Length of Stay, Readmission Rates, and Mortality Are Similar Between Hospitalized Individuals With Sickle Cell Disease With and Without COVID-19.

Background We sought to clarify the impact of COVID-19 on clinical outcomes in sickle cell disease patients, given their baseline hypercoagulable state in combination with COVID-19-related coagulopathies and other complications.  Methods Retrospective chart review of two groups of sickle cell disease patients hospitalized between March 2020 to December 2021: Group 1 did not have COVID-19 (n = 95) and Group 2 did (n = 73).  Results Groups 1 and 2 were similar in terms of age, race, sex, comorbid illnesses, genotype, hydroxyurea use, and opioid use. Group 1 and 2 patients had a mean hospital length of stay of 7.05 and 7.64 days, respectively (p = 0.981). ICU-level care was required for six (6.3%) Group 1 patients and four (5.5%) Group 2 patients (p = 1.000). Readmissions within 30 days occurred for 25 (26.3%) Group 1 patients, and 18 (24.7%) Group 2 patients (p = 0.807). Death occurred for one (1.05%) Group 1 patient and one (1.4%) Group 2 patient (p = 1.000). There were no significant differences in commonly ordered initial laboratory values (total bilirubin, hemoglobin, hematocrit, creatinine, lactate dehydrogenase, and D-dimer) between Group 1 and Group 2 patients.  Conclusions We observed no significant differences in clinical outcomes among sickle cell disease patients hospitalized due to COVID-19 compared to those without COVID-19.

Galactosemia among Positive-screened Patients who Underwent Lactose Challenge: A Review of Records of the Newborn Screening Program.

Background: Newborns screened positive for Galactosemia through Expanded Newborn Screening (ENBS) with borderline levels undergo lactose challenge that requires interruption of breastfeeding temporarily then shifting to soy-based formula. Objective: To determine the percentage of Classical Galactosemia (CGal), Non-classical Galactosemia (NCGal), probable mild variant form, and negative Galactosemia among newborns screened positive for Galactosemia who underwent lactose challenge. Methods: This is a retrospective study. NBS records were reviewed and data were collected from January 2015 to December 2020. Results: Out of the 117 newborns screened positive for Galactosemia, 58 underwent lactose challenge. Majority were male, term with a birth weight of 2500-4000g and received a final disposition in 4-6 months. Fifteen patients underwent 1-week lactose challenge wherein six reached a resolution on first challenge. Majority, 35 (60.3%) were negative for Galactosemia, six (10.3%) probable mild variant Galactosemia, three (5.2%) NCGal, and no CGal were observed. Fourteen suspected cases (24.1%) are pending final disposition. Conclusion: This study describes the demographics of newborns flagged for Galactosemia who underwent lactose challenge. A 1-week lactose challenge may be recommended to further detect patients who are negative for Galactosemia.

Canadian College of Medical Geneticists: clinical practice advisory document - responsibility to recontact for reinterpretation of clinical genetic testing.

BACKGROUND: Advances in technology and knowledge have facilitated both an increase in the number of patient variants reported and variants reclassified. While there is currently no duty to recontact for reclassified genetic variants, there may be a responsibility. The purpose of this clinical practice advisory document is to provide healthcare practitioners guidance for recontact of previously identified and classified variants, suggest methods for recontact, and principles to consider, taking account patient safety, feasibility, ethical considerations, health service capacity and resource constraints. The target audience are practitioners who order genetic testing, follow patients who have undergone genetic testing and those analysing and reporting genetic testing. METHODS: A multidisciplinary group of laboratory and ordering clinicians, patient representatives, ethics and legal researchers and a genetic counsellor from the Canadian Association of Genetic Counsellors reviewed the existing literature and guidelines on responsibility to recontact in a clinical context to make recommendations. Comments were collected from the Canadian College of Medical Geneticists (CCMG) Education, Ethics, and Public Policy, Clinical Practice and Laboratory Practice committees, and the membership at large. RESULTS: Following incorporation of feedback, and external review by the Canadian Association of Genetic Counsellors and patient groups, the document was approved by the CCMG Board of Directors. The CCMG is the Canadian organisation responsible for certifying laboratory and medical geneticists who provide medical genetics services, and for establishing professional and ethical standards for clinical genetics services in Canada. CONCLUSION: The document describes the ethical and practical factors and suggests a shared responsibility between patients, ordering clinician and laboratory practitioners.

Considerations for Using Phage Display Technology in Therapeutic Antibody Drug Discovery.

Phage display is a versatile and effective platform for the identification and engineering of biologic-based therapeutics. Using standard molecular biology laboratory techniques, one can create a highly diverse and functional antibody phage-displayed library, and rapidly identify antibody fragments that bind to a target of interest with exquisite specificity and high affinity. Here, we discuss key aspects for the development of an antibody discovery strategy to harness the power of phage display technology to obtain molecules that can successfully be developed into therapeutics, including target validation, antibody design goals, and considerations for preparing and executing phage panning campaigns. Careful design and implementation of discovery campaigns-regardless of the target-provides the best chance of identifying desirable antibody fragments for further therapeutic development, so these principles can be applied to any new discovery project.

Semisynthetic Phage Display Library Construction: Generation of Filtered Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the second of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Gold Plus Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library (FL) construction, and (3) secondary library construction. The second step, described here, involves display of the PLs as single-chain variable fragment (scFv) fusions to protein pIII of the M13 phage, as well as heat shock treatment and subsequent selection of well-folded and thermostable scFvs via protein L binding, whereas unstable and defective scFvs are removed by washing steps and centrifugation. The quality of the filtration process is assessed by sequencing clones chosen at random from the FLs. These libraries, enriched with thermostable antibodies, are then ready to be used for the third and final step of the process: generation of secondary libraries.

Semisynthetic Phage Display Library Construction: Design and Synthesis of Diversified Single-Chain Variable Fragments and Generation of Primary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the first of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library (PL) construction, (2) filtered library construction, and (3) secondary library construction. The first step, described here, entails design, synthesis, and cloning of four PLs. These PLs are designed with specific properties amenable to therapeutic antibody development using one universal variable heavy (VH) scaffold and four distinct variable light (VL) scaffolds. The scaffolds are diversified in positions that bind both protein and peptide targets identified in antibody-antigen complexes of known structure using the amino acid frequencies found in those positions in known human antibody sequences, avoiding residues that may lead to developability liabilities. The diversified scaffolds are combined with 90 synthetic neutral HCDR3 sequences designed with developable human diversity genes (IGHD) and joining heavy genes (IGHJ) in germline configuration, and assembled as single-chain variable fragments (scFvs) in a VL-linker-VH orientation. The four designed PLs are synthesized using trinucleotide phosphoramidites (TRIMs) and cloned independently into a phagemid vector for M13 pIII display. Quality control of the cloning of the four PLs is also described, which involves sequencing scFvs in each library.

Semisynthetic Phage Display Library Construction: Generation of Single-Chain Variable Fragment Secondary Libraries.

Display of antibody fragments on the surface of M13 filamentous bacteriophages is a well-established approach for the identification of antibodies binding to a target of interest. Here, we describe the third and final step of a three-step method to construct Antibody Libraries for Therapeutic Antibody Discovery (ALTHEA) Libraries. The three-step method involves (1) primary library construction, (2) filtered library (FL) construction, and (3) secondary library (SL) construction. In the third step, described here, the nucleotide sequences encoding the single-chain variable fragments (scFvs) of FLs are amplified by PCR and combined with the heavy- chain CDR3 region (HCDR3) and joining fragments (H3J) obtained from a pool of donors to maximize diversity ("natural H3J fragments"). These natural H3J fragments are amplified with a set of primers designed to capture >95% of the natural H3J repertoire. The resultant fragments replace the neutral H3J fragments of the FLs, resulting in the final semisynthetic secondary libraries. The quality of these libraries is assessed by sequencing clones chosen at random from the libraries, typically 96 clones. These libraries are then ready to be used for phage selections on targets of interest, providing a robust antibody discovery platform.

Inference of the genetic basis of fruit texture in highbush blueberries using genome-wide association analyses.

The global production and consumption of blueberry (Vaccinium spp.), a specialty crop known for its abundant bioactive and antioxidant compounds, has more than doubled over the last decade. To hold this momentum, plant breeders have begun to use quantitative genetics and molecular breeding to guide their decisions and select new cultivars that are improved for fruit quality. In this study, we leveraged our inferences on the genetic basis of fruit texture and chemical components by surveying large breeding populations from northern highbush blueberries (NHBs) and southern highbush blueberries (SHBs), the two dominant cultivated blueberries. After evaluating 1065 NHB genotypes planted at the Oregon State University, and 992 SHB genotypes maintained at the University of Florida for 17 texture-related traits, evaluated over multiple years, our contributions consist of the following: (i) we drew attention to differences between NHB and SHB materials and showed that both blueberry types can be differentiated using texture traits; (ii) we computed genetic parameters and shed light on the genetic architecture of important texture attributes, indicating that most traits had a complex nature with low to moderate heritability; (iii) using molecular breeding, we emphasized that prediction could be performed across populations; and finally (iv) the genomic association analyses pinpointed some genomic regions harboring potential candidate genes for texture that could be used for further validation studies. Altogether, the methods and approaches used here can guide future breeding efforts focused on maximizing texture improvements in blueberries.

Mouse Corneal Immune Cell Heterogeneity Revealed by Single-Cell RNA Sequencing.

Purpose: This study aimed to define the heterogeneity, spatial localization, and functional roles of immune cells in the mouse cornea using single-cell RNA sequencing (scRNA-seq) and immunofluorescent staining. Methods: Enriched mouse corneal immune cells (C57BL/6 strain, age 16-20 weeks) underwent single-cell RNA sequencing library preparation, sequencing, and analysis with Seurat, Monocle 3, and CellChat packages in R. Pathway analysis used Qiagen Ingenuity Pathway Analysis software. Immunostaining confirmed cell distribution. Results: We identified 14 distinct immune cell clusters (56% myeloid and 44% lymphoid). Myeloid populations included resident macrophages, conventional dendritic cells (cDC2s), Langerhans cells, neutrophils, monocytes, and mast cells. Additionally, lymphocyte subsets (B, CD8, CD4, γδT, natural killer, natural killer T, and group 2 innate lymphoid cells) were found. We also found three new subtypes of resident macrophages in the cornea. Trajectory analysis suggested a differentiation pathway from monocytes to conventional dendritic cells, resident macrophages, and LCs. Intercellular communication network analysis using cord diagram identified amyloid precursor protein, chemokine (C-C motif) ligands (2, 3, 4, 6, 7, 9, and 12), Cxcl2, Mif, Tnf, Tgfb1, Igf1, and Il10 as prominent ligands involved in these interactions. Sexually dimorphic gene expression patterns were observed, with male myeloid cells expressing genes linked to immune regulation (Egr1, Foxp1, Mrc1, and Il1rn) and females showing higher expression of antigen presentation genes (Clic1, Psmb8, and Psmb9). Finally, immunostaining confirmed the spatial distribution of these cell populations within the cornea. Conclusions: This study unveils a diverse immune landscape in the mouse cornea, with evidence for cell differentiation and sex-based differences. Immunostaining validates the spatial distribution of these populations, furthering our knowledge of corneal immune function.

ROCK Inhibitor Enhances Neurite Outgrowth In Vitro and Corneal Sensory Nerve Reinnervation In Vivo.

Purpose: The intraepithelial corneal nerves are essential to corneal health. Rho kinase or ROCK inhibitors (RIs) have been reported to play a role in neuron survival after injury. Here we assess integrin and extracellular matrix expression in primary mouse neurons and determine whether treating cells with RI impacts neurite outgrowth in vitro and reinnervation after trephine and debridement injury in mice in vivo. Methods: Cocultures of human corneal limbal epithelial cells and E11.5 mouse trigeminal neurons and neurons alone were grown on glass coverslips. High-resolution imaging was performed to localize integrins and laminin on neurons and to determine whether RI impacts neurite outgrowth in vitro and in vivo after both 1.5-mm trephine and 1.5-mm debridement injuries. Results: Several integrin α (α3, α6, αv) chains as well as ß4 integrin are expressed on neuron axons and growth cones in cocultures. RI treatment of isolated neurons, cocultures, and in conditioned media increases neurite outgrowth. In vivo, RI positively impacts sensory nerve reinnervation after trephine and debridement injury. Conclusions: These studies are the first to demonstrate expression of ß4 integrin on trigeminal sensory neurons and preferential adhesion of neurons to the laminin-enriched matrices found in footprints deposited by human corneal limbal epithelial cells. In addition, we also document for the first time the positive impact of RI on neurite outgrowth in vitro and reinnervation in vivo.

Physical and Mental Health of Informal Carers from Culturally and Linguistically Diverse (CALD) and Non-CALD Groups in Australia.

INTRODUCTION: Empirical evidence shows that many family carers, especially immigrants, experience considerable health disadvantages and poorer quality of life. Australia has a rapidly increasing multicultural population, officially referred to as Culturally and Linguistically Diverse (CALD) people. This paper explores similarities and differences in the carer profile and physical and mental health of CALD and non-CALD family carers. METHODS: A cross-sectional anonymous survey was conducted of self-reported family carers aged 18 years and older. Identical paper and online survey modes were provided to enable choice. Key variables included demographic and carer profile, diagnosed chronic physical health conditions, and validated scales such as CESD-12 and MOS-SF12, including derivative composite Physical and Mental Component Summary (PCS and MCS, respectively) scores. The sample comprised 649 participants (CALD = 347, non-CALD = 302). The analyses included univariate, bivariate, and multivariable linear regression analyses for three outcome variables: PCS, MCS, and CESD-12. RESULTS: CALD carers were comparatively younger and married, and 54% had university-level education (29% in the gfvnon-CALD group). Women were primary carers in both groups (67.4% versus 72.2%). The weekly care hours were higher for non-CALD carers. Both groups had below population-referenced scores for mean PCS and MCS values. For CESD-12, non-CALD respondents had higher scores (17.5 vs. 11.2, p < 0.022). Regression analyses showed significant differences for demographic, carer, and physical health variables across the three outcome variables. DISCUSSION AND CONCLUSION: Women have a higher domestic workload, which, when combined with high care hours, adversely impacts physical and mental health. The need for improved and culturally aligned care support systems is required.

Breast Milk Feeding for Infants Who Required Major Surgery.

OBJECTIVE: To identify barriers or facilitators that influenced mothers to provide mother's own milk (MOM) for 6 months to their infants who were hospitalized in the NICU after major surgery. DESIGN: Descriptive qualitative. SETTING: An 80-bed, Level 4 NICU of a regional pediatric hospital in the western United States. PARTICIPANTS: Fourteen mothers who provided MOM for their infants who required surgery within 1 week of age. METHODS: We conducted in-person interviews upon admission and discharge of the infant, phone interviews 1 and 2 weeks after discharge, and phone interviews monthly for 6 months or until discontinuance of the provision of MOM. We analyzed interviews using the Brooks thematic template analysis method. RESULTS: Eleven infants received exclusive MOM at discharge, and nine infants remained on exclusive MOM at 6 months. We generated four principal themes from the participants' comments: Value of Breast Milk, Challenges of Providing MOM, Emotional Fluctuation, and Coping With Reality of Circumstances. CONCLUSION: Internalizing the value of MOM, family support, and coping with barriers were key factors that influenced participants to provide MOM for at least 4 months. Findings of this study suggest that prenatal education with anticipatory guidance and lactation support in the NICU can help mothers achieve the goal of extended provision of MOM to infants with serious conditions that require surgery. Education and support may be especially helpful for young, first-time mothers.

Effectiveness of community-based Baduanjin exercise intervention for older adults with varying frailty status: a randomized controlled trial.

BACKGROUND: Due to poorer exercise tolerance, it may be challenging for frail older adults to engage in moderate- or vigorous-intensity exercise. While low-intensity exercise interventions may be more feasible, its effectiveness for such population group remains unclear. We examined the effectiveness and implementation of community-based Baduanjin Qigong, a low-intensity exercise program in older adults with varying frailty status. METHODS: A two-arm, multicenter assessor-blind parallel group randomized controlled trial was conducted at three local senior activity centers. Fifty-six community-dwelling older adults with low handgrip strength were randomly allocated to either the intervention (IG) or wait-list control (CG) group. The IG underwent a supervised 16-week Baduanjin exercise program at a frequency of 2-3 × 60 min sessions/week. The CG was instructed to maintain their usual activity and received a monthly health education talk. The primary outcome measures were knee extension strength, vital exhaustion, and fear of falling. Secondary outcome measures include physiological falls risk, handgrip strength, gait speed, timed up and go test, 30-second sit-to-stand, quality of life, depression, and frailty. All outcome measures were assessed at baseline and 4-month follow-up. RESULTS: Overall, there were no statistically significant differences in all outcome measures between CG and IG at 4-month follow-up. However, in exploratory compliance analysis, a statistically significant group x time interaction was found for vital exhaustion (B = -3.65, 95% CI [-7.13, -0.16], p = .047) among participants with at least 75% attendance. In post-hoc within-group comparisons, IG showed improved vital exhaustion by 4.31 points (95% CI [1.41, 7.20], d = 0.60). The average participant attendance rate was 81.3%. No major adverse events occurred, and all participants reported positive experiences with the exercise intervention. CONCLUSIONS: Our study demonstrated that Baduanjin is a safe, feasible, and acceptable exercise program that can be successfully implemented in community settings for older adults with varying frailty status. With good adherence, Baduanjin exercise could potentially be effective in alleviating vital exhaustion. However, the effectiveness of Baduanjin on physical performance, psychological measures and frailty in community-dwelling older adults remains equivocal. TRIAL REGISTRATION: ClinicalTrials.gov NCT04549103. Registered September 16, 2020.

Correction: Gut-derived wild blueberry phenolic acid metabolites modulate extrinsic cutaneous damage.

Correction for 'Gut-derived wild blueberry phenolic acid metabolites modulate extrinsic cutaneous damage' by John Ivarsson et al., Food Funct., 2024, 15, 7849-7864, https://doi.org/10.1039/D4FO01874E.

Zinc deficiency impairs the development of human regulatory B cells from purified B cells.

Zinc is a vital trace element, important for many different immune processes and adequate functionality. B cell development is known to be dependent on sufficient zinc supply. Recently a regulatory B cell (Breg) population has been identified, as CD19+IL-10+ B cells, able to regulate immune responses by secretion of anti-inflammatory cytokines, such as IL-10. Due to their promotion of an anti-inflammatory milieu, Bregs could reduce or might even prevent excessive pro-inflammatory responses. Hence, having and maintaining Bregs could be interesting for patients suffering from allergies, asthma, and autoimmune diseases. Therefore, understanding Breg generation, required signaling, and their developmental requirements are important. Since our group could previously show that zinc is important for regulatory T cells, we aimed to determine the effect of zinc deficiency on Breg development from human peripheral blood CD19+ B cells. We observed highest Breg generation with a combined stimulus of CD40L and the toll like receptor (TLR) ligand, CpG-ODN2006. Using this stimulus, we observed that zinc deficient medium significantly decreased Breg generation from purified B cells. This was not seen in Bregs generated from peripheral blood mononuclear cells (PBMCs) without B cell enrichment suggesting a compensatory mechanism. In line with literature, our data also confirms Bregs develop from CD19+ B cells, since total CD19+ frequencies remained unchanged, while Breg frequencies varied between stimuli and zinc media conditions. Our study shows for the first time that zinc deficiency significantly impairs Breg development, which provides an important new perspective for clinical applications and therapeutic strategies.

Model-Informed Approach to Recommend Burosumab Dosing Regimens for Pediatric and Adult Patients With the Ultrarare Disease Tumor-Induced Osteomalacia.

Burosumab is approved for the treatment of hypophosphatemia in persistent tumor-induced osteomalacia. This work exemplifies a model-informed drug development approach that evaluated burosumab pharmacokinetics and pharmacokinetic/pharmacodynamics in the ultrarare tumor-induced osteomalacia population to support adult and pediatric dosing. Data from tumor-induced osteomalacia participants were combined with data from X-linked hypophosphatemia to understand pharmacokinetic and pharmacokinetic/pharmacodynamic characteristics and covariates specific to tumor-induced osteomalacia. Pharmacokinetic and pharmacokinetic/pharmacodynamic simulations were performed using final models to support dosing recommendations for adults and extrapolation to pediatric patients. Burosumab pharmacokinetics were described using a one-compartment model with first-order absorption and body weight as a significant covariate. Pharmacokinetic/pharmacodynamic relationships were described using a sigmoidal Emax model with significant covariates of baseline fibroblast growth factor 23 on baseline fasting serum phosphate and potency of burosumab response and tumor-induced osteomalacia disease state resulting in a steep slope of response; however, the covariates are not clinically meaningful. Simulations demonstrated that, in pediatric patients, starting doses of burosumab 0.3 and 0.4 mg/kg every 2 weeks at steady state would achieve normal serum phosphate levels in ≥ 30% of patients with relatively low risk of hyperphosphatemia (< 3%). In adults, burosumab 0.3 and 0.5 mg/kg every 4 weeks achieves similar percentages of responders and a relative low risk of hyperphosphatemia (< 7%). Serum phosphate titration-based burosumab dosing increased the probability of achieving normal serum phosphate levels. The models supported a model-informed drug development approach for global approvals of titration-based burosumab dosing, guided by monitoring fasting serum phosphate levels.

Venetoclax Dose Escalation Rapidly Activates a BAFF/BCL-2 Survival Axis in Chronic Lymphocytic Leukemia.

Venetoclax, a first-in-class BH3 mimetic drug targeting BCL-2, has improved outcomes for patients with chronic lymphocytic leukemia (CLL). Early measurements of the depth of the venetoclax treatment response, assessed by minimal residual disease, are strong predictors of long-term clinical outcomes. Yet, there are limited data concerning the early changes induced by venetoclax treatment that might inform strategies to improve responses. To address this gap, we conducted longitudinal mass cytometric profiling of blood cells from patients with CLL during the first five weeks of venetoclax monotherapy. At baseline, we resolved CLL heterogeneity at the single-cell level to define multiple subpopulations in all patients distinguished by proliferative, metabolic and cell survival proteins. Venetoclax induced significant reduction in all CLL subpopulations coincident with rapid upregulation of pro-survival BCL-2, BCL-XL and MCL-1 proteins in surviving cells, which had reduced sensitivity to the drug. Mouse models recapitulated the venetoclax-induced elevation of survival proteins in B cells and CLL-like cells that persisted in vivo, with genetic models demonstrating that extensive apoptosis and access to the B cell cytokine, BAFF, were essential. Accordingly, analysis of patients with CLL that were treated with venetoclax or the anti-CD20 antibody obinutuzumab exhibited marked elevation of BAFF and increased pro-survival proteins in leukemic cells that persisted. Overall, these data highlight the rapid adaptation of CLL cells to targeted therapies via homeostatic factors and support co-targeting of cytokine signals to achieve deeper and more durable long-term responses.