Update on "Cell Therapy" Clinics Offering Treatments of Ocular Conditions Using Direct-To-Consumer Marketing Websites in the U.S.

PURPOSE: To assess the scope of U.S.-based companies advertising and administering non-Federal Drug Administration (FDA) approved cell-based therapy (herein called NFACT) for ocular conditions based on information from companies' public websites after the FDA's legal actions against specific NFACT clinics in 2018 and 2019. Current findings are compared to previously published data from 2017. DESIGN: Trend study looking at U.S.-based companies that use direct-to-consumer marketing and have websites advertising therapy for ocular conditions. METHODS: A systematic and extensive keyword-based Internet search was utilized to identify, document, and analyze U.S. business websites offering NFACT for ocular conditions as of August 2022. Main outcomes measured include, clinic locations, marketed ocular conditions, types of NFACT offered, source of stem cells used, routes of administration, and treatment costs. RESULTS: From the prior analysis in 2017 to the 2019 analysis, there was a decrease in the number of NFACT clinics from 76 to 62 and companies from 40 to 39. Given the concerning persistence of NFACTs in August 2019 an additional analysis was performed in 2022 which showed a drastic decrease in NFACT clinics from 62 in 2019 to 18 in 2023 and from 39 companies to 13 in 2023. In both 2019 and 2022, the most commonly referenced ocular condition was age-related macular degeneration (2019 - 72%, 2022 - 92%). The state with the most clinics was in Texas (2019 - 12; 2022 - 5). Autologous adipose-derived stem cells were the most common cell type used in both analyses. CONCLUSIONS: In 2019 U.S.-based direct-to-consumer companies marketing NFACT persisted despite (1) a lack of high-quality clinical evidence supporting the efficacy of these procedures, (2) the association of some of these treatments with severe vision loss, and (3) increasing FDA oversight and recent legal action. In 2022 the number of clinics and companies decreased, but their persistence is a reminder that continued concern is necessary and ophthalmic associations need to continue advocacy efforts to protect patients from these potentially predatory organizations.

Influence of global warming and industrialization on coral reefs: A 600-year record of elemental changes in the Eastern Red Sea.

The Red Sea has been recognized as a coral reef refugia, but it is vulnerable to warming and pollution. Here we investigated the spatial and temporal trends of 15 element concentrations in 9 coral reef sediment cores (aged from the 1460s to the 1980s AD) to study the influence of global warming and industrialization on the Eastern Red Sea coral reefs. We found Na, Ca, Cr, Fe, Co, Ni, and Sr concentrations were higher in the northern Red Sea (i.e., Yanbu), whereas Mg, P, S, Mn, and Cd concentrations were higher in the southern Red Sea (i.e., Thuwal & Al Lith) reef sediments. In the central (i.e., Thuwal) to southern (i.e., Al Lith) Red Sea, the study revealed diverse temporal trends in element concentrations. However, both reef sedimentation rates (-36.4 % and -80.5 %, respectively) and elemental accumulation rates (-49.4 % for Cd to -12.2 % for Zn in Thuwal, and -86.2 % for Co to -61.4 % for Cu in Al Lith) exhibited a declining pattern over time, possibly attributed to warming-induced thermal bleaching. In the central to northern Red Sea (i.e., Yanbu), the severity of thermal bleaching is low, while the reef sedimentation rates (187 %), element concentrations (6.7 % for S to 764 % for Co; except Na, Mg, Ca, Sr, and Cd), and all elemental accumulation rates (190 % for Mg to 2697 % for Co) exponentially increased from the 1970s, probably due the rapid industrialization in Yanbu. Our study also observed increased trace metal concentrations (e.g., Cu, Zn, and Ni) in the Thuwal and Al Lith coral reefs with severe bleaching histories, consistent with previous reports that trace metals might result in decreased resistance of corals to thermal stress under warming scenarios. Our study points to the urgent need to reduce the local discharge of trace metal pollutants to protect this biodiversity hotspot.

McCannel Suture Technique Resolves Persistent Dysphotopsia Following Laser Peripheral Iridotomy in Phakic Eyes.

A 57-year-old woman presented with photophobia and complaint of a persistent white light in the inferior field of her left eye for 18 months following laser peripheral iridotomy both eyes. In primary gaze, the upper lid margin was noted to bisect the iridotomy at 2 o'clock left eye (OS). She relates that the light moves with her eyelid OS only and resolves by lifting the lid. A 54-year-old male presented with complaint of seeing 2 horizontal lines in his field of vision immediately following laser iridotomy OS that have persisted for a duration of 7 years. He notes they are constant and resolve by lifting up the eyelid. In primary position, the left upper lid margin was noted to bisect the iridotomy at 12 o'clock OS. Given the presence of nonresolving symptomatic dysphotopsia, each patient underwent surgical repair of the iridotomy using a McCannel suture technique with complete resolution of their symptoms. Dysphotopsias are an uncommon complication that can occur following laser peripheral iridotomy. If they persist and conservative measures are ineffective, iris suture repair can provide a definitive intervention in resolving them. Laser iridotomies located in the far periphery pose a surgical challenge with respect to accessibility. A McCannel suture technique provides a feasible approach in suturing them with minimal iris manipulation. Furthermore, it is prudent to assess the upper lid position in primary gaze before creating an iridotomy in order to avoid interference with the lid margin tear film meniscus that can lead to the formation of symptomatic dysphotopsia.

Corneal neovascularization.

The optical clarity of the cornea is essential for maintaining good visual acuity. Corneal neovascularization, which is a major cause of vision loss worldwide, leads to corneal opacification and often contributes to a cycle of chronic inflammation. While numerous factors prevent angiogenesis within the cornea, infection, inflammation, hypoxia, trauma, corneal degeneration, and corneal transplantation can all disrupt these homeostatic safeguards to promote neovascularization. Here, we summarize its etiopathogenesis and discuss the molecular biology of angiogenesis within the cornea. We then review the clinical assessment and diagnostic evaluation of corneal neovascularization. Finally, we describe current and emerging therapies.

Force production of human cytoplasmic dynein is limited by its processivity.

Cytoplasmic dynein is a highly complex motor protein that generates forces toward the minus end of microtubules. Using optical tweezers, we demonstrate that the low processivity (ability to take multiple steps before dissociating) of human dynein limits its force generation due to premature microtubule dissociation. Using a high trap stiffness whereby the motor achieves greater force per step, we reveal that the motor's true maximal force ("stall force") is ~2 pN. Furthermore, an average force versus trap stiffness plot yields a hyperbolic curve that plateaus at the stall force. We derive an analytical equation that accurately describes this curve, predicting both stall force and zero-load processivity. This theoretical model describes the behavior of a kinesin motor under low-processivity conditions. Our work clarifies the true stall force and processivity of human dynein and provides a new paradigm for understanding and analyzing molecular motor force generation for weakly processive motors.

Adaptation to temperature stress by Vibrio fischeri facilitates this microbe's symbiosis with the Hawaiian bobtail squid (Euprymna scolopes).

For microorganisms cycling between free-living and host-associated stages, where reproduction occurs in both of these lifestyles, an interesting inquiry is whether adaptation to stress during the free-living stage can impact microbial fitness in the host. To address this topic, the mutualism between the Hawaiian bobtail squid (Euprymna scolopes) and the marine bioluminescent bacterium Vibrio fischeri was utilized. Using microbial experimental evolution, V. fischeri was selected to low (8°C), high (34°C), and fluctuating temperature stress (8°C/34°C) for 2000 generations. The temperatures 8°C and 34°C were the lower and upper growth limits, respectively. V. fischeri was also selected to benign temperatures (21°C and 28°C) for 2000 generations, which served as controls. V. fischeri demonstrated significant adaptation to low, high, and fluctuating temperature stress. V. fischeri did not display significant adaptation to the benign temperatures. Adaptation to stressful temperatures facilitated V. fischeri's ability to colonize the squid host relative to the ancestral lines. Bioluminescence levels also increased. Evolution to benign temperatures did not manifest these results. In summary, microbial adaptation to stress during the free-living stage can promote coevolution between hosts and microorganisms.

Molecular mechanism of cytoplasmic dynein tension sensing.

Cytoplasmic dynein is the most complex cytoskeletal motor protein and is responsible for numerous biological functions. Essential to dynein's function is its capacity to respond anisotropically to tension, so that its microtubule-binding domains bind microtubules more strongly when under backward load than forward load. The structural mechanisms by which dynein senses directional tension, however, are unknown. Using a combination of optical tweezers, mutagenesis, and chemical cross-linking, we show that three structural elements protruding from the motor domain-the linker, buttress, and stalk-together regulate directional tension-sensing. We demonstrate that dynein's anisotropic response to directional tension is mediated by sliding of the coiled-coils of the stalk, and that coordinated conformational changes of dynein's linker and buttress control this process. We also demonstrate that the stalk coiled-coils assume a previously undescribed registry during dynein's stepping cycle. We propose a revised model of dynein's mechanochemical cycle which accounts for our findings.

Is C-reactive protein elevated in obstructive sleep apnea? a systematic review and meta-analysis.

Purpose: This study examined whether circulating C-reactive protein (CRP) is elevated in obstructive sleep apnoea (OSA) independent of the confounding effects of comorbidities, smoking, body mass index (BMI), age and gender. Methods: A systematic review of the literature was performed using PubMed, Embase and Cochrane databases from 1 January 1997 to 1 November 2017 using the key words obstructive sleep apnoea and C-Reactive protein to identify full text English language studies that compared CRP in adult non-smoking OSA participants without comorbidities and adult healthy non-smoking control participants matched for BMI, age and gender. Data from eligible studies were subjected to meta-analysis using RevMan version 5.3. Results: Five studies (219 OSA participants, 116 controls) met the selection criteria. The total standard mean difference for circulating high sensitivity CRP was 0.61 mg/dL higher in OSA participants than in control participants (confidence interval: 0.38 to 0.84, p < 0.00001), with low between-studies heterogeneity (df = 7, p = 0.16, I2 = 33%) and minimal evidence of publication bias. Conclusions: CRP levels in non-smoking OSA participants without comorbidities were increased relative to levels in healthy matched non-smoking control participants, suggesting that pharyngeal or systemic inflammatory effects attributable to OSA may elevate CRP.

Increasing the stability of Lumbricus terrestris erythrocruorin via poly(acrylic acid) conjugation.

Since donated red blood cells must be constantly refrigerated, they are often unavailable in remote areas and battlefields. The goal of this study was to synthesize a highly stable blood substitute that does not require refrigeration. Specifically, the extracellular haemoglobin (a.k.a. erythrocruorin, Ec) of the earthworm Lumbricus terrestris erythrocruororin (LtEc) was cross-linked with poly(acrylic acid) (PAA) and ethylene diamine (EDA). PAGE analysis of the LtEc nanoparticles reveals cross-linking between subunits, while dynamic light scattering and scanning electron microscopy show that cross-linking significantly increases the size of the LtEc nanoparticles (164 ± 13.9 nm). Cross-linking also significantly increased the thermal stability of the LtEc nanoparticles by 10 °C (Tm = 72 ± 0.84 °C) relative to native LtEc (Tm = 62 ± 0.6 °C). In addition, while native LtEc rapidly dissociates at pH 9, the LtEc nanoparticles resist subunit dissociation up to pH 10. The oxygen affinity of the LtEc nanoparticles (P50 = 6.85 ± 0.13 mm Hg) is much higher than native LtEc (P50 = 26.67 ± 0.4 mm Hg), but the cooperativity (n = 2.43 ± 0.12) is not affected. Altogether, these results show that cross-linking LtEc with PAA and EDA provides a potential blood substitute with increased stability and oxygen affinity.

Using continuous quantitative capnography for emergency department procedural sedation: a systematic review and cost-effectiveness analysis.

End-tidal CO2 has been advocated to improve safety of emergency department (ED) procedural sedation by decreasing hypoxia and catastrophic outcomes. This study aimed to estimate the cost-effectiveness of routine use of continuous waveform quantitative end-tidal CO2 monitoring for ED procedural sedation in prevention of catastrophic events. Markov modeling was used to perform cost-effectiveness analysis to estimate societal costs per prevented catastrophic event (death or hypoxic brain injury) during routine ED procedural sedation. Estimates for efficacy of capnography and safety of sedation were derived from the literature. This model was then applied to all procedural sedations performed in US EDs with assumptions selected to maximize efficacy and minimize cost of implementation. Assuming that capnography decreases the catastrophic adverse event rate by 40.7% (proportional to efficacy in preventing hypoxia), routine use of capnography would decrease the 5-year estimated catastrophic event rate in all US EDs from 15.5 events to 9.2 events (difference 6.3 prevented events per 5 years). Over a 5-year period, implementing routine end-tidal CO2 monitoring would cost an estimated $2,830,326 per prevented catastrophic event, which translates into $114,007 per quality-adjusted life-year. Sensitivity analyses suggest that reasonable assumptions continue to estimate high costs of prevented catastrophic events. Continuous waveform quantitative end-tidal CO2 monitoring is a very costly strategy to prevent catastrophic complications of procedural sedation when applied routinely in ED procedural sedations.

Immune responses following experimental infection with Ascaridia galli and necrotic enteritis in broiler chickens.

Broilers commonly suffer from necrotic enteritis (NE). Other gastrointestinal infectious diseases affect poultry, including nematode infections which are considered a re-emerging disease in barn and free-range systems. The aim of this study was to characterize the immune response of broilers after artificial infection with NE and contrast these with responses to the nematode Ascaridia galli and determine whether immune parameters measured during the course of infection can be used to distinguish infected from uninfected birds. A total of 96 one-day-old male Ross 308 broiler chickens were used in this study. At 10 days of age, broilers were randomly assigned to one of the following treatment groups: control birds (n = 32), A. galli infected birds (n = 32), or NE infected birds (n = 32) and inoculated with the appropriate infective agents. The immune response of birds was monitored through evaluation of haematology parameters, acute phase protein production, and intraepithelial intestinal lymphocyte population changes at 11, 16, 20, and 32 days of age. T-helper cells (CD4+CD8-) increased significantly over time, and were significantly higher in A. galli and NE compared to day 10 controls. In conclusion, α-1 glycoprotein levels can distinguish birds with NE from other birds, including those infected with A. galli; also T-helper cell numbers can distinguish both NE and A. galli from uninfected birds and thirdly, 10 days post infection is the best time point to evaluate the bird's immune response for A. galli infections.

Cytoplasmic dynein regulates its attachment to microtubules via nucleotide state-switched mechanosensing at multiple AAA domains.

Cytoplasmic dynein is a homodimeric microtubule (MT) motor protein responsible for most MT minus-end-directed motility. Dynein contains four AAA+ ATPases (AAA: ATPase associated with various cellular activities) per motor domain (AAA1-4). The main site of ATP hydrolysis, AAA1, is the only site considered by most dynein motility models. However, it remains unclear how ATPase activity and MT binding are coordinated within and between dynein's motor domains. Using optical tweezers, we characterize the MT-binding strength of recombinant dynein monomers as a function of mechanical tension and nucleotide state. Dynein responds anisotropically to tension, binding tighter to MTs when pulled toward the MT plus end. We provide evidence that this behavior results from an asymmetrical bond that acts as a slip bond under forward tension and a slip-ideal bond under backward tension. ATP weakens MT binding and reduces bond strength anisotropy, and unexpectedly, so does ADP. Using nucleotide binding and hydrolysis mutants, we show that, although ATP exerts its effects via binding AAA1, ADP effects are mediated by AAA3. Finally, we demonstrate "gating" of AAA1 function by AAA3. When tension is absent or applied via dynein's C terminus, ATP binding to AAA1 induces MT release only if AAA3 is in the posthydrolysis state. However, when tension is applied to the linker, ATP binding to AAA3 is sufficient to "open" the gate. These results elucidate the mechanisms of dynein-MT interactions, identify regulatory roles for AAA3, and help define the interplay between mechanical tension and nucleotide state in regulating dynein motility.

Control of cytoplasmic dynein force production and processivity by its C-terminal domain.

Cytoplasmic dynein is a microtubule motor involved in cargo transport, nuclear migration and cell division. Despite structural conservation of the dynein motor domain from yeast to higher eukaryotes, the extensively studied S. cerevisiae dynein behaves distinctly from mammalian dyneins, which produce far less force and travel over shorter distances. However, isolated reports of yeast-like force production by mammalian dynein have called interspecies differences into question. We report that functional differences between yeast and mammalian dynein are real and attributable to a C-terminal motor element absent in yeast, which resembles a 'cap' over the central pore of the mammalian dynein motor domain. Removal of this cap increases the force generation of rat dynein from 1 pN to a yeast-like 6 pN and greatly increases its travel distance. Our findings identify the CT-cap as a novel regulator of dynein function.

Cep192 controls the balance of centrosome and non-centrosomal microtubules during interphase.

Cep192 is a centrosomal protein that contributes to the formation and function of the mitotic spindle in mammalian cells. Cep192's mitotic activities stem largely from its role in the recruitment to the centrosome of numerous additional proteins such as gamma-tubulin and Pericentrin. Here, we examine Cep192's function in interphase cells. Our data indicate that, as in mitosis, Cep192 stimulates the nucleation of centrosomal microtubules thereby regulating the morphology of interphase microtubule arrays. Interestingly, however, cells lacking Cep192 remain capable of generating normal levels of MTs as the loss of centrosomal microtubules is augmented by MT nucleation from other sites, most notably the Golgi apparatus. The depletion of Cep192 results in a significant decrease in the level of centrosome-associated gamma-tubulin, likely explaining its impact on centrosome microtubule nucleation. However, in stark contrast to mitosis, Cep192 appears to maintain an antagonistic relationship with Pericentrin at interphase centrosomes. Interphase cells depleted of Cep192 display significantly higher levels of centrosome-associated Pericentrin while overexpression of Cep192 reduces the levels of centrosomal Pericentrin. Conversely, depletion of Pericentrin results in elevated levels of centrosomal Cep192 and enhances microtubule nucleation at centrosomes, at least during interphase. Finally, we show that depletion of Cep192 negatively impacts cell motility and alters normal cell polarization. Our current working hypothesis is that the microtubule nucleating capacity of the interphase centrosome is determined by an antagonistic balance of Cep192, which promotes nucleation, and Pericentrin, which inhibits nucleation. This in turn determines the relative abundance of centrosomal and non-centrosomal microtubules that tune cell movement and shape.

Covalent immobilization of microtubules on glass surfaces for molecular motor force measurements and other single-molecule assays.

Rigid attachment of microtubules (MTs) to glass cover slip surfaces is a prerequisite for a variety of microscopy experiments in which MTs are used as substrates for MT-associated proteins, such as the molecular motors kinesin and cytoplasmic dynein. We present an MT-surface coupling protocol in which aminosilanized glass is formylated using the cross-linker glutaraldehyde, fluorescence-labeled MTs are covalently attached, and the surface is passivated with highly pure beta-casein. The technique presented here yields rigid MT immobilization while simultaneously blocking the remaining glass surface against nonspecific binding by polystyrene optical trapping microspheres. This surface chemistry is straightforward and relatively cheap and uses a minimum of specialized equipment or hazardous reagents. These methods provide a foundation for a variety of optical tweezers experiments with MT-associated molecular motors and may also be useful in other assays requiring surface-immobilized proteins.

An improved optical tweezers assay for measuring the force generation of single kinesin molecules.

Numerous microtubule-associated molecular motors, including several kinesins and cytoplasmic dynein, produce opposing forces that regulate spindle and chromosome positioning during mitosis. The motility and force generation of these motors are therefore critical to normal cell division, and dysfunction of these processes may contribute to human disease. Optical tweezers provide a powerful method for studying the nanometer motility and piconewton force generation of single motor proteins in vitro. Using kinesin-1 as a prototype, we present a set of step-by-step, optimized protocols for expressing a kinesin construct (K560-GFP) in Escherichia coli, purifying it, and studying its force generation in an optical tweezers microscope. We also provide detailed instructions on proper alignment and calibration of an optical trapping microscope. These methods provide a foundation for a variety of similar experiments.

The yeast dynein Dyn2-Pac11 complex is a dynein dimerization/processivity factor: structural and single-molecule characterization.

Cytoplasmic dynein is the major microtubule minus end-directed motor. Although studies have probed the mechanism of the C-terminal motor domain, if and how dynein's N-terminal tail and the accessory chains it binds regulate motor activity remain to be determined. Here, we investigate the structure and function of the Saccharomyces cerevisiae dynein light (Dyn2) and intermediate (Pac11) chains in dynein heavy chain (Dyn1) movement. We present the crystal structure of a Dyn2-Pac11 complex, showing Dyn2-mediated Pac11 dimerization. To determine the molecular effects of Dyn2 and Pac11 on Dyn1 function, we generated dyn2Δ and dyn2Δpac11Δ strains and analyzed Dyn1 single-molecule motor activity. We find that the Dyn2-Pac11 complex promotes Dyn1 homodimerization and potentiates processivity. The absence of Dyn2 and Pac11 yields motors with decreased velocity, dramatically reduced processivity, increased monomerization, aggregation, and immobility as determined by single-molecule measurements. Deleting dyn2 significantly reduces Pac11-Dyn1 complex formation, yielding Dyn1 motors with activity similar to Dyn1 from the dyn2Δpac11Δ strain. Of interest, motor phenotypes resulting from Dyn2-Pac11 complex depletion bear similarity to a point mutation in the mammalian dynein N-terminal tail (Loa), highlighting this region as a conserved, regulatory motor element.

Design of a measurement system for temperature distribution in continuous-flow microwave heating of pumpable fluids using infrared imaging and fiber optic technology.

Continuous microwave heating has the potential to revolutionize processing operations in which high energy densities per unit volume are required. The adoption of microwave heating for large scale commercial application has been hampered by a lack of reliable information on the temperature distribution during heating of pumpable fluids. This study represents an initial attempt to design a system that quantifies the temperature distribution during continuous microwave heating and its relationship with processing tube surface temperature distribution. The novel measurement system designed consisted of a semi-rigid thin axial support system on which fiber optic probes were attached at different lengths in the microwave heating cavity. Inlet, outlet and in-line longitudinal temperatures were monitored during continuous flow microwave heating (1.5 kW, 2.5 kW, 3.5 kW, and 4.5 kW) of water of three different salinities (0 ppt, 15 ppt, and 30 ppt) at a flow rate of 1.6 l/min. Inlet and exit temperatures were measured using standard thermocouples; in line temperatures were determined using fiber optic temperature probes. The surface temperature distribution of the applicator tube was measured using an infrared camera. Results showed that fiber optic-based internal temperatures were 10 degrees C higher than observed external infrared temperatures for fresh water and 14.6 degrees C higher for 30 ppt salt water. The measurement system and method presented are intended to provide experimental validation of numerical models of continuous flow microwave heating, as well as to help develop empirical models of temperature distribution under varying processing parameters.