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Parent-teacher relationships are germane to child outcomes and are especially important when children experience social-behavioral difficulties. We used dyadic methods to assess levels and congruence in parent-teacher relationship joining (affective quality) and communication-to-other (communication behaviors). The study also examined whether teacher beliefs about parent involvement and years of experience and parents' educational attainment and ethnic-racial identity related to relationship qualities. Data come from three randomized controlled trials of a family-school partnership intervention for children with social-behavioral difficulties. Results indicated dyad levels of joining were higher than communication-to-other and identified a degree of mismatch in parent-teacher relationships. Parents reported more positive joining and communication-to-other than teachers. Parent-teacher dyads reported higher-quality relationships when parents identified as White and teachers held positive beliefs about parent involvement. Congruence was greater when teachers held positive beliefs about parent involvement and when parents had higher educational attainment. Implications for school-based family engagement efforts are discussed.

Today's college students increasingly rely on technology in their daily activities, and a basic awareness of data privacy is becoming essential from both a personal and professional standpoint. This article describes the design of an interdisciplinary undergraduate course focused on data privacy law. It outlines the key content areas that encompass the foundational principles of this emerging field and includes descriptions of hands-on exercises that illustrate the technical and managerial elements of a comprehensive privacy compliance program. The course framework and sample assignments can serve as a model for other faculty seeking to teach data privacy law to undergraduate students.

Conjoint Behavioral Consultation (CBC) is an evidence-based, indirect service delivery model that addresses children's behavioral concerns across home and school. However, to date, researchers have not yet examined specific aspects of the intervention that maximize the effects of CBC. The current study examined whether a foundational aspect of consultation--the framing of target behaviors (i.e., positive behaviors to increase versus negative behaviors to decrease)--moderated the effects of CBC on children's problem and adaptive behaviors. Participants were 267 children in Kindergarten through third grade (n[subscript CBC] = 159, n[subscript Control] = 108) and their parents and teachers. Results revealed positively framed target behaviors were associated with fewer behavior concerns at school for children who received CBC relative to other child participants. Children who received CBC and whose target behaviors were framed negatively showed more noncompliant behavior relative to all other participants. Implications of these findings for practice, study limitations, and areas for future research are discussed.

Low-level jets (LLJs) occur under a variety of atmospheric conditions and influence the available wind resource for wind energy projects. In 2020, lidar-mounted buoys owned by the US Department of Energy (DOE) were deployed off the California coast in two wind energy lease areas administered by the Bureau of Ocean Energy Management: Humboldt and Morro Bay. The wind profile observations from the lidars and collocated near-surface meteorological stations (4–240 m) provide valuable year-long analyses of offshore LLJ characteristics at heights relevant to wind turbines. At Humboldt, LLJs were associated with flow reversals and north-northeasterly winds, directions that are more aligned with terrain influences than the predominant northerly flow. At Morro Bay, coastal LLJs were observed primarily during northerly flow as opposed to the predominant north-northwesterly flow. LLJs were observed more frequently in colder seasons within the lowest 250 m a.s.l. (above sea level), in contrast with the summertime occurrence of the higher-altitude California coastal jet influenced by the North Pacific High, which typically occurs at heights of 300–400 m. The lidar buoy observations also validate LLJ representation in atmospheric models that estimate potential energy yield of offshore wind farms. The European Centre for Medium-Range Weather Forecasts Reanalysis version 5 (ERA5) was unsuccessful at identifying all observed LLJs at both buoy locations within the lowest 200 m. An extension of the National Renewable Energy Laboratory (NREL) 20-year wind resource dataset for the Outer Continental Shelf off the coast of California (CA20-Ext) yielded marginally greater captures of observed LLJs using the Mellor–Yamada–Nakanishi–Niino (MYNN) planetary boundary layer (PBL) scheme than the 2023 National Offshore Wind dataset (NOW-23), which uses the Yonsei University (YSU) scheme. However, CA20-Ext also produced the most LLJ false alarms, which are instances when a model identified an LLJ but no LLJ was observed. CA20-Ext and NOW-23 exhibited a tendency to overestimate the duration of LLJ events and underestimate LLJ core heights.

Abstract Patients with chronic kidney disease (CKD) are often regarded as experiencing wasting of muscle mass and declining muscle strength and function, collectively termed sarcopenia. The extent of skeletal muscle wasting in clinical and preclinical CKD populations is unclear. We evaluated skeletal muscle atrophy in preclinical and clinical models of CKD, with multiple sub‐analyses for muscle mass assessment methods, CKD severity, sex and across the different preclinical models of CKD. We performed a systematic literature review of clinical and preclinical studies that measured muscle mass/size using the following databases: Ovid Medline, Embase and Scopus. A random effects meta‐analysis was utilized to determine standard mean difference (SMD; Hedges' g) between healthy and CKD. Heterogeneity was evaluated using the I2 statistic. Preclinical study quality was assessed via the Systematic Review Centre for Laboratory Animal Experimentation and clinical studies quality was assessed via the Newcastle‐Ottawa Scale. This study was registered in PROSPERO (CRD42020180737) prior to initiation of the search. A total of 111 studies were included in this analysis using the following subgroups: 106 studies in the primary CKD analysis, 18 studies that accounted for diabetes and 7 kidney transplant studies. Significant atrophy was demonstrated in 78% of the preclinical studies and 49% of the clinical studies. The random effects model demonstrated a medium overall SMD (SMD = 0.58, 95% CI = 0.52–0.64) when combining clinical and preclinical studies, a medium SMD for the clinical population (SMD = 0.48, 95% CI = 0.42–0.55; all stages) and a large SMD for preclinical CKD (SMD = 0.95, 95% CI = 0.76–1.14). Further sub‐analyses were performed based upon assessment methods, disease status and animal model. Muscle atrophy was reported in 49% of the clinical studies, paired with small mean differences. Preclinical studies reported significant atrophy in 78% of studies, with large mean differences. Across multiple clinical sub‐analyses such as severity of CKD, dialysis modality and diabetes, a medium mean difference was found. Sub‐analyses in both clinical and preclinical studies found a large mean difference for males and medium for females suggesting sex‐specific implications. Muscle atrophy differences varied based upon assessment method for clinical and preclinical studies. Limitations in study design prevented conclusions to be made about the extent of muscle loss with disease progression, or the impact of dialysis. Future work would benefit from the use of standardized measurement methods and consistent clinical staging to improve our understanding of atrophy changes in CKD progression, and analysis of biological sex differences.

Two buoys equipped with Doppler lidars owned by the US Department of Energy (DOE) were deployed off the coast of California in autumn of 2020 by Pacific Northwest National Laboratory. The buoys collected data for an entire annual cycle at two offshore locations proposed for offshore wind development by the Bureau of Ocean Energy Management. One of the buoys was deployed approximately 50 km off the coast near Morro Bay in central California in 1100 m of water. The second buoy was deployed approximately 40 km off Humboldt County in northern California in 625 m of water. The buoys provided the first-ever continuous measurements of the air–sea transition zone off the coast of California. The atmospheric and oceanographic characteristics of the area and estimates of annual energy production at both the Morro Bay and Humboldt wind energy areas show that both locations have a high wind energy yield and are prime locations for future floating offshore wind turbines. This article provides a description and comprehensive analysis of the data collected by the buoys, and a final post-processed dataset is uploaded to a data archive maintained by the DOE. Additional analysis was conducted to show the value of the data collected by the DOE buoys. All post-processed data from this study are available on the Wind Data Hub website: https://a2e.energy.gov/data# (last access: 14 September 2023). Near-surface, wave, current, and cloud datasets for Humboldt and Morro Bay are provided at https://doi.org/10.21947/1783807 (Krishnamurthy and Sheridan, 2023b) and https://doi.org/10.21947/1959715 (Krishnamurthy and Sheridan, 2023a), respectively. Lidar datasets for Humboldt and Morro Bay are provided at https://doi.org/10.21947/1783809 (Krishnamurthy and Sheridan, 2023d) and https://doi.org/10.21947/1959721 (Krishnamurthy and Sheridan, 2023c), respectively.

The California Pacific coast is characterized by considerable wind resource and areas of dense population, propelling interest in offshore wind energy as the United States moves toward a sustainable and decarbonized energy future. Reanalysis models continue to serve the wind energy community in a multitude of ways, and the need for validation in locations where observations have been historically limited, such as offshore environments, is strong. The U.S. Department of Energy (DOE) owns two lidar buoys that collect wind speed observations across the wind turbine rotor layer along with meteorological and oceanographic data near the surface to characterize the wind resource. Lidar buoy data collected from recent deployments off the northern California coast near Humboldt County and the central California coast near Morro Bay allow for validation of commonly used reanalysis products. In this article, wind speeds from the Modern-Era Retrospective analysis for Research and Applications version 2 (MERRA-2), the Climate Forecast System version 2 (CFSv2), the North American Regional Reanalysis (NARR), the European Centre for Medium-Range Weather Forecasts Reanalysis version 5 (ERA5), and the analysis system of the Rapid Refresh (RAP) are validated at heights within the wind turbine rotor layer ranging from 50 to 100 m. The validation results offer guidance on the performance and uncertainty associated with utilizing reanalyses for offshore wind resource characterization, providing the offshore wind energy community with information on the conditions that lead to reanalysis error. At both California coast locations, the reanalyses tend to underestimate the observed rotor-level wind resource. Occasions of large reanalysis error occur in conjunction with stable atmospheric conditions, wind speeds associated with peak turbine power production (> 10 m s−1), and mischaracterization of the diurnal wind speed cycle in summer months.

Growth in adoption of distributed wind turbines for energy generation is significantly impacted by challenges associated with siting and accurate estimation of the wind resource. Small turbines, at hub heights of 40 m or less, are greatly impacted by terrestrial obstacles such as built structures and vegetation that can cause complex wake effects. While some progress in high-fidelity complex fluid dynamics (CFD) models has increased the potential accuracy for modelling the impacts of obstacles on turbulent wind flow, these models are too computationally expensive for practical siting and resource assessment applications. To understand the efficacy of available models in situ, this study evaluates classic and commonly used methods alongside new state-of-the-art lower-order models derived from CFD simulations and machine learning approaches. This evaluation is conducted using a subset of an extensive original dataset of measurements from more than 300 operational wind turbines in the northern Netherlands. The results show that data-driven methods (e.g. machine learning and statistical modelling) are most effective at predicting production at real sites with an average error in annual energy production of 2.5 %. When sufficient data may not be available de novo to support these data-driven approaches, models derived from high-fidelity simulations show promise and reliably outperform classic methods. On average these models have 6.3 %–11.5 % error compared with 26 % for classic methods and 27 % baseline error for reanalysis data without obstacle correction. While more performant on average, these methods are also sensitive to the quality of obstacle descriptions and reanalysis inputs.

Due to financial and temporal limitations, the small wind community relies upon simplified wind speed models and energy production simulation tools to assess site suitability and produce energy generation expectations. While efficient and user-friendly, these models and tools are subject to errors that have been insufficiently quantified at small wind turbine heights. This study leverages observations from meteorological towers and sodars across the United States to validate wind speed estimates from the Wind Integration National Dataset (WIND) Toolkit, the European Centre for Medium-Range Weather Forecasts (ECMWF) Reanalysis v5 (ERA5), and the Modern-Era Retrospective Analysis for Research and Applications, version 2 (MERRA-2), revealing average biases within ±0.5 m s−1 at small wind hub heights. Observations from small wind turbines across the United States provide references for validating energy production estimates from the System Advisor Model (SAM), Wind Report, MyWindTurbine.com, and Global Wind Atlas 3 (GWA3), which are seen to overestimate actual annual capacity factors by 2.5, 4.2, 11.5, and 7.3 percentage points, respectively. In addition to quantifying the error metrics, this paper identifies sources of model and tool discrepancies, noting that interannual fluctuation in the wind resource, wind speed class, and loss assumptions produces more variability in estimates than different horizontal and vertical interpolation techniques. The results of this study provide small wind installers and owners with information about these challenges to consider when making performance estimates and thus possible adjustments accordingly. Looking to the future, recognizing these error metrics and sources of discrepancies provides model and tool researchers and developers with opportunities for product improvement that could positively impact small wind customer confidence and the ability to finance small wind projects.

The objectives of this study were to: (1) assess the intensification of chemical oxygen demand (COD) and phosphate (PO4-P) removal; and (2) generate a set of rate constants of COD degradation (kCOD) and phosphate (kPO4-P) removal for the treatment of industrial wastewater (WW) using intensified adsorption beds. Two horizontal subsurface flow constructed wetlands (HSSFCWs) filled with coal ash and alum sludge and two conventional HSSFCWs packed with gravels were operated with different loadings of COD and PO4-P at a hydraulic retention time (HRT) of 24 hrs at water depth of 0.40 m. The bed performance was analysed for COD and PO4-P removal efficiency. The intensified HSSFCWs outperformed the control beds by a mean COD and PO4-P removal efficiency of 43 and 49%, respectively. The progression of COD and PO4-P removal along the system was fitted into the first-order plug flow model (K-C model). In this study the kCOD values ranged from 0.36 to 0.65 m/d with a mean of 0.46 ± 0.08 m/d (n = 30). The kPO4-P values ranged from 0.74 to 1.76 m/d and averaged to 1.23 ± 0.37 m/d (n = 30), irrespective of the condition applied. Hence, these data can be used for future projects using HSSFCWs to treat industrial wastewater. HIGHLIGHTS Industrial wastewaters are strong organic sources of pollution.; Industrial effluents are not commonly treated using conventional HSSFCW.; Addition of an adsorption mechanism using coal ash and alum sludge in the system intensifies bed performance.; Such intensified HSSFCWs are a sustainable and low-cost treatment system.; Rate constants are important design parameters to determine the size of a HSSFCW.;

Human trabecular meshwork is a sieve-like tissue with large pores, which plays a vital role in aqueous humor outflow. Dysfunction of this tissue can occur, which leads to glaucoma and permanent vision loss. Replacement of trabecular meshwork with a tissue-engineered device is the ultimate objective. This study aimed to create a biomimetic structure of trabecular meshwork using electrospinning. Conventional electrospinning was compared to cryogenic electrospinning, the latter being an adaptation of conventional electrospinning whereby dry ice is incorporated in the fiber collector system. The dry ice causes ice crystals to form in-between the fibers, increasing the inter-fiber spacing, which is retained following sublimation. Structural characterization demonstrated cryo-scaffolds to have closer recapitulation of the trabecular meshwork, in terms of pore size, porosity, and thickness. The attachment of a healthy, human trabecular meshwork cell line (NTM5) to the scaffold was not influenced by the fabrication method. The main objective was to assess cell infiltration. Cryo-scaffolds supported cell penetration deep within their structure after seven days, whereas cells remained on the outer surface for conventional scaffolds. This study demonstrates the suitability of cryogenic electrospinning for the close recapitulation of trabecular meshwork and its potential as a 3D in vitro model and, in time, a tissue-engineered device.

Abstract Background Internationally, elective spinal surgery rates in workers’ compensation populations are high, as are reoperation rates, while return-to-work rates following spinal surgery are low. Little information is available from Australia. The aim of this study was to describe the rates, costs, return to work and reoperation following elective spinal surgery in the workers’ compensation population in New South Wales (NSW), Australia. Methods This retrospective cohort study used administrative data from the State Insurance Regulatory Authority, the government organisation responsible for regulating and administering workers’ compensation insurance in NSW. These data cover all workers’ compensation-insured workers in New South Wales (over 3 million workers/year). We identified a cohort of insured workers who underwent elective spinal surgery (fusion or decompression) between January 1, 2010 and December 31, 2018. People who underwent surgery for spinal fracture or dislocation, or who had sustained a traumatic brain injury were excluded. The main outcome measures were annual spinal surgery rates, cost of the surgical episode, cumulative costs (surgical, hospital, medical and physical therapy) to 2 years post-surgery, and reoperation and return-to-work rates 2 years post-surgery. Results There were 9343 eligible claims (39.1 % fusion; 59.9 % decompression); claimants were predominantly male (75 %) with a mean age of 43 (range 18 to 75) years. Spinal surgery rates ranged from 15 to 29 surgeries per 100,000 workers per year, fell from 2011-12 to 2014-15 and rose thereafter. The average cost in Australian dollars for a surgical episode was $46,000 for a spinal fusion and $20,000 for a decompression. Two years post-fusion, only 19 % of people had returned to work at full capacity; 39 % after decompression. Nineteen percent of patients underwent additional spinal surgery within 2 years of the index surgery, to a maximum of 5 additional surgeries. Conclusion Rates of workers’ compensation-funded spinal surgery did not rise significantly during the study period, but reoperation rates are high and return-to-work rates are low in this population at 2 years post- surgery. In the context of the poor evidence base supporting lumbar fusion surgery, the high cost, increasing rates, and the increased likelihood of poor outcomes in the workers’ compensation population, we question the value of this procedure in this setting.

About 5% of B cells in healthy mice and humans are allelically or isotypically included and hence co-express two different antibodies. In mice, dual antibody B cells (B2R) expand with systemic autoimmunity, co-express autoreactive and non-autoreactive antibodies, and participate in immune responses, but this phenomenon is strain dependent. This study was developed with two goals: 1) to establish the contribution of TLR and IFN receptor signaling to the development of germinal center B cells that express two antibodies in MRL/lpr mice; and 2) to determine whether B2R B cells are increased and particularly activated in a subset of adult patients diagnosed with systemic lupus erythematosus (SLE). Results from the MRL/lpr studies indicate that the enhanced differentiation of dual-κ B cells into germinal center B cells is due to a heightened response to TLR7 and TLR9 signaling, further fueled by an increased response to type II IFN. To understand the clinical and translational implications of our observations in mouse B2R B cells, cohorts of SLE patients and healthy controls were recruited and evaluated for expression of dual BCRs. Results from flow cytometry and microscopy revealed supraphysiological frequencies of κ+λ+ B2R cells in one fourth of the SLE patients. Abnormal numbers of κ+λ+ B cells correlated with higher frequencies of activated naïve B cells and age-associated B cells, and a lower proportion of “B cells that are naïve IgD+” (BND). However, results from single cell V(D)J sequencing demonstrated that these high κ+λ+ SLE patients harbored normal frequencies of κ+λ+ and other B2R B cells. and we further show that their B cells were instead decorated by κ and λ VH4-34 autoantibodies. Thus, our findings indicate that elevated flow cytometric detection of isotypically-included B cells can identify patients with high titers of B cell-reactive VH4-34 autoantibodies and abnormal distribution of B cell subsets relevant to autoimmunity.

The primary cause of failure for minimally invasive glaucoma surgery (MIGS) is fibrosis in the trabecular meshwork (TM) that regulates the outflow of aqueous humour, and no anti-fibrotic drug is available for intraocular use in MIGS. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a promising anti-fibrotic target. This study aims to utilise a novel lipid nanoparticle (LNP) to deliver MRTF-B siRNA into human TM cells and to compare its effects with those observed in human conjunctival fibroblasts (FF). Two LNP formulations were prepared with and without the targeting peptide cΥ, and with an siRNA concentration of 50 nM. We examined the biophysical properties and encapsulation efficiencies of the LNPs, and evaluated the effects of MRTF-B silencing on cell viability, key fibrotic genes expression and cell contractility. Both LNP formulations efficiently silenced MRTF-B gene and were non-cytotoxic in TM and FF cells. The presence of cΥ made the LNPs smaller and more cationic, but had no significant effect on encapsulation efficiency. Both TM and FF cells also showed significantly reduced contractibility after transfection with MRTF-B siRNA LNPs. In TM cells, LNPs with cΥ achieved a greater decrease in contractility compared to LNPs without cΥ. In conclusion, we demonstrate that the novel CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. LNPs can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS.

The detection of foreign antigens in vivo has relied on fluorescent conjugation or indirect read-outs such as antigen presentation. In our studies, we found that these widely used techniques had several technical limitations that have precluded a complete picture of antigen trafficking or retention across lymph node cell types. To address these limitations, we developed a ‘molecular tracking device’ to follow the distribution, acquisition, and retention of antigen in the lymph node. Utilizing an antigen conjugated to a nuclease-resistant DNA tag, acting as a combined antigen-adjuvant conjugate, and single-cell mRNA sequencing, we quantified antigen abundance in the lymph node. Variable antigen levels enabled the identification of caveolar endocytosis as a mechanism of antigen acquisition or retention in lymphatic endothelial cells. Thus, these molecular tracking devices enable new approaches to study dynamic tissue dissemination of antigen-adjuvant conjugates and identify new mechanisms of antigen acquisition and retention at cellular resolution in vivo.

Glaucoma is the second leading cause of irreversible blindness worldwide. Glaucoma is a progressive optic neuropathy in which permanent loss of peripheral vision results from neurodegeneration in the optic nerve head. The trabecular meshwork is responsible for regulating intraocular pressure, which to date, is the only modifiable risk factor associated with the development of glaucoma. Lowering intraocular pressure reduces glaucoma progression and current surgical approaches for glaucoma attempt to reduce outflow resistance through the trabecular meshwork. Many surgical approaches use minimally invasive glaucoma surgeries (MIGS) to control glaucoma. In this progress report, biomaterials currently employed to treat glaucoma, such as MIGS, and the issues associated with them are described. The report also discusses innovative biofabrication approaches that aim to revolutionise glaucoma treatment through tissue engineering and regenerative medicine (TERM). At present, there are very few applications targeted towards TM engineering in vivo, with a great proportion of these biomaterial structures being developed for in vitro model use. This is a consequence of the many anatomical and physiological attributes that must be considered when designing a TERM device for microscopic tissues, such as the trabecular meshwork. Ongoing advancements in TERM research from multi-disciplinary teams should lead to the development of a state-of-the-art device to restore trabecular meshwork function and provide a bio-engineering solution to improve patient outcomes.

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr

Assignment of cell types from single-cell RNA sequencing (scRNA-seq) data remains a time-consuming and error-prone process. Current packages for identity assignment use limited types of reference data and often have rigid data structure requirements. We developed the clustifyr R package to leverage several external data types, including gene expression profiles to assign likely cell types using data from scRNA-seq, bulk RNA-seq, microarray expression data, or signature gene lists. We benchmark various parameters of a correlation-based approach and implement gene list enrichment methods. clustifyr is a lightweight and effective cell-type assignment tool developed for compatibility with various scRNA-seq analysis workflows. clustifyr is publicly available at https://github.com/rnabioco/clustifyr

Glaucoma is linked to raised intraocular pressure (IOP). The trabecular meshwork (TM) plays a major role in regulating IOP by enabling outflow of aqueous humor from the eye through its complex 3D structure. A lack of therapies targeting the dysfunctional TM highlights the need to develop biomimetic scaffolds that provide 3D in vitro models for glaucoma research or as implantable devices to regenerate TM tissue. To artificially mimic the TM’s structure, we assessed methods for its decellularization and outline an optimized protocol for cell removal and structural retention. Using bovine TM, we trialed 2 lysing agents—Trypsin (0.05% v/v) and Ammonium Hydroxide (NH4OH; 2% v/v). Twenty-four hours in Trypsin caused significant structural changes. Shorter exposure (2 h) reduced this disruption whilst decellularizing the tissue (dsDNA 26 ± 14 ng/mL (control 1970 ± 146 ng/mL)). In contrast, NH4OH lysed all cells (dsDNA 25 ± 21 ng/mL), and the TM structure remained intact. For human TM, 2% v/v NH4OH similarly removed cells (dsDNA 52 ± 4 ng/mL (control 1965 ± 233 ng/mL)), and light microscopy and SEM presented no structural damage. X-ray computed tomography enabled a novel 3D reconstruction of decellularized human TM and observation of the tissue’s intricate architecture. This study provides a new, validated method using NH4OH to decellularize delicate human TM without compromising tissue structure.

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM’s structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

The anterior segment of the eye is a complex set of structures that collectively act to maintain the integrity of the globe and direct light towards the posteriorly located retina. The eye is exposed to numerous physical and environmental insults such as infection, UV radiation, physical or chemical injuries. Loss of transparency to the cornea or lens (cataract) and dysfunctional regulation of intra ocular pressure (glaucoma) are leading causes of worldwide blindness. Whilst traditional therapeutic approaches can improve vision, their effect often fails to control the multiple pathological events that lead to long-term vision loss. Regenerative medicine approaches in the eye have already had success with ocular stem cell therapy and ex vivo production of cornea and conjunctival tissue for transplant recovering patients’ vision. However, advancements are required to increase the efficacy of these as well as develop other ocular cell therapies. One of the most important challenges that determines the success of regenerative approaches is the preservation of the stem cell properties during expansion culture in vitro. To achieve this, the environment must provide the physical, chemical and biological factors that ensure the maintenance of their undifferentiated state, as well as their proliferative capacity. This is likely to be accomplished by replicating the natural stem cell niche in vitro. Due to the complex nature of the cell microenvironment, the creation of such artificial niches requires the use of bioengineering techniques which can replicate the physico-chemical properties and the dynamic cell–extracellular matrix interactions that maintain the stem cell phenotype. This review discusses the progress made in the replication of stem cell niches from the anterior ocular segment by using bioengineering approaches and their therapeutic implications.

Summary: Evidence suggests that a stem-cell-driven differentiation hierarchy maintains the dynamic thymic epithelial cell (TEC) network that governs T lymphocyte development. The identification of TEC stem/progenitor cells has been a major focus in the field, and several candidates with contrasting phenotypes have been described. We sought to determine the provenance and function of the only population reported to exhibit TEC stem cell properties in the adult, a Foxn1− EpCAM− cell that generates so-called thymospheres. We provide evidence that the thymosphere-forming cell (TSFC) is not a TEC stem cell but can incorporate bystander TECs into thymospheres, providing an explanation for the epithelial activity ascribed to these structures. TSFCs were found to share a phenotype, transcriptional profile, and developmental origin with thymic fibroblasts and can generate adipocytes. In summary, this study redefines the nature of bipotent TEC stem/progenitor cells in the adult thymus and highlights a potentially important mesenchymal progenitor population. : The phenotype of putative bipotent epithelial stem/progenitor cells in the adult thymus has been controversial. Sheridan et al. find that a prominent candidate, the FoxN1−EpCAM− sphere-forming cell, harbors no epithelial stem/progenitor capacity and present evidence that they are a unique intrathymic mesenchymal stromal/stem cell population with adipocyte differentiation capacity. Keywords: thymus, thymic epithelial cell, stem cell, progenitor, thymosphere, MSC, mesenchyme, adipocyte

LUBAC is a ubiquitin ligase complex of HOIL-1, HOIP and SHARPIN important for signal transduction of a range of stimuli. Here the authors define the function of all three LUBAC components in T cell development and homeostasis.

Manipulation of protein stability with ligand-regulated degron fusions is a powerful method for investigating gene function. We developed a selectable cassette for easy C-terminal tagging of endogenous human proteins with the E. coli dihydrofolate reductase (eDHFR) degron using CRISPR/Cas9 genome editing. This cassette permits high-efficiency recovery of correct integration events using an in-frame self-cleaving 2A peptide and the puromycin resistance gene. PCR amplified donor eDHFR cassette fragments with 100 bases of homology on each end are integrated by homology-directed repair (HDR) of guide RNA (gRNA)-targeted double-stranded DNA breaks at the 3′ ends of open reading frames (ORFs). As proof of principle, we generated cell lines in which three endogenous proteins were tagged with the eDHFR degron. When the antibiotic trimethoprim is removed from the media, each of the eDHFR-tagged proteins was depleted by >90% within 2–4 h, and this depletion was reversed by re-addition of trimethoprim. Since puromycin selection permits recovery of in-frame degron fusions with high efficiency using only 100-bp long regions of homology, this method should be applicable on a genome-wide scale for generating libraries of conditional mutant cell lines.

Gene set enrichment analysis is a popular approach for prioritising the biological processes perturbed in genomic datasets. The Bioconductor project hosts over 80 software packages capable of gene set analysis. Most of these packages search for enriched signatures amongst differentially regulated genes to reveal higher level biological themes that may be missed when focusing only on evidence from individual genes. With so many different methods on offer, choosing the best algorithm and visualization approach can be challenging. The EGSEA package solves this problem by combining results from up to 12 prominent gene set testing algorithms to obtain a consensus ranking of biologically relevant results.This workflow demonstrates how EGSEA can extend limma-based differential expression analyses for RNA-seq and microarray data using experiments that profile 3 distinct cell populations important for studying the origins of breast cancer. Following data normalization and set-up of an appropriate linear model for differential expression analysis, EGSEA builds gene signature specific indexes that link a wide range of mouse or human gene set collections obtained from MSigDB, GeneSetDB and KEGG to the gene expression data being investigated. EGSEA is then configured and the ensemble enrichment analysis run, returning an object that can be queried using several S4 methods for ranking gene sets and visualizing results via heatmaps, KEGG pathway views, GO graphs, scatter plots and bar plots. Finally, an HTML report that combines these displays can fast-track the sharing of results with collaborators, and thus expedite downstream biological validation. EGSEA is simple to use and can be easily integrated with existing gene expression analysis pipelines for both human and mouse data.

New tools for reproducible exploratory data analysis of large datasets are important to address the rising size and complexity of genomic data. We developed the valr R package to enable flexible and efficient genomic interval analysis. valr leverages new tools available in the ”tidyverse”, including dplyr. Benchmarks of valr show it performs similar to BEDtools and can be used for interactive analyses and incorporated into existing analysis pipelines.