals beneficial effects of normoglycemic erythrocyte storage solutions and a novel rejuvenating solution.

A set of 3D-printed analytical devices were developed to investigate erythrocytes (ERYs) processed in conventional and modified storage solutions used in transfusion medicine. During storage, prior to transfusion into a patient recipient, ERYs undergo many chemical and physical changes that are not completely understood. However, these changes are thought to contribute to an increase in post-transfusion complications, and even an increase in mortality rates. Here, a reusable fluidic device (fabricated with additive manufacturing technologies) enabled the evaluation of ERYs prior to, and after, introduction into a stream of flowing fresh ERYs, thus representing components of an in vivo ERY transfusion on an in vitro platform. Specifically, ERYs stored in conventional and glucose-modified solutions were assayed by chemiluminescence for their ability to release flow-induced ATP. The ERY's deformability was also determined throughout the storage duration using a novel membrane transport approach housed in a 3D-printed scaffold. Results show that hyperglycemic conditions permanently alter ERY deformability, which may explain the reduced ATP release, as this phenomenon is related to cell deformability. Importantly, the reduced deformability and ATP release were reversible in an in vitro model of transfusion; specifically, when stored cells were introduced into a flowing stream of healthy cells, the ERY-derived release of ATP and cell deformability both returned to states similar to that of non-stored cells. However, after 1-2 weeks of storage, the deleterious effects of the storage were permanent. These results suggest that currently approved hyperglycemic storage solutions are having adverse effects on stored ERYs used in transfusion medicine and that normoglycemic storage may reduce the storage lesion, especially for cells stored for longer than 14 days.

Ancient DNA Methods Improve Forensic DNA Profiling of Korean War and World War II Unknowns.

The integration of massively parallel sequencing (MPS) technology into forensic casework has been of particular benefit to the identification of unknown military service members. However, highly degraded or chemically treated skeletal remains often fail to provide usable DNA profiles, even with sensitive mitochondrial (mt) DNA capture and MPS methods. In parallel, the ancient DNA field has developed workflows specifically for degraded DNA, resulting in the successful recovery of nuclear DNA and mtDNA from skeletal remains as well as sediment over 100,000 years old. In this study we use a set of disinterred skeletal remains from the Korean War and World War II to test if ancient DNA extraction and library preparation methods improve forensic DNA profiling. We identified an ancient DNA extraction protocol that resulted in the recovery of significantly more human mtDNA fragments than protocols previously used in casework. In addition, utilizing single-stranded rather than double-stranded library preparation resulted in increased attainment of reportable mtDNA profiles. This study emphasizes that the combination of ancient DNA extraction and library preparation methods evaluated here increases the success rate of DNA profiling, and likelihood of identifying historical remains.

Contact Lens Prescribing Trends for Keratoconus at an Academic Medical Center: Increased Utilization of Scleral Lenses for Severe Disease.

OBJECTIVES: To describe contact lens prescription trends for patients with keratoconus. METHODS: Demographics, corneal curvature, visual acuity, and type of contact lens prescribed were reviewed for all patients with keratoconus evaluated in the contact lens service in 2010 and 2020. RESULTS: There were 292 patients in 2010 and 217 in 2020. In 2010, 69% were using corneal gas-permeable lenses (GP), 16% soft toric, 13% hybrid, and 2% soft sphere with no scleral lens (SL). In 2020, 60% were using corneal GP, 22% SL, 12% soft toric, 5% hybrid, and 1% soft sphere. Mean log of minimum angle of resolution visual acuities with manifest refraction were (represented in mean [SD, range]) 0.42 (0.33, 0-1.3) in 2010 and 0.35 (0.33, 0-1.6) in 2020 improving to 0.19 (0.18, 0-1.3; P≤0.01) and 0.13 (0.14, 0-0.60; P<0.01) with contact lenses. Mean keratometry measurement in patients using SLs was 53.0 diopters (D) (9.9, 42.1-84.5), which was steeper than 46.6 D in patient's using all other lens types in 2010 and 2020 (3.9, 40.9-57.9; P<0.01). CONCLUSIONS: Scleral lens prescription increased during the past decade; however, corneal GP lenses remain the most frequent lens prescribed for patients with keratoconus in this cohort. Despite being prescribed for patients with the most advanced disease, SLs provide good visual acuity.

Extended kinship analysis of historical remains using SNP capture.

DNA-assisted identification of historical remains requires the genetic analysis of highly degraded DNA, along with a comparison to DNA from known relatives. This can be achieved by targeting single nucleotide polymorphisms (SNPs) using a hybridization capture and next-generation sequencing approach suitable for degraded skeletal samples. In the present study, two SNP capture panels were designed to target ~ 25,000 (25 K) and ~ 95,000 (95 K) nuclear SNPs, respectively, to enable distant kinship estimation (up to 4th degree relatives). Low-coverage SNP data were successfully recovered from 14 skeletal elements 75 years postmortem using an Illumina MiSeq benchtop sequencer. All samples contained degraded DNA but were of varying quality with mean fragment lengths ranging from 32 bp to 170 bp across the 14 samples. SNP comparison with DNA from known family references was performed in the Parabon Fx Forensic Analysis Platform, which utilizes a likelihood approach for kinship prediction that was optimized for low-coverage sequencing data with cytosine deamination. The 25 K panel produced 15,000 SNPs on average, which allowed for accurate kinship prediction with strong statistical support in 16 of the 21 pairwise comparisons. The 95 K panel increased the average SNPs to 42,000 and resulted in an additional accurate kinship prediction with strong statistical support (17 of 21 pairwise comparisons). This study demonstrates that SNP capture combined with massively parallel sequencing on a benchtop platform can yield sufficient SNP recovery from compromised samples, enabling accurate, extended kinship predictions.

Application of Defined Approaches to Assess Skin Sensitization Potency of Isothiazolinone Compounds.

Introduction: Isothiazolinones (ITs) are widely used as antimicrobial preservatives in cosmetics and as additives for preservation of consumer and industrial products to control bacteria, fungi, and algae. Although they are effective biocides, they have the potential to produce skin irritation and sensitization, which poses a human health hazard. In this project, we evaluated nonanimal defined approaches (DAs) for skin sensitization that can provide point-of-departure estimates for use in quantitative risk assessment for ITs. Materials and Methods: The skin sensitization potential of six ITs was evaluated using three internationally harmonized nonanimal test methods: the direct peptide reactivity assay, KeratinoSens™, and the human cell line activation test. Results from these test methods were then applied to two versions of the Shiseido Artificial Neural Network DA. Results: Sensitization hazard or potency predictions were compared with those of the in vivo murine local lymph node assay (LLNA). The nonanimal methods produced skin sensitization hazard and potency classifications concordant with those of the LLNA. EC3 values (the estimated concentration needed to produce a stimulation index of three, the threshold positive response) generated by the DAs had less variability than LLNA EC3 values, and confidence limits from the DAs overlapped those of the LLNA EC3 for most substances. Conclusion: The application of in silico models to in chemico and in vitro skin sensitization data is a promising data integration procedure for DAs to support hazard and potency classification and quantitative risk assessment.

Generation and Export of Red Blood Cell ATP in Health and Disease.

Metabolic homeostasis in animals depends critically on evolved mechanisms by which red blood cell (RBC) hemoglobin (Hb) senses oxygen (O2) need and responds accordingly. The entwined regulation of ATP production and antioxidant systems within the RBC also exploits Hb-based O2-sensitivity to respond to various physiologic and pathophysiologic stresses. O2 offloading, for example, promotes glycolysis in order to generate both 2,3-DPG (a negative allosteric effector of Hb O2 binding) and ATP. Alternatively, generation of the nicotinamide adenine dinucleotide phosphate (NADPH) critical for reducing systems is favored under the oxidizing conditions of O2 abundance. Dynamic control of ATP not only ensures the functional activity of ion pumps and cellular flexibility, but also contributes to the availability of vasoregulatory ATP that can be exported when necessary, for example in hypoxia or upon RBC deformation in microvessels. RBC ATP export in response to hypoxia or deformation dilates blood vessels in order to promote efficient O2 delivery. The ability of RBCs to adapt to the metabolic environment via differential control of these metabolites is impaired in the face of enzymopathies [pyruvate kinase deficiency; glucose-6-phosphate dehydrogenase (G6PD) deficiency], blood banking, diabetes mellitus, COVID-19 or sepsis, and sickle cell disease. The emerging availability of therapies capable of augmenting RBC ATP, including newly established uses of allosteric effectors and metabolite-specific additive solutions for RBC transfusates, raises the prospect of clinical interventions to optimize or correct RBC function via these metabolite delivery mechanisms.

Pannexin 1 channels control the hemodynamic response to hypoxia by regulating O2-sensitive extracellular ATP in blood.

Pannexin 1 (Panx1) channels export ATP and may contribute to increased concentration of the vasodilator ATP in plasma during hypoxia in vivo. We hypothesized that Panx1 channels and associated ATP export contribute to hypoxic vasodilation, a mechanism that facilitates the matching of oxygen delivery to metabolic demand of tissue. Male and female mice devoid of Panx1 (Panx1-/-) and wild-type controls (WT) were anesthetized, mechanically ventilated, and instrumented with a carotid artery catheter or femoral artery flow transducer for hemodynamic and plasma ATP monitoring during inhalation of 21% (normoxia) or 10% oxygen (hypoxia). ATP export from WT vs. Panx1-/-erythrocytes (RBC) was determined ex vivo via tonometer experimentation across progressive deoxygenation. Mean arterial pressure (MAP) was similar in Panx1-/- (n = 6) and WT (n = 6) mice in normoxia, but the decrease in MAP in hypoxia seen in WT was attenuated in Panx1-/- mice (-16 ± 9% vs. -2 ± 8%; P < 0.05). Hindlimb blood flow (HBF) was significantly lower in Panx1-/- (n = 6) vs. WT (n = 6) basally, and increased in WT but not Panx1-/- mice during hypoxia (8 ± 6% vs. -10 ± 13%; P < 0.05). Estimation of hindlimb vascular conductance using data from the MAP and HBF experiments showed an average response of 28% for WT vs. -9% for Panx1-/- mice. Mean venous plasma ATP during hypoxia was 57% lower in Panx1-/- (n = 6) vs. WT mice (n = 6; P < 0.05). Mean hypoxia-induced ATP export from RBCs from Panx1-/- mice (n = 8) was 82% lower than that from WT (n = 8; P < 0.05). Panx1 channels participate in hemodynamic responses consistent with hypoxic vasodilation by regulating hypoxia-sensitive extracellular ATP levels in blood.NEW & NOTEWORTHY Export of vasodilator ATP from red blood cells requires pannexin 1. Blood plasma ATP elevations in response to hypoxia in mice require pannexin 1. Hemodynamic responses to hypoxia are accompanied by increased plasma ATP in mice in vivo and require pannexin 1.

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulic.

Sister Marija Krucifiksa Kozulic (1852-1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including her biological sister, Tereza Kozulic (1861-1933). When the remains were exhumed in 2011, they were found in a deteriorated state and commingled with several other sets of remains. Thus, mitochondrial genome sequencing of the long bones was performed to sort the remains by mitochondrial haplotype. Two similar but unique haplotypes belonging to haplogroup H1bu were identified, and samples from these bones were subjected to autosomal short tandem repeat (STR) and single nucleotide polymorphism (SNP) sequencing. Although only partial profiles were obtained, the data were sufficient for kinship analysis with the profile of a paternal niece of Sister Marija (Fides Kozulic). The data indicate that it is 574,195-fold more likely that the two sets of skeletal remains represent 2nd-degree relatives of Fides than sisters who are unrelated to Fides. Although it is impossible to discern which set of remains belongs to Marija and which belongs to Tereza, forensic genomics methods have enabled identification of the sisters.

HGT in the human and skin commensal Malassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction.

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn's disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

Mitochondrial DNA haplogrouping to assist with the identification of unknown service members from the World War II Battle of Tarawa.

The World War II Battle of Tarawa, 1943, was a devastating conflict that resulted in losses of more than 1100 American and 4690 Japanese troops. The United States government aims to identify and repatriate the remains of all missing American service members through the Defense Prisoner of War/Missing in Action (POW/MIA) Accounting Agency (DPAA) and its partners such as the Armed Forces Medical Examiner System's Armed Forces DNA Identification Laboratory (AFMES-AFDIL). Remains associated with the Battle of Tarawa have been recovered from field excavations conducted by History Flight, a DPAA strategic partner, as well as from the National Memorial Cemetery of the Pacific (NMCP) in Hawaii where unknowns have been disinterred for identification. DNA testing at the AFMES-AFDIL has produced mitochondrial DNA (mtDNA) sequences from 1027 case samples to date. Haplogroup assignments indicate that more than one third (36.2 %) of field-collected samples are likely of Asian maternal ancestry. Therefore the field collections from the Tarawa battlefield comprise the remains of American service members but also those of foreign nationals from Asia. The mtDNA of the NMCP unknowns is similar in ancestry proportion to the family reference sample distribution. The DPAA uses the ancestry information gleaned from mtDNA sequence data in conjunction with anthropological evidence to make foreign national determinations. In this way, mtDNA haplogrouping is used to sort the commingled and fragmentary remains recovered from Tarawa between Americans and foreign nationals, which are then repatriated to their country of origin.

Comparison of International Ovarian Tumor Analysis Simple Rules to Society of Radiologists in Ultrasound Guidelines for Detection of Malignancy in Adnexal Cysts.

OBJECTIVE. The purpose of this study was to evaluate the International Ovarian Tumor Analysis (IOTA) simple rules and the Society of Radiologists in Ultrasound (SRU) guidelines for detecting ovarian malignancy in a general population of women presenting to radiology departments with adnexal cystic lesions. MATERIALS AND METHODS. A retrospective multicenter study of ultrasound-detected adnexal cystic lesions with appropriate follow-up was conducted. Lesions were classified into benign, indeterminate, or malignant categories according to criteria based on the IOTA simple rules and the SRU guidelines. The prevalence of nonneoplastic cysts, neoplasms, and malignant tumors was calculated. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated, and ROC analysis for the diagnosis of malignancy was performed. RESULTS. A total of 697 women with 764 cystic lesions were included; 85.2% (651/764) of the lesions were nonneoplastic, 12.2% (93/764) were benign neoplasms, and 2.6% (20/764) were malignant neoplasms. Nearly all malignancies were classified into indeterminate and malignant categories. The prevalence of malignancy in the indeterminate category was 4.8% (7/145) (SRU) to 10.7% (7/65) (IOTA) and in the malignant category was 18.1% (13/72) (SRU) to 34.3% (12/35) (IOTA). Only one malignancy was misclassified as benign by the IOTA simple rules. The sensitivity of the IOTA simple rules for malignancy was 90.0%; specificity, 96.5%; PPV, 29.0%; NPV, 99.8%; and accuracy, 96.4%. The corresponding values for the SRU guidelines were 100%, 89.6%, 14.9%, 100%, and 89.8%. In ROC analysis, the IOTA simple rules were slightly more accurate than the SRU guidelines (AUC, 0.9805 versus 0.9713; p = 0.0003). CONCLUSION. Both imaging characterization methods were sensitive for identifying ovarian malignancies, but the PPV was low among women presenting to radiology departments, and the indeterminate classification harbored one-third of the total malignancies. Exploration of varied clinical settings and inclusion of secondary tests may help to refine these systems.

Prevalence of Ocular Surface Disease and Corneal Irregularity and Outcomes in Patients Using Therapeutic Scleral Lenses at a Tertiary Care Center.

OBJECTIVES: To describe indications for scleral contact lens (ScCL) evaluation, previous treatments, and outcomes of patients prescribed ScCL at a tertiary referral center. METHODS: This retrospective study reviewed 133 patients evaluated for ScCL between January 1, 2010, and December 31, 2015, at the University of Illinois at Chicago (UIC) Contact Lens Service. Patient demographics, ocular history, indications for evaluation, previous treatments, presence of punctate epithelial erosions, number of lenses ordered, follow-up visits, best-corrected visual acuity before ScCL, and visual acuity with ScCL were evaluated. Patients were categorized based on primary indication for ScCL evaluation as ocular surface disease (OSD) or corneal irregularity (CI). The primary outcome was visual acuity. RESULTS: Visual acuity improved from logarithm of the minimal angle of resolution 0.3±0.4 at presentation to 0.1±0.2 with ScCL in all eyes (n=223, P=0.0001), and from 0.3±0.5 to 0.1±0.2 (n=164, P=0.001) in the OSD group and 0.5±0.4 to 0.2±0.2 (n=59, P=0.0001) in the CI group. Seventy percent of patients were evaluated for bilateral ScCL evaluation. Indication for ScCL evaluation was OSD in 71% (n=95) of patients, with 20% having a secondary diagnosis of CI. Corneal irregularity was the primary diagnosis in 29% of patients (n=38), with 50% having a secondary diagnosis of OSD. CONCLUSIONS: Ocular surface disease was common in this tertiary referral patient population and was a primary or secondary indication for ScCL evaluation in 85.7% (n=114) of patients evaluated for ScCL. Scleral contact lenses improved visual outcomes in patients with both primary diagnoses of CI and OSD.

Red Blood Cell Deformability, Vasoactive Mediators, and Adhesion.

Healthy red blood cells (RBCs) deform readily in response to shear stress in the circulation, facilitating their efficient passage through capillaries. RBCs also export vasoactive mediators in response to deformation and other physiological and pathological stimuli. Deoxygenation of RBC hemoglobin leads to the export of vasodilator and antiadhesive S-nitrosothiols (SNOs) and adenosine triphosphate (ATP) in parallel with oxygen transport in the respiratory cycle. Together, these mediated responses to shear stress and oxygen offloading promote the efficient flow of blood cells and in turn optimize oxygen delivery. In diseases including sickle cell anemia and conditions including conventional blood banking, these adaptive functions may be compromised as a result, for example, of limited RBC deformability, impaired mediator formation, or dysfunctional mediator export. Ongoing work, including single cell approaches, is examining relevant mechanisms and remedies in health and disease.

Nitric oxide loading reduces sickle red cell adhesion and vaso-occlusion in vivo.

Sickle red blood cells (SSRBCs) are adherent to the endothelium, activate leukocyte adhesion, and are deficient in bioactive nitric oxide (NO) adducts such as S-nitrosothiols (SNOs), with reduced ability to induce vasodilation in response to hypoxia. All these pathophysiologic characteristics promote vascular occlusion, the hallmark of sickle cell disease (SCD). Loading hypoxic SSRBCs in vitro with NO followed by reoxygenation significantly decreased epinephrine-activated SSRBC adhesion to the endothelium, the ability of activated SSRBCs to mediate leukocyte adhesion in vitro, and vessel obstruction in vivo. Because transfusion is frequently used in SCD, we also determined the effects of banked (SNO-depleted) red blood cells (RBCs) on vaso-occlusion in vivo. Fresh or 14-day-old normal RBCs (AARBCs) reduced epinephrine-activated SSRBC adhesion to the vascular endothelium and prevented vaso-occlusion. In contrast, AARBCs stored for 30 days failed to decrease activated SSRBC adhesivity or vaso-occlusion, unless these RBCs were loaded with NO. Furthermore, NO loading of SSRBCs increased S-nitrosohemoglobin and modulated epinephrine's effect by upregulating phosphorylation of membrane proteins, including pyruvate kinase, E3 ubiquitin ligase, and the cytoskeletal protein 4.1. Thus, abnormal SSRBC NO/SNO content both contributes to the vaso-occlusive pathophysiology of SCD, potentially by affecting at least protein phosphorylation, and is potentially amenable to correction by (S)NO repletion or by RBC transfusion.

DNA Testing Reveals the Putative Identity of JB55, a 19th Century Vampire Buried in Griswold, Connecticut.

In 1990 in Griswold, Connecticut, archaeologists excavated a burial found in a "skull and crossbones" orientation. The lid of the 19th century coffin had brass tacks that spelled "JB55", the initials of the person lying there and age at death. JB55 had evidence of chronic pulmonary infection, perhaps tuberculosis. It is possible that JB55 was deemed a vampire due to his disease, and therefore had to be "killed" by mutilating his corpse. In an attempt to reveal the identity of JB55, DNA testing was performed. Ancestry informative single nucleotide polymorphism (SNP) analysis using the Precision ID Ancestry Panel indicated European ancestry. A full Y-chromosomal short tandem repeat (Y-STR) profile was obtained, belonging to haplogroup R1b. When the Y-STR profile was searched in the publicly accessible FamilyTreeDNA R1b Project website, the two closest matches had the surname "Barber". A search of historical records led to a death notice mentioning John Barber, whose son Nathan Barber was buried in Griswold in 1826. The description of Nathan Barber closely fits the burial of "NB13," found near JB55. By applying modern forensic DNA tools to a historical mystery, the identity of JB55 as John Barber, the 19th century Connecticut vampire, has been revealed.

Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12-15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).