Validation of a Caco-2 microfluidic Chip model for predicting intestinal absorption of BCS Class I-IV drugs.

Oral delivery is considered the most patient preferred route of drug administration, however, the drug must be sufficiently soluble and permeable to successfully formulate an oral formulation. There have been advancements in the development of more predictive solubility and dissolution tools, but the tools that has been developed for permeability assays have not been validated as extensively as the gold-standard Caco-2 Transwell assay. Here, we evaluated Caco-2 intestinal permeability assay in Transwells and a commercially available microfluidic Chip using 19 representative Biopharmaceutics Classification System (BCS) Class I-IV compounds. For each selected compound, we performed a comprehensive viability test, quantified its apparent permeability (Papp), and established an in vitro in vivo correlation (IVIVC) to the human fraction absorbed (fa) in both culture conditions. Permeability differences were observed across the models as demonstrated by antipyrine (Transwell Papp: 38.5 ± 6.1 × 10-8 cm/s vs Chip Papp: 32.9 ± 11.3 × 10-8 cm/s) and nadolol (Transwell Papp: 0.6 ± 0.1 × 10-7 cm/s vs Chip Papp: 3 ± 1.2 × 10-7 cm/s). The in vitro in vivo correlation (IVIVC; Papp vs. fa) of the Transwell model (r2 = 0.59-0.83) was similar to the Chip model (r2 = 0.41-0.79), highlighting similar levels of predictivity. Comparing to historical data, our Chip Papp data was more closely aligned to native tissues assessed in Ussing chambers. This is the first study to comprehensively validate a commercial Gut-on-a-Chip model as a predictive tool for assessing oral absorption to further reduce our reliance on animal models.

Zebrafish raptor mutation inhibits the activity of mTORC1, inducing craniofacial defects due to autophagy-induced neural crest cell death.

The mechanistic target of rapamycin (mTOR) coordinates metabolism and cell growth with environmental inputs. mTOR forms two functional complexes: mTORC1 and mTORC2. Proper development requires both complexes but mTORC1 has unique roles in numerous cellular processes, including cell growth, survival and autophagy. Here, we investigate the function of mTORC1 in craniofacial development. We created a zebrafish raptor mutant via CRISPR/Cas9, to specifically disrupt mTORC1. The entire craniofacial skeleton and eyes were reduced in size in mutants; however, overall body length and developmental timing were not affected. The craniofacial phenotype associates with decreased chondrocyte size and increased neural crest cell death. We found that autophagy is elevated in raptor mutants. Chemical inhibition of autophagy reduced cell death and improved craniofacial phenotypes in raptor mutants. Genetic inhibition of autophagy, via mutation of the autophagy gene atg7, improved facial phenotypes in atg7;raptor double mutants, relative to raptor single mutants. We conclude that finely regulated levels of autophagy, via mTORC1, are crucial for craniofacial development.

Adding an extension piece to the end of the purge side arm of the Impella device can prevent the incidence of the cassette breaking and decrease the Impella device failure rate: Impact of practice change on patient outcome.

Background: Impella 5.5® with Smart Assist is a minimally invasive Left Ventricular Assist Devices (LVAD) approved by the Food and Drug Administration (FDA) for treating ongoing cardiogenic shock for up to 14 days. The Impella® intends to reduce ventricular workload and provide the circulatory support necessary for myocardial recovery.Research Question: Compared to standard practice, does adding an extension piece to the purge tube side arm of the Impella® Device decrease the incidence of device failure and positively impact the health outcome of adult patients receiving Impella® support?Study Design and Methods: A retrospective chart review of ICU patients was done at a tertiary care center from August 2018 to August 2022 to assess the differences in patient outcomes related to Impella® Device utilization before and after the implementation of the extension piece to the purge tube sidearm. Among patients reviewed, a total of 20 were included in our review, with seven not having the purge tube side arm extension added, while 13 patients had the extension.Results: The two study groups had no significant difference in patient health outcomes. Additionally, there were no instances of device failure requiring explanation without the extension tubing. However, there were no cases of the purge cassette cracking with the addition of the extension tubing.Conclusion: The addition of extension tubing to the purge cassette of the Impella® Device did not impact patient health outcomes or the incidence of device failure. There was a complete reduction in the incidence of the purge cassette cracking, which could reduce the potential for infection or device failure over a long period of mechanical support. There is a need for long-term prospective studies to confirm the results.

Central corneal distortion after small-incision lenticule extraction.

A 33-year-old woman with a prior history of small-incision lenticule extraction (SMILE) presented with gradual deterioration of vision in her right eye since the surgery. She had undergone bilateral SMILE for myopic correction (-7.00 diopters [D] in the right eye and -6.00 D in the left eye) 3 weeks prior. SMILE was performed with a Zeiss VisuMax femtosecond laser system, with a cap thickness of 120 µm, a cap diameter of 7.50 mm, and a lenticule diameter of 6.50 mm. At the completion of the surgery, loose epithelium was noted at the SMILE incision bilaterally, and bandage contact lenses were placed in both eyes. On the first postoperative day, the patient's uncorrected distance visual acuity (UDVA) was 20/40 in both eyes. The bandage lenses were removed from both eyes, with the epithelium intact. At the first-week postoperative visit, her visual acuity was recorded as 20/30 in the right eye and 20/20 in the left eye. She noticed her vision in the right eye was not as sharp as that in her left eye. She denied experiencing any pain, redness, or ocular surface irritations. She was advised to return to the clinic for a 1-month postoperative visit and continue with aggressive lubrication in both eyes. However, a week later, the patient returned for an emergency visit, citing significant central visual distortion in the right eye and difficulty working on the computer. At this visit, her UDVA and corrected distance visual acuity (CDVA) was 20/50 in the right eye and 20/15 in the left eye at both near and far distances. A slitlamp examination revealed mild central changes in the right eye. She once again denied any pain, redness, or irritation. She was advised to continue with artificial tears and return to the office in 1 week for further observation of the central distortion in her right eye. Upon returning to the clinic at the third postoperative week, the patient still complained of central visual changes in the right eye, with a visual acuity of 20/70. Further slitlamp examination revealed a nonspecific central haze in the same eye, but there was no corneal staining or signs of epithelial defects. Anterior segment ocular coherence tomography (AS-OCT) and NIDEK topography were performed, showing the same central distortion in the right eye (Figures 1 and 2JOURNAL/jcrs/04.03/02158034-202311000-00016/figure1/v/2023-10-18T004638Z/r/image-tiffJOURNAL/jcrs/04.03/02158034-202311000-00016/figure2/v/2023-10-18T004638Z/r/image-tiff). Based on the examination and images provided, what is your working medical diagnosis? What other medical conditions are in your differential diagnosis? What medical and/or surgical interventions would you recommend, if any?

Incidence and Management of Epithelial-Related Complications After SMILE.

Purpose: To investigate the incidence and management of only epithelial-related complications following small incision lenticule extraction (SMILE). Patients and Methods: A retrospective, single-site study analyzed patients who underwent SMILE at Hoopes Vision Clinic in Draper, Utah, from June 2017 to February 2023. Demographic data and preoperative parameters were reviewed. Postoperatively, patients were assessed for visual acuity and complications at different time points. Statistical analyses were conducted between the control and complication groups. Results: Four hundred and thirty-two eyes of 220 patients received SMILE. Postoperative epithelial-related complications were indicated in 68 (15.7%) eyes, including anterior basement membrane (ABM) changes (five [1.2%]) eyes), epithelial ingrowth (nine [2.1%] eyes), erosion (two [0.5%] eyes), rough epithelium (18 [4.2%] eyes), epithelial defect (12 [2.8%] eyes), diffuse lamellar keratitis (DLK) secondary to epitheliopathy (two [0.5%] eyes), microstriae secondary to epitheliopathy (four [0.9%] eyes), interface debris (21 [4.9%] eyes), and incisional fibrosis (one [0.2%] eye). There was a statistically significant difference in age, with older patients more likely to develop epitheliopathy postoperatively (P = 0.001). Additionally, patients with epithelial-related complications were more likely to receive photorefractive keratectomy (PRK) enhancement after SMILE than the control (P = 0.001). However, there was no statistical difference in uncorrected distance visual acuity (UDVA) better than 20/20 and corrected distance visual acuity (CDVA) between the complications group and the control at the last postoperative visit (P = 0.974 and 0.310, respectively). There was no statistically significant difference in the safety and efficacy indices between the complications and control group (P = 0.281 and 0.617, respectively). Conclusion: In our study, epithelial-related complications were more prevalent in older patients and predisposed patients to require PRK enhancements after recovery from SMILE. Despite the incidence of epithelial-related complications, visual prognoses were favorable and achieved through various management strategies.

Ultrasensitive Detection of Circulating LINE-1 ORF1p as a Specific Multicancer Biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. Although proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible expression in normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 mol/L) ORF1p concentrations in plasma across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multianalyte panel, provides early therapeutic response monitoring in gastroesophageal cancers, and is prognostic for overall survival in gastroesophageal and colorectal cancers. Together, these observations nominate ORF1p as a multicancer biomarker with potential utility for disease detection and monitoring. SIGNIFICANCE: The LINE-1 ORF1p transposon protein is pervasively expressed in many cancers and is a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 µL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring. See related commentary by Doucet and Cristofari, p. 2502. This article is featured in Selected Articles from This Issue, p. 2489.

Molecular Crowding Facilitates Ribozyme-Catalyzed RNA Assembly.

Catalytic RNAs or ribozymes are considered to be central to primordial biology. Most ribozymes require moderate to high concentrations of divalent cations such as Mg2+ to fold into their catalytically competent structures and perform catalysis. However, undesirable effects of Mg2+ such as hydrolysis of reactive RNA building blocks and degradation of RNA structures are likely to undermine its beneficial roles in ribozyme catalysis. Further, prebiotic cell-like compartments bounded by fatty acid membranes are destabilized in the presence of Mg2+, making ribozyme function inside prebiotically relevant protocells a significant challenge. Therefore, we sought to identify conditions that would enable ribozymes to retain activity at low concentrations of Mg2+. Inspired by the ability of ribozymes to function inside crowded cellular environments with <1 mM free Mg2+, we tested molecular crowding as a potential mechanism to lower the Mg2+ concentration required for ribozyme-catalyzed RNA assembly. Here, we show that the ribozyme-catalyzed ligation of phosphorimidazolide RNA substrates is significantly enhanced in the presence of the artificial crowding agent polyethylene glycol. We also found that molecular crowding preserves ligase activity under denaturing conditions such as alkaline pH and the presence of urea. Additionally, we show that crowding-induced stimulation of RNA-catalyzed RNA assembly is not limited to phosphorimidazolide ligation but extends to the RNA-catalyzed polymerization of nucleoside triphosphates. RNA-catalyzed RNA ligation is also stimulated by the presence of prebiotically relevant small molecules such as ethylene glycol, ribose, and amino acids, consistent with a role for molecular crowding in primordial ribozyme function and more generally in the emergence of RNA-based cellular life.

Anti-fibrotic strategies and pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) results from the dysregulated process of injury and repair, which promotes scarring of the lung tissue and deposition of collagen-rich extracellular matrix (ECM) components, that make the lung unphysiologically stiff. IPF presents a serious concern as its pathogenesis remains elusive, and current anti-fibrotic treatments are only effective in slowing rather than halting disease progression. The IPF disease pathogenesis is incompletely defined, complex and incorporates interplay between different fibrogenesis signaling pathways. Preclinical IPF experimental models used to validate drug candidates present significant limitations in modeling IPF pathobiology, with their limited time frame, simplicity and inaccurate representation of the disease and the mechanical influences of IPF. Potentially more accurate mimetic disease models that capture the cell-cell and cell-matrix interaction, such as 3D cultures, organoids and precision-cut lung slices (PCLS), may yield more meaningful clinical predictions for drug candidates. Recent advances in developing anti-fibrotic compounds have positioned drug towards targeting components of the fibrogenesis signaling pathway of IPF or the extracellular microenvironment. The major goals in this area of research focus on finding ways to reverse or halt the disease progression by utilizing more disease-relevant experimental models to improve the qualification of potential drug targets for treating pulmonary fibrosis.

Passive endocytosis in model protocells.

Semipermeable membranes are a key feature of all living organisms. While specialized membrane transporters in cells can import otherwise impermeable nutrients, the earliest cells would have lacked a mechanism to import nutrients rapidly under nutrient-rich circumstances. Using both experiments and simulations, we find that a process akin to passive endocytosis can be recreated in model primitive cells. Molecules that are too impermeable to be absorbed can be taken up in a matter of seconds in an endocytic vesicle. The internalized cargo can then be slowly released over hours, into the main lumen or putative cytoplasm. This work demonstrates a way by which primitive life could have broken the symmetry of passive permeation prior to the evolution of protein transporters.

RNA-Catalyzed RNA Ligation within Prebiotically Plausible Model Protocells.

Demonstrating RNA catalysis within prebiotically relevant models of primordial cells (protocells) remains a challenge in origins of life research. Fatty acid vesicles encapsulating genomic and catalytic RNAs (ribozymes) are attractive models for protocells; however, RNA catalysis has largely been incompatible with fatty acid vesicles due to their instability in the presence of Mg2+ at the concentrations required for ribozyme function. Here, we report a ribozyme that catalyzes template-directed RNA ligation at low Mg2+ concentrations and thus remains active within stable vesicles. Ribose and adenine, both prebiotically relevant molecules, were found to greatly reduce Mg2+ -induced RNA leakage from vesicles. When we co-encapsulated the ribozyme, substrate, and template within fatty acid vesicles, we observed efficient RNA-catalyzed RNA ligation upon subsequent addition of Mg2+ . Our work shows that RNA-catalyzed RNA assembly can occur efficiently within prebiotically plausible fatty acid vesicles and represents a step toward the replication of primordial genomes within self-replicating protocells.

Passive endocytosis in model protocells.

Semipermeable membranes are a key feature of all living organisms. While specialized membrane transporters in cells can import otherwise impermeable nutrients, the earliest cells would have lacked a mechanism to import nutrients rapidly under nutrient-rich circumstances. Using both experiments and simulations, we find that a process akin to passive endocytosis can be recreated in model primitive cells. Molecules that are too impermeable to be absorbed can be taken up in a matter of seconds in an endocytic vesicle. The internalized cargo can then be slowly released over hours, into the main lumen or putative cytoplasm. This work demonstrates a way by which primitive life could have broken the symmetry of passive permeation prior to the evolution of protein transporters.

Enhanced nonenzymatic RNA copying with in-situ activation of short oligonucleotides.

The nonenzymatic copying of RNA is thought to have been necessary for the transition between prebiotic chemistry and ribozyme-catalyzed RNA replication in the RNA World. We have previously shown that a potentially prebiotic nucleotide activation pathway based on phospho-Passerini chemistry can lead to the efficient synthesis of 2-aminoimidazole activated mononucleotides when carried out under freeze-thaw cycling conditions. Such activated nucleotides react with each other to form 5'-5' 2-aminoimidazolium bridged dinucleotides, enabling template-directed primer extension to occur within the same reaction mixture. However, mononucleotides linked to oligonucleotides by a 5'-5' 2-aminoimidazolium bridge are superior substrates for nonenzymatic primer extension; their higher intrinsic reactivity and their higher template affinity enable faster template copying at lower substrate concentrations. Here we show that eutectic phase phospho-Passerini chemistry efficiently activates short oligonucleotides and promotes the formation of monomer-bridged-oligonucleotide species during freeze-thaw cycles. We then demonstrate that in-situ generated monomer-bridged-oligonucleotides lead to efficient nonenzymatic template copying in the same reaction mixture. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life.

The absence of a prebiotically plausible pathway for the efficient nonenzymatic copying of RNAs remains a major obstacle towards constructing self-replicating protocells that emulate early lifeforms. We demonstrate the activation of short oligonucleotides and the subsequent formation of monomer-bridged-oligonucleotide species, leading to efficient nonenzymatic template copying in the same reaction mixture. Our findings suggest that in-situ activated mixtures of mono- and oligo-nucleotides would significantly outperform mononucleotides in driving the copying of arbitrary RNA sequences. Our demonstration that multiple steps in the pathway from activation chemistry to RNA copying can occur together in a single complex environment simplifies this aspect of the origin of life.

Ultrasensitive detection of circulating LINE-1 ORF1p as a specific multi-cancer biomarker.

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17 M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.

A glomerulus and proximal tubule microphysiological system simulating renal filtration, reabsorption, secretion, and toxicity.

Microphysiological systems (MPS) are powerful predictive tools for assessing drug-induced kidney injuries. Previous MPS have examined single regions of the nephron, but lack simultaneous filtration, reabsorption, and secretion functionality. Here, we developed a partially open MPS that structurally and functionally recapitulated the glomerular filtration barrier, proximal tubular reabsorption, and secretion for seven days. The system introduced a recirculation circuit and an open filtrate output as a source of functional testing. As a proof-of-concept, a tri-culture of immortalized podocytes, umbilical vein endothelial cells, and proximal tubule (PCT) cells were housed in a single MPS: T-junction, glomerulus housing unit, and PCT chip. The MPS successfully retained blood serum protein, reabsorbed glucose, secreted creatinine, and expressed cell-type specific proteins (VE-cadherin, nephrin, and ZO-1). To simulate drug-induced kidney injuries, the system was perfused with cisplatin and adriamycin, and then tested using serum albumin filtration, glucose clearance, and lactate dehydrogenase release. The glomerulus and PCT MPS demonstrated a complex, dynamic microenvironment and recreated some in vivo-like functions in basal and drug-induced conditions, offering a novel prototype for preclinical testing.

The first reptilian allergen and major allergen for fish-allergic patients: Crocodile ß-parvalbumin.

BACKGROUND: Clinical cross-reactivity between bony fish, cartilaginous fish, frog, and chicken muscle has previously been demonstrated in fish-allergic patients. In indicative studies, two reports of anaphylaxis following the consumption of crocodile meat and IgE-cross-binding were linked to the major fish allergen parvalbumin (PV). This study investigates IgE-binding proteins in crocodile meat with a focus on PV and their clinical relevance. METHODS: Proteins were extracted from muscle tissue of crocodile, three bony fish, and two cartilaginous fish. A cohort of fish-allergic pediatric patients (n = 77) underwent allergen skin prick testing (SPT) to three fish preparations (n = 77) and crocodile (n = 12). IgE-binding proteins were identified and quantified by SDS-PAGE, mass spectrometric analyses, and immunoblotting using commercial and in-house antibodies, as well as individual and pooled patients' serum. PV isoforms were purified or recombinantly expressed before immunological analyses, including human mast cell degranulation assay. RESULTS: Of the tissues analyzed, PV was most abundant in heated crocodile preparation, triggering an SPT of ≥3 mm in 8 of 12 (67%) fish-allergic patients. Seventy percent (31 of 44) of fish PV-sensitized patients demonstrated IgE-binding to crocodile PV. Crocodile ß-PV was the major IgE-binding protein but 20-fold less abundant than α-PV. Cellular reactivity was demonstrated for ß-PV and epitopes predicted, explaining frequent IgE-cross-binding of ß-PVs. Both PV isoforms are now registered as the first reptile allergens with the WHO/IUIS (ß-PV as Cro p 1 and α-PV as Cro p 2). CONCLUSION: Fish-allergic individuals may be at risk of an allergy to crocodile and should seek specialist advice before consuming crocodilian meat.

A Systematic Review on the Effect of Common Medications on Platelet Count and Function: Which Medications Should Be Stopped Before Getting a Platelet-Rich Plasma Injection?

Background: Platelet-rich plasma (PRP) exerts its effect through the release of growth factors and cytokines from the platelet concentrate. Certain medications may affect platelet count or function, resulting in decreased efficacy of PRP injections. Purpose: To systematically review the literature regarding common medications and their effects on platelets to establish guidelines for which medications should be stopped before obtaining a PRP injection. Study Design: Systematic review; Level of evidence, 2. Methods: This review was performed in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. A search for studies assessing the effect of common medications on platelet count or platelet function was performed of the PubMed, Cochrane Library, Web of Science, and OpenGrey databases. Inclusion criteria were as follows: drug studied was aspirin, acetaminophen, a nonsteroidal anti-inflammatory drug (NSAID), a statin, or gabapentin; human participants; and article in the English language. Risk of bias was assessed using the Cochrane Risk of Bias tool and the Risk of Bias in Non-randomised Studies-of Interventions tool. Results: A total of 1711 studies were identified through the initial search, with 20 studies meeting all inclusion criteria. No studies involving gabapentin met all inclusion criteria. Patients treated with aspirin (268 patients) or acetaminophen (13 patients) showed a significant decrease in platelet aggregation. Statin therapy (73 patients) did not result in a significant decrease in platelet aggregation. Patients who took NSAIDs (172 patients) demonstrated significantly decreased platelet aggregation only when treated with nonselective formulations. Those treated with cyclooxygenase (COX)-2-selective NSAIDs showed no significant difference in platelet aggregation. Treatment with aspirin, acetaminophen, statins, or NSAIDs did not lead to a significant decrease in platelet count. Conclusion: Aspirin, acetaminophen, and nonselective NSAIDs should be considered for suspension before a PRP injection because of their potential to diminish the effects of the injection. COX-2-selective NSAIDs and statins do not need to be withheld before a PRP injection.

Freeze-thaw cycles enable a prebiotically plausible and continuous pathway from nucleotide activation to nonenzymatic RNA copying.

Nonenzymatic template-directed RNA copying using chemically activated nucleotides is thought to have played a key role in the emergence of genetic information on the early Earth. A longstanding question concerns the number and nature of different environments that might have been necessary to enable all of the steps from nucleotide synthesis to RNA copying. Here we explore three sequential steps from this overall pathway: nucleotide activation, synthesis of imidazolium-bridged dinucleotides, and template-directed RNA copying. We find that all three steps can take place in one reaction mixture undergoing multiple freeze-thaw cycles. Recent experiments have demonstrated a potentially prebiotic methyl isocyanide-based nucleotide activation chemistry. However, the original version of this approach is incompatible with nonenzymatic RNA copying because the high required concentration of the imidazole activating group prevents the accumulation of the essential imidazolium-bridged dinucleotide. Here we report that ice eutectic phase conditions facilitate not only the methyl isocyanide-based activation of ribonucleotide 5'-monophosphates with stoichiometric 2-aminoimidazole, but also the subsequent conversion of these activated mononucleotides into imidazolium-bridged dinucleotides. Furthermore, this one-pot approach is compatible with template-directed RNA copying in the same reaction mixture. Our results suggest that the simple and common environmental fluctuation of freeze-thaw cycles could have played an important role in prebiotic nucleotide activation and nonenzymatic RNA copying.