Unskilled and unaware: second-order judgments increase with miscalibration for low performers.

Overestimation and miscalibration increase with a decrease in performance. This finding has been attributed to a common factor: participants' knowledge and skills about the task performed. Researchers proposed that the same knowledge and skills needed for performing well in a test are also required for accurately evaluating one's performance. Thus, when people lack knowledge about a topic they are tested on, they perform poorly and do not know they did so. This is a compelling explanation for why low performers overestimate themselves, but such increases in overconfidence can also be due to statistical artifacts. Therefore, whether overestimation indicates lack of awareness is debatable, and additional studies are needed to clarify this issue. The present study addressed this problem by investigating the extent to which students at different levels of performance know that their self-estimates are biased. We asked 653 college students to estimate their performance in an exam and subsequently rate how confident they were that their self-estimates were accurate. The latter judgment is known as second-order judgments (SOJs) because it is a judgment of a metacognitive judgment. We then looked at whether miscalibration predicts SOJs per quartile. The findings showed that the relationship between miscalibration and SOJs was negative for high performers and positive for low performers. Specifically, for low performers, the less calibrated their self-estimates were the more confident they were in their accuracy. This finding supports the claim that awareness of what one knows and does not know depends in part on how much one knows.

Proteogenomic Reprogramming to a Functional Human Totipotent Stem Cell State via a PARP-DUX4 Regulatory Axis.

PARP1 (ARTD1) and Tankyrases (TNKS1/TNKS2; PARP5a/5b) are poly-ADP-ribose polymerases (PARPs) with catalytic and non-catalytic functions that regulate both the genome and proteome during zygotic genome activation (ZGA), totipotent, and pluripotent embryonic stages. Here, we show that primed, conventional human pluripotent stem cells (hPSC) cultured continuously under non-specific TNKS1/TNKS2/PARP1-inhibited chemical naive reversion conditions underwent epigenetic reprogramming to clonal blastomere-like stem cells. TIRN stem cells concurrently expressed hundreds of gene targets of the ZGA-priming pioneer factor DUX4, as well as a panoply of four-cell (4C)-specific (e.g., TPRXL, HOX clusters), eight-cell (8C)-specific (e.g., DUXA, GSC, GATA6), primitive endoderm-specific (e.g., GATA4, SOX17), trophectoderm-specific (e.g., CDX2, TFAP2C), and naive epiblast-specific (e.g., DNMT3L, NANOG, POU5F1(OCT4)) factors; all in a hybrid, combinatorial single-cell manner. Mapping of proteomic and single-cell expressions of TIRN cells against human preimplantation embryo references identified them as relatively homogenous 4C-8C stage populations. Injection of TIRN cells into murine 8C-16C-staged embryos resulted in efficient totipotent-like single cell contributions of human cells to both extra-embryonic (trophectoderm, placenta) and embryonic (neural, fetal liver, hematopoietic) lineages in human-murine blastocyst and fetal chimeras. Pairing of proteome with ubiquitinome analyses of TIRN cells revealed a global shutdown of ADP-ribosylation, and a perturbed TNKS/PARP1 equilibrium which not only impacted the protein levels of hundreds of TNKS/PARP1 substrates via a rewiring of the ubiquitin-proteosome system (UPS), but also de-repressed expression of hundreds of developmental genes associated with PARP1 suppression. ChIP-Seq analysis of core NANOG-SOX2-OCT4 (NSO) pluripotency factors in TIRN cells identified reprogrammed DUX4-accessible distal and cis-regulatory enhancer regions that were co-bound by PARP1 (NSOP). These NSOP enhancer regions possessed co-binding motifs for hundreds of the same ZGA-associated, embryonic, and extraembryonic lineage-specifying pioneer factors (e.g., HOX, FOX, GATA, SOX, TBX, CDX families) that were concurrently co-expressed in TIRN cells; suggesting that PARP1 and DUX4 cooperate with NSO pluripotency core factors to regulate the epigenetic plasticity of a human totipotency program. These findings provide the first demonstration that global, proteome-wide perturbations of post-translational modifications (i.e., ADP-ribosylation, ubiquitination) can regulate epigenetic reprogramming during human embryogenesis. Totipotent TIRN stem cells will provide a valuable cell culture model for studying the proteogenomic regulation of lineage specification from human blastomere stages and may facilitate the efficient generation of human organs in interspecies chimeras.

Breaking New Ground in Interventional Pulmonology: Integrating Cone Beam CT and Robotic-Assisted Bronchoscopy for High-Risk Pneumothorax in Peripherally Located Solitary Pulmonary Nodules.

Lung cancer, a leading cause of global cancer-related deaths, necessitates the development of innovative diagnostic techniques. Traditional bronchoscopy, while useful, has limitations in diagnosing peripheral pulmonary lesions (PPLs) and carries a higher risk of complications such as pneumothorax. However, the field of interventional pulmonology has seen significant advancements, including the introduction of robotic-assisted bronchoscopy (RAB), cone-beam computed tomography (CBCT), radial endobronchial ultrasound (R-EBUS), and rapid on-site evaluation (ROSE). These advancements have greatly improved the precision of diagnosing high-risk PPLs. This report presents the case of a 60-year-old female smoker with chronic obstructive pulmonary disease and extensive centrilobular emphysema, who had a peripherally located high-risk pulmonary nodule. She was successfully diagnosed with metastatic adenocarcinoma using an integrated approach, despite the challenging location of the lesion and high risk of pneumothorax. The integration of RAB with CBCT and augmented fluoroscopy offers a groundbreaking approach for diagnosing and managing difficult-to-reach, high-risk pulmonary nodules, marking a significant stride in the field of interventional pulmonology.

The elucidation of species-specific receptor pharmacology: a case study using subtype selective para- and meta-carborane estrogen receptor agonists.

Estrogen receptors are essential pharmacological targets for treating hormonal disorders and estrogen-dependent malignancies. Selective activation of estrogen receptor (ER) ß is hypothesized to provide therapeutic benefit with reduced risk of unwanted estrogenic side-effects associated with ERα activity. However, activating ERß without activating α is challenging due to the high sequence and structural homology between the receptor subtypes. We assessed the impact of structural modifications to the parent compound OSU-ERß-12 on receptor subtype binding selectivity using cell-free binding assays. Functional selectivity was evaluated by transactivation in HEK-293 cells overexpressing human or murine estrogen receptors. In vivo selectivity was examined through the uterotrophic effects of the analogs after oral administration in estrogen-naïve female mice. Furthermore, we evaluated the in vivo pharmacokinetics of the analogs following single dose IV and oral administration. Regarding selectivity, a single compound exhibited greater functional selectivity than OSU-ERß-12 for human ERß. However, like others in the meta-carborane series, its poor in vivo pharmacokinetics limit its suitability for further development. Surprisingly, and at odds with their pharmacokinetic and in vitro human activity data, most analogs potently induced uterotrophic effects in estrogen-naïve female mice. Further investigation of activity in HEK293 cells expressing murine estrogen receptors revealed species-specific differences in the ER-subtype selectivity of these analogs. Our findings highlight species-specific receptor pharmacology and the challenges it poses to characterizing developmental therapeutics in preclinical species. Significance Statement This study investigates para- and meta-substituted carborane analogs targeting estrogen receptors, revealing the greater selectivity of carborane analogs for human ERß compared to the mouse homolog. These findings shed light on the intricacies of using preclinical species in drug development to predict human pharmacology. The report also provides insights for the refinement and optimization of carborane analogs as potential therapeutic agents for estrogen-related disease states.

Predicting the clinical subcutaneous absorption rate constant of monoclonal antibodies using only the primary sequence: a machine learning approach.

Subcutaneous injections are an increasingly prevalent route of administration for delivering biological therapies including monoclonal antibodies (mAbs). Compared with intravenous delivery, subcutaneous injections reduce administration costs, shorten the administration time, and are strongly preferred from a patient experience point of view. An understanding of the absorption process of a mAb from the injection site to the systemic circulation is critical to the process of subcutaneous mAb formulation development. In this study, we built a model to predict the absorption rate constant (ka), which denotes how fast a mAb is absorbed from the site of administration. Once trained, our model (enabled by the XGBoost algorithm in machine learning) can predict the ka of a mAb following a subcutaneous injection using in silico molecular properties alone (generated from the primary sequence). Our model does not need clinically observed plasma concentration-time data; this is a novel capability not previously achieved in predictive pharmacokinetic models. The model also showed improved performance when benchmarked against a recently reported mechanistic model that relied on clinical data to predict subcutaneous absorption of mAbs. We further interpreted the model to understand which molecular properties affect the absorption rate and showed that our findings are consistent with previous studies evaluating subcutaneous absorption through direct experimentation. Taken altogether, this study reports the development, validation, benchmarking, and interpretation of a model that can predict the clinical ka of a mAb using its primary sequence as the only input.

Unique presentation of an endemic opportunistic fungal infection: Disseminated coccidioidomycosis mimicking metastatic lung cancer with endotracheal and endobronchial involvement.

Coccidioidomycosis is a fungal infection primarily Endemic in the Southwest United States. Disseminated Coccidioidomycosis is a life-threatening variant that mainly occurs in an immunocompromised host. This report describes an unusual presentation of disseminated Coccidioidomycosis in an immunocompetent individual. The patient was admitted with a subacute cough, progressively worsening shortness of breath, significant weight loss, nodular skin lesions in upper extremities, and acute hypoxemic respiratory failure. Chest imaging revealed extensive nodularity and mass-like lesions. What sets this case apart is the significant endotracheal and endobronchial involvement, which mimicked metastatic lung cancer. The diagnosis was confirmed through serology and bronchoscopy biopsy. This case underscores the critical importance of considering detailed travel history and maintaining a high index of suspicion for fungal infections in patients with endobronchial lesions, particularly in regions where Coccidioidomycosis is endemic.

The Metaverse, Religious Practice and Wellbeing: A Narrative Review.

The metaverse is touted as the next phase in the evolution of the Internet. This emerging digital ecosystem is widely conceptualized as a persistent matrix of interconnected multiuser, massively scaled online environments optimally experienced through immersive digital technologies such as virtual reality (VR). Much of the prognostication about the social implications of the metaverse center on secular activities. For example, retail, entertainment (gaming/concerts), and social networking. Little attention has been given to how the metaverse might impact religion. This narrative review explores contemporary research into online religious practice and the use of immersive digital technologies for religious purposes. This focus informs a discussion about how the metaverse, an online and immersive technology, might impact religion/religious practices. For billions worldwide, religion is an essential aspect of social identity and a cornerstone of psychological wellbeing. The emergence of the metaverse may represent a new way of connecting with an ancient source of human flourishing.

Religious coping and levels of posttraumatic stress disorder symptomatology after the Beirut explosion.

OBJECTIVE: Religious coping has implications for the development of psychopathology in the aftermath of traumatic events. This study explored the relationship between religious coping (positive and negative) and posttraumatic stress disorder (PTSD) symptomology among survivors of a large industrial explosion that devastated parts of Beirut in August of 2020. METHOD: Three months after the disaster, 996 residents of Beirut and Lebanon completed validated measures of religious coping (RCOPE) and PTSD symptomatology (Impact of Events Scale-Revised) in either English or Arabic. The majority of participants were young adults aged between 18 and 25 years. RESULTS: Results indicated that higher levels of negative religious coping were a significant predictor of higher levels of PTSD symptomatology and were associated with a two-fold risk of meeting the criteria for probable PTSD. Other significant predictors included female gender, being a resident of Beirut at the time of the explosion, having personally sustained an injury, or knowing a person injured in the explosion. Effects sizes ranged from .34 to .68. CONCLUSIONS: Higher scores on measures of negative religious coping were associated with higher levels of PTSD symptomatology. However, negative religious coping may be better construed as a set of religious-based appraisals of event causality and may represent a form of peritraumatic appraisal in the wake of traumatic events. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Antibody Drug Clearance: An Underexplored Marker of Outcomes with Checkpoint Inhibitors.

Immune-checkpoint inhibitor (ICI) therapy has dramatically changed the clinical landscape for several cancers, and ICI use continues to expand across many cancer types. Low baseline clearance (CL) and/or a large reduction of CL during treatment correlates with better clinical response and longer survival. Similar phenomena have also been reported with other monoclonal antibodies (mAb) in cancer and other diseases, highlighting a characteristic of mAb clinical pharmacology that is potentially shared among various mAbs and diseases. Though tempting to attribute poor outcomes to low drug exposure and arguably low target engagement due to high CL, such speculation is not supported by the relatively flat exposure-response relationship of most ICIs, where a higher dose or exposure is not likely to provide additional benefit. Instead, an elevated and/or increasing CL could be a surrogate marker of the inherent resistant phenotype that cannot be reversed by maximizing drug exposure. The mechanisms connecting ICI clearance, therapeutic efficacy, and resistance are unclear and likely to be multifactorial. Therefore, to explore the potential of ICI CL as an early marker for efficacy, this review highlights the similarities and differences of CL characteristics and CL-response relationships for all FDA-approved ICIs, and we compare and contrast these to selected non-ICI mAbs. We also discuss underlying mechanisms that potentially link mAb CL with efficacy and highlight existing knowledge gaps and future directions where more clinical and preclinical investigations are warranted to clearly understand the value of baseline and/or time-varying CL in predicting response to ICI-based therapeutics.

Decoupling FcRn and tumor contributions to elevated immune checkpoint inhibitor clearance in cancer cachexia.

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia.

The spectrum of cerebrospinal fluid findings in tuberculous meningitis and their relation to severity, radiological features, and outcome.

Objectives: The aim of the study was to evaluate cerebrospinal fluid (CSF) findings in tuberculous meningitis (TBM) and correlate it with severity, radiological features, and outcome of TBM. Materials and Methods: In a retrospective study, data from admitted TBM patients were analyzed, and findings of CSF examinations were recorded. The CSF was categorized as typical (protein 50-500 mg/dL, cells 50-500/mm3, and glucose 50% or lower of blood sugar); those above and below these values were categorized as increased or decreased, respectively. The CSF findings were correlated with stage of TBM, and 3-month outcome and radiological features. Paradoxical response was also noted. Results: There were 111 patients with TBM (definite 34, highly probable 77). On admission, 20 patients were in Stage I, 63 in Stage II, and 28 in Stage III TBM. CSF cells were in typical range in 73, low in 27 and increased in 11 patients. Protein was in typical range in 92 patients decreased in 11 patients and increased in eight patients. Sugar was normal in 41 and reduced in 70 patients. CSF cells, glucose, and protein did not correlate with the severity of meningitis. Fifteen patients had normal initial magnetic resonance imaging (MRI). Tuberculomas were present in 53 patients, hydrocephalus in 43 patients, basal exudates in 43 patients, and infarction in 44 patients. Mixed findings were present in 65 patients. The MRI features did not correlate with CSF. Second CSF was available after a median duration of 26 (13-276) days in 50 patients. The CSF cells were decreased in 20 and increased in 30 patients, protein increased in 30 and sugars decreased in 16 patients. Paradoxical worsening occurred in 27 patients. Fifty-one patients recovered completely, 41 partially, 15 had poor, three patients were lost to follow-up, and one died. CSF parameters did not correlate with 3-month outcome or paradoxical worsening. CSF parameters do not differ significantly between baseline and 1 month CSF, but cells and lymphocytes changed significantly between 1st month and 3rd month CSF. Conclusion: Typical CSF findings were present in 66% and did not correlate severity of TBM, radiological features paradoxical worsening or 3-month outcome. CSF cell count decreased within 3 months of treatment.

Hydrogen bonding in duplex DNA probed by DNP enhanced solid-state NMR N-H bond length measurements.

Numerous biological processes and mechanisms depend on details of base pairing and hydrogen bonding in DNA. Hydrogen bonds are challenging to quantify by X-ray crystallography and cryo-EM due to difficulty of visualizing hydrogen atom locations but can be probed with site specificity by NMR spectroscopy in solution and the solid state with the latter particularly suited to large, slowly tumbling DNA complexes. Recently, we showed that low-temperature dynamic nuclear polarization (DNP) enhanced solid-state NMR is a valuable tool for distinguishing Hoogsteen base pairs (bps) from canonical Watson-Crick bps in various DNA systems under native-like conditions. Here, using a model 12-mer DNA duplex containing two central adenine-thymine (A-T) bps in either Watson-Crick or Hoogsteen confirmation, we demonstrate DNP solid-state NMR measurements of thymine N3-H3 bond lengths, which are sensitive to details of N-H···N hydrogen bonding and permit hydrogen bonds for the two bp conformers to be systematically compared within the same DNA sequence context. For this DNA duplex, effectively identical TN3-H3 bond lengths of 1.055 ± 0.011 Å and 1.060 ± 0.011 Å were found for Watson-Crick A-T and Hoogsteen A (syn)-T base pairs, respectively, relative to a reference amide bond length of 1.015 ± 0.010 Å determined for N-acetyl-valine under comparable experimental conditions. Considering that prior quantum chemical calculations which account for zero-point motions predict a somewhat longer effective peptide N-H bond length of 1.041 Å, in agreement with solution and solid-state NMR studies of peptides and proteins at ambient temperature, to facilitate direct comparisons with these earlier studies TN3-H3 bond lengths for the DNA samples can be readily scaled appropriately to yield 1.083 Å and 1.087 Å for Watson-Crick A-T and Hoogsteen A (syn)-T bps, respectively, relative to the 1.041 Å reference peptide N-H bond length. Remarkably, in the context of the model DNA duplex, these results indicate that there are no significant differences in N-H···N A-T hydrogen bonds between Watson-Crick and Hoogsteen bp conformers. More generally, high precision measurements of N-H bond lengths by low-temperature DNP solid-state NMR based methods are expected to facilitate detailed comparative analysis of hydrogen bonding for a range of DNA complexes and base pairing environments.

Treating black patients as "knowers".

Recent trends in healthcare policy from high-volume service models to "high-value" delivery systems have refocused the need for patient-centered approaches to quality care. However, benchmarks of how to define and evaluate successful patient-centeredness have not been sufficiently established. Such ill-defined evaluation criteria can further exacerbate systemic inequities in maximum quality health care delivery, especially based on the intersectional diversity of various patient populations. In this context, applying a phenomenology of medicine framework or perspective-driven analysis is useful in defining cross-cultural patient-centeredness. This reframing from a naturalistic or objective/biological viewpoint to a phenomenological viewpoint may aid in placing greater epistemic or knowledge authority in the hands of vulnerable and/or marginalized patients- allowing these patients to become key "knowers" in the clinical interaction. Moreover, treating Black patients as "knowers" emphasizes the prioritization of patient values at the core of providing valuable healthcare. Such an academic, policy, and clinical approach to medicine agrees with well-established principles of medical ethics. In addition, the framework of a phenomenology of medicine can better facilitate physician-patient communication and interaction by delineating often muddled hermeneutics.

Now you see me, now you don't: New approaches to sparing nonmalignant cells from CAR T cell therapies.

While new immunotherapies have revolutionized the field of oncology, they have been limited by their inability to distinguish between cancerous cells and healthy HSPCs. Work by Casirati et al.1 and Wellhausen et al.2 in epitope editing antigens commonly expressed on AML and HSPCs has unlocked several new targets for immunotherapies.

Sepsis as the Grand Mimic of Secondary Hemophagocytic Lymphohistiocytosis: Serratia marcescens Bacteremia with Concomitant Decompensated Cirrhotic Liver Disease.

Secondary hemophagocytic lymphohistiocytosis (HLH) is an elusive entity with sequelae that may be confused with sepsis. We discuss a 45-year-old man with decompensated liver cirrhosis with sepsis treated with broad-spectrum intravenous antibiotics. Further work-up initially supported sepsis-HLH overlap syndrome (SHLHOS) and corticosteroids were added. Ongoing refractory hypotension ensued, and the patient passed within 31 hours of presentation. Based on the patient's overwhelming immune activation and clinical course likely unsalvageable by cytotoxic immunosuppressive agents, the patient was diagnosed with sepsis with acute end organ dysfunction. This case report illustrates both the diagnostic challenge of sepsis versus HLH, which both require very different treatments, and the potential for rapid clinical decline without swift recognition and management of the true pathology.

Nanotopography by chromatic confocal microscopy of the endothelium in Fuchs endothelial corneal dystrophy, pseudophakic bullous keratopathy and healthy corneas.

AIM: To investigate the interest of chromatic confocal microscopy (CCM) to characterise guttae in Fuchs endothelial corneal dystrophy (FECD). METHODS: Descemet's membranes (DM) were obtained during endothelial keratoplasty in patients with FECD and pseudophakic bullous keratopathy (PBK). They were compared with healthy samples obtained from body donation to science. Samples were fixed in 0.5% paraformaldehyde and flat mounted. Surface roughness of DMs was quantified using CCM and the AltiMap software that provided the maximum peak (Sp) and valley (Sv) heights, the mean square roughness (Rq) and the asymmetry coefficient (Ssk). RESULTS: The physiological roughness of healthy samples was characterised by an Rq of 0.12±0.05 µm, which was two times rougher than in PBK (Rq=0.06±0.03 µm), but both were still flat with a symmetrical distribution between peaks and valleys (Ssk close to 0, npeaks=nvalleys), smaller than 1 µm. In FECD, the maximum peak height was 5.10±2.40 µm, up to 5.8 and 8.3 times higher than the control and PBK, respectively. The maximum valley depth was half than the peak (2.28±0.89 µm). The surface with guttae was very rough (Rq=0.45±0.14 µm) and the Ssk=1.84± 0.43 µm, greater than 0, confirms an asymmetric surface with high peaks and low valleys (npeaks>nvalleys). Moreover, the CCM provided quantitative parameters allowing to distinguish different types of guttae from different patients. CONCLUSIONS: CCM is an innovative approach to describe and quantify different morphologies of guttae. It could be useful to analyse the different stages of FECD and define subgroups of patients.

Modelling the impact of forest management and CO2-fertilisation on growth and demography in a Sitka spruce plantation.

Afforestation and reforestation to meet 'Net Zero' emissions targets are considered a necessary policy by many countries. Their potential benefits are usually assessed through forest carbon and growth models. The implementation of vegetation demography gives scope to represent forest management and other size-dependent processes within land surface models (LSMs). In this paper, we evaluate the impact of including management within an LSM that represents demography, using both in-situ and reanalysis climate drivers at a mature, upland Sitka spruce plantation in Northumberland, UK. We compare historical simulations with fixed and variable CO2 concentrations, and with and without tree thinning implemented. Simulations are evaluated against the observed vegetation structure and carbon fluxes. Including thinning and the impact of increasing CO2 concentration ('CO2 fertilisation') gave more realistic estimates of stand-structure and physical characteristics. Historical CO2 fertilisation had a noticeable effect on the Gross Primary Productivity seasonal-diurnal cycle and contributed to approximately 7% higher stand biomass by 2018. The net effect of both processes resulted in a decrease of tree density and biomass, but an increase in tree height and leaf area index.

CD90-targeted lentiviral vectors for HSC gene therapy.

Hematopoietic stem cell (HSC) gene therapy is currently performed on CD34+ hematopoietic stem and progenitor cells containing less than 1% true HSCs and requiring a highly specialized infrastructure for cell manufacturing and transplantation. We have previously identified the CD34+CD90+ subset to be exclusively responsible for short- and long-term engraftment. However, purification and enrichment of this subset is laborious and expensive. HSC-specific delivery agents for the direct modification of rare HSCs are currently lacking. Here, we developed novel targeted viral vectors to specifically transduce CD90-expressing HSCs. Anti-CD90 single chain variable fragments (scFvs) were engineered onto measles- and VSV-G-pseudotyped lentiviral vectors that were knocked out for native targeting. We further developed a custom hydrodynamic titration methodology to assess the loading of surface-engineered capsids, measure antigen recognition of the scFv, and predict the performance on cells. Engineered vectors formed with minimal impairment in the functional titer, maintained their ability to fuse with the target cells, and showed highly specific recognition of CD90 on cells ex vivo. Most important, targeted vectors selectively transduced human HSCs with secondary colony-forming potential. Our novel HSC-targeted viral vectors have the potential to significantly enhance the feasibility of ex vivo gene therapy and pave the way for future in vivo applications.

Investigating the Role of TGF-ß Signaling Pathways in Human Corneal Endothelial Cell Primary Culture.

Corneal endothelial diseases are the leading cause of corneal transplantation. The global shortage of donor corneas has resulted in the investigation of alternative methods, such as cell therapy and tissue-engineered endothelial keratoplasty (TEEK), using primary cultures of human corneal endothelial cells (hCECs). The main challenge is optimizing the hCEC culture process to increase the endothelial cell density (ECD) and overall yield while preventing endothelial-mesenchymal transition (EndMT). Fetal bovine serum (FBS) is necessary for hCEC expansion but contains TGF-ßs, which have been shown to be detrimental to hCECs. Therefore, we investigated various TGF-ß signaling pathways using inhibitors to improve hCEC culture. Initially, we confirmed that TGF-ß1, 2, and 3 induced EndMT on confluent hCECs without FBS. Using this TGF-ß-induced EndMT model, we validated NCAM as a reliable biomarker to assess EndMT. We then demonstrated that, in a culture medium containing 8% FBS for hCEC expansion, TGF-ß1 and 3, but not 2, significantly reduced the ECD and caused EndMT. TGF-ß receptor inhibition had an anti-EndMT effect. Inhibition of the ROCK pathway, notably that of the P38 MAPK pathway, increased the ECD, while inhibition of the ERK pathway decreased the ECD. In conclusion, the presence of TGF-ß1 and 3 in 8% FBS leads to a reduction in ECD and induces EndMT. The use of SB431542 or LY2109761 may prevent EndMT, while Y27632 or Ripasudil, and SB203580 or SB202190, can increase the ECD.