Protocol to correlate electron microscopy with electrophysiology in single-cell autaptic microcultures.

Single-cell microcultures (SCMs) form a monosynaptic circuit that allows stimulation and recording of postsynaptic responses using a single electrode. Here, we present a protocol to establish autaptic cultures from rat superior cervical ganglion neurons. We describe the steps for preparing SCMs, recording synaptic currents, and identifying and processing the recorded neurons for electron microscopy. We then detail procedures for visualizing synapses. This protocol is illustrated by correlating evoked and spontaneous neurotransmitter release with the ultrastructural features of synapses recorded. For complete details on the use and execution of this protocol, please refer to Velasco et al.1.

Magnetic Resonance Imaging-Guided Frameless Stereotactic Injections of the Bilateral Cerebellar Dentate Nuclei in Nonhuman Primates: Technical Note.

BACKGROUND AND OBJECTIVES: Nonhuman primates (NHPs) are important preclinical models for evaluating therapeutics because of their anatomophysiological similarities to humans, and can be especially useful for testing new delivery targets. With the growing promise of cell and gene therapies for the treatment of neurological diseases, it is important to ensure the accurate and safe delivery of these agents to target structures in the brain. However, a standard guideline or method has not been developed for stereotactic targeting in NHPs. In this article, we describe the safe use of a magnetic resonance imaging-guided frameless stereotactic system to target bilateral cerebellar dentate nuclei for accurate, real-time delivery of viral vector in NHPs. METHODS: Seventeen rhesus macaques (Macaca mulatta) underwent stereotactic surgery under real-time MRI guidance using the ClearPoint® system. Bilateral cerebellar dentate nuclei were targeted through a single parietal entry point with a transtentorial approach. Fifty microliters of contrast-impregnated infusate was delivered to each dentate nucleus, and adjustments were made as necessary according to real-time MRI monitoring of delivery. Perioperative clinical outcomes and postoperative volumes of distribution were recorded. RESULTS: All macaques underwent bilateral surgery successfully. Superficial pin site infection occurred in 4/17 (23.5%) subjects, which resolved with antibiotics. Two episodes of transient neurological deficit (anisocoria and unilateral weakness) were recorded, which did not require additional postoperative treatment and resolved over time. Volume of distribution of infusate achieved satisfactory coverage of target dentate nuclei, and only 1 incidence (2.9%) of cerebrospinal fluid penetration was recorded. Mean volume of distribution was 161.22 ± 39.61 mm3 (left, 173.65 ± 48.29; right, 148.80 ± 23.98). CONCLUSION: MRI-guided frameless stereotactic injection of bilateral cerebellar dentate nuclei in NHPs is safe and feasible. The use of this technique enables real-time modification of the surgical plan to achieve adequate target coverage and can be readily translated to clinical use.

Recent Developments in Synthesis, Properties, Applications and Recycling of Bio-Based Elastomers.

In 2021, global plastics production was 390.7 Mt; in 2022, it was 400.3 Mt, showing an increase of 2.4%, and this rising tendency will increase yearly. Of this data, less than 2% correspond to bio-based plastics. Currently, polymers, including elastomers, are non-recyclable and come from non-renewable sources. Additionally, most elastomers are thermosets, making them complex to recycle and reuse. It takes hundreds to thousands of years to decompose or biodegrade, contributing to plastic waste accumulation, nano and microplastic formation, and environmental pollution. Due to this, the synthesis of elastomers from natural and renewable resources has attracted the attention of researchers and industries. In this review paper, new methods and strategies are proposed for the preparation of bio-based elastomers. The main goals are the advances and improvements in the synthesis, properties, and applications of bio-based elastomers from natural and industrial rubbers, polyurethanes, polyesters, and polyethers, and an approach to their circular economy and sustainability. Olefin metathesis is proposed as a novel and sustainable method for the synthesis of bio-based elastomers, which allows for the depolymerization or degradation of rubbers with the use of essential oils, terpenes, fatty acids, and fatty alcohols from natural resources such as chain transfer agents (CTA) or donors of the terminal groups in the main chain, which allow for control of the molecular weights and functional groups, obtaining new compounds, oligomers, and bio-based elastomers with an added value for the application of new polymers and materials. This tendency contributes to the development of bio-based elastomers that can reduce carbon emissions, avoid cross-contamination from fossil fuels, and obtain a greener material with biodegradable and/or compostable behavior.

Recent developments in bio-based polyethylene: Degradation studies, waste management and recycling.

Nowadays, the tendency to replace conventional fossil-based plastics is increasing considerably; there is a growing trend towards alternatives that involve the development of plastic materials derived from renewable sources, which are compostable and biodegradable. Indeed, only 1.5 % of whole plastic production is part of the small bioplastics market, even when these materials with a partial or full composition from biomass are rapidly expanding. A very interesting field of investigation is currently being developed in which the disposal and processing of the final products are evaluated in terms of reducing environmental harm. This review presents a compilation of polyethylene (PE) types, their uses, and current problems in the waste management of PE and recycling. Particularly, this review is based on the capabilities to synthesize bio-based PE from natural and renewable sources as a replacement for the raw material derived from petroleum. In addition to recent studies in degradation on different types of PE with weight loss ranges from 1 to 47 %, the techniques used and the main changes observed after degradation. Finally, perspectives are presented in the manuscript about renewable and non-renewable polymers, depending on the non-degradable, biodegradable, and compostable behavior, including composting recent studies in PE. In addition, it contributes to the 3R approaches to responsible waste management of PE and advancement towards an environmentally friendly PE.

The histologic outcomes of indeterminate thyroid nodules with rat sarcoma mutations: A case series.

Molecular testing is an adjunct test for thyroid fine needle aspirations with indeterminate diagnoses, with certain mutations showing a greater risk of malignancy (ROM). Rat sarcoma (RAS) point mutations are the most common alterations in indeterminate thyroid nodules. While they can have a high ROM, they are also found in benign disease. This study describes the histologic outcomes of indeterminate nodules with RAS mutations. Bethesda III and IV thyroid nodules with ThyroSeq results showing RAS mutations (NRAS, KRAS, and HRAS) were identified between November 1, 2018 and February 28, 2023. Baseline patient characteristics, ThyroSeq results, and surgical diagnoses were collected. We identified 18 nodules with RAS mutations from 17 patients. Fourteen were NRAS (isolated NRAS in 6; NRAS with other abnormalities [NRAS+] in 8); one was isolated KRAS; and three were HRAS with other abnormalities (HRAS+). NRAS Q16R was the most common amino acid change. Twelve cases had follow-up. Two were malignant, a minimally invasive follicular carcinoma (NRAS+) and a papillary thyroid carcinoma, follicular variant (HRAS+). Three were noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP), 2 HRAS+ and 1 NRAS+. Four were follicular adenomas, one being atypical (3 NRAS+ and one isolated NRAS). One was an oncocytic adenoma (isolated NRAS). Two were nodular hyperplasias (isolated NRAS and NRAS+, respectively). Twenty-eight percent of our RAS-mutated nodules were malignant or NIFTP. All three HRAS-mutated nodules were malignant or NIFTP. The three isolated RAS mutations with follow up were benign (adenomas or nodular hyperplasia). These findings were in line with the literature.

Microtubule depolymerization contributes to spontaneous neurotransmitter release in vitro.

Microtubules are key to multiple neuronal functions involving the transport of organelles, however, their relationship to neurotransmitter release is still unresolved. Here, we show that microtubules present in the presynaptic compartment of cholinergic autaptic synapses are dynamic. To investigate how the balance between microtubule growth and shrinkage affects neurotransmission we induced synchronous microtubule depolymerization by photoactivation of the chemical inhibitor SBTub3. The consequence was an increase in spontaneous neurotransmitter release. An analogous effect was obtained by dialyzing the cytosol with Kif18A, a plus-end-directed kinesin with microtubule depolymerizing activity. Kif18A also inhibited the refilling of the readily releasable pool of synaptic vesicles during high frequency stimulation. The action of Kif18A was associated to one order of magnitude increases in the numbers of exo-endocytic pits and endosomes present in the presynaptic terminal. An enhancement of spontaneous neurotransmitter release was also observed when neurons were dialyzed with stathmin-1, a protein with a widespread presence in the nervous system that induces microtubule depolymerization. Taken together, these results support that microtubules restrict spontaneous neurotransmitter release as well as promote the replenishment of the readily releasable pool of synaptic vesicles.

Effect of low dietary concentrations of Acacia mearnsii tannin extract on chewing, ruminal fermentation, digestibility, nitrogen partition, and performance of dairy cows.

The supplementation of dairy cows with tannins can reduce the ruminal degradation of dietary protein and urine N excretion, but high concentration in the diet can impair ruminal function, diet digestibility, feed intake, and milk yield. This study evaluated the effect of low concentrations (0, 0.14, 0.29, or 0.43% of diet in DM basis) of a tannin extract from the bark of Acacia mearnsii (TA) on milking performance, dry matter intake (DMI), digestibility, chewing behavior, ruminal fermentation, and N partition of dairy cows. Twenty Holstein cows (34.7 ± 4.8 kg/d, 590 ± 89 kg, and 78 ± 33 d in lactation) were individually fed a sequence of 4 treatments in 5, 4 × 4 Latin squares (with 21-d treatment periods, each with a 14-d adaptation period). The TA replaced citrus pulp in the total mixed ration and other feed ingredients were kept constant. Diets had 17.1% crude protein, mostly from soybean meal and alfalfa haylage. The TA had no detected effect on DMI (22.1 kg/d), milk yield (33.5 kg/d), and milk components. The proportions in milk fat of mixed origin fatty acids (16C and 17C) and the daily secretion of unsaturated fatty acids were linearly reduced and the proportion of de novo fatty acids was increased by TA. Cows fed TA had linear increase in the molar proportion of butyrate and linear reduction in propionate in ruminal fluid, whereas acetate did not differ. There was a tendency for the ratio of acetate to propionate to be linearly increased by TA. Cows fed TA had a linear reduction in the relative ruminal microbial yield, estimated by the concentrations of allantoin and creatinine in urine and body weight. The total-tract apparent digestibility of neutral detergent fiber, starch, and crude protein also did not differ. The TA induced a linear increase in meal size and duration of the first daily meal and reduced meal frequency. Rumination behavior did not differ with treatment. Cows fed 0.43% TA selected against feed particles >19 mm in the morning. There were tendencies for linear decreases in milk urea N (16.1-17.3 mg/dL), urine N (153-168 g/d and 25.5-28.7% of N intake), and plasma urea N at 6, 18, and 21 h postmorning feeding, and plasma urea N 12 h postfeeding was reduced by TA. The proportion of N intake in milk (27.1%) and feces (21.4%) did not differ with treatment. Reductions in urine N excretion and milk and plasma urea N suggest that TA reduced ruminal AA deamination, whereas lactation performance did not differ. Overall, TA up to 0.43% of DM did not affect DMI and lactation performance, while there was a tendency to reduce urine N excretion.

Experimental Analysis of Fiber Reinforcement Rings' Effect on Tensile and Flexural Properties of Onyx™-Kevlar® Composites Manufactured by Continuous Fiber Reinforcement.

Additive manufacturing of composite materials is progressing in the world of 3D printing technologies; composite materials allow the combination of the physical and mechanical properties of two or more constituents to create a new material that meets the required properties of several applications. In this research, the impact of adding Kevlar® reinforcement rings on the tensile and flexural properties of the Onyx™ (nylon with carbon fibers) matrix was analyzed. Parameters such as infill type, infill density and fiber volume percentage were controlled to determine the mechanical response in tensile and flexural tests of the additive manufactured composites. The tested composites showed an increment of four times the tensile modulus and 1.4 times the flexural modulus of pure Onyx™ matrix when compared with that of the Onyx™-Kevlar®. The experimental measurements demonstrated that Kevlar® reinforcement rings can increase the tensile and flexural modulus of Onyx™-Kevlar® composites using low fiber volume percentages (lower than 19% in both samples) and 50% of rectangular infill density. However, the appearance of some defects, such as delamination, was observed and should be further analyzed to obtain products that are errorless and can be reliable for real functions as in automotive or aeronautical industries.

Cyst of Hattori: A Rare Cyst in the Posterior Mediastinum.

Sixteen years since their initial description in the literature, posterior mediastinal Mullerian cysts - otherwise known as cysts of Hattori, after their discoverer - remain rare, with only 40 patients reported as of 2020. We report a 43 year old obese female that presented with a 2.6 cm cyst found in the posterior mediastinum by endoscopic ultrasound (EUS) and previously by chest magnetic resonance imaging (MRI) originally diagnosed as a congenital enteric duplication cyst of the esophagus radiologically. Upon surgical excision via a DaVinci thoracoscopy, the cyst was confirmed to be of Mullerian origin by PAX8, WT1, and ER staining. It is possible that the majority of cysts of Hattori remain unrecognized and undiagnosed, given their rarity and resemblance to other pathologies. It is important that this entity become a part of every pathologist's differential for a posterior mediastinal cyst in a female.

Synthesis of Biobased Hydroxyl-Terminated Oligomers by Metathesis Degradation of Industrial Rubbers SBS and PB: Tailor-Made Unsaturated Diols and Polyols.

Biobased hydroxyl-terminated polybutadiene (HTPB) was successfully synthesized in a one-pot reaction via metathesis degradation of industrial rubbers. Thus, polybutadiene (PB) and poly(styrene-butadiene-styrene) (SBS) were degraded via metathesis with high yields (>94%), using the fatty alcohol 10-undecen-1-ol as a chain transfer agent (CTA) and the second-generation Grubbs−Hoveyda catalyst. The identification of the hydroxyl groups (-OH) and the formation of biobased HTPB were verified by FT-IR and NMR. Likewise, the molecular weight and properties of the HTPB were controlled by changing the molar ratio of rubber to CTA ([C=C]/CTA) from 1:1 to 100:1, considering a constant molar ratio of the catalyst ([C=C]/Ru = 500:1). The number average molecular weight (Mn) ranged between 583 and 6580 g/mol and the decomposition temperatures between 134 and 220 °C. Moreover, the catalyst optimization study showed that at catalyst loadings as low as [C=C]/Ru = 5000:1, the theoretical molecular weight is in good agreement with the experimental molecular weight and the expected diols and polyols are formed. At higher ratios than those, the difference between theoretical and experimental molecular weight is wide, and there is no control over HTPB. Therefore, the rubber/CTA molar ratio and the amount of catalyst play an important role in PB degradation and HTPB synthesis. Biobased HTPB can be used to synthesize engineering design polymers, intermediates, fine chemicals, and in the polyurethane industry, and contribute to the development of environmentally friendly raw materials.

Estimation of the Effects of CO2 and Temperature on the Swelling of PS-CO2 Mixtures at Supercritical Conditions on Rheological Testing.

The use of supercritical CO2 as a blowing agent for polymeric foams instead of traditional blowing agents has been a trend in recent years. To achieve the final desired properties of the polymeric foams, the rheological behavior of the material needs to be reliable. The polymer swelling in the samples for rheological testing affects the results of the viscoelastic properties of the material. This study proposes a new testing methodology to control the accuracy and repeatability of the rheological characterization for PS-SCO2 samples. To develop this methodology, three polystyrene resins with different molecular weight distribution were studied at three temperatures (170, 185 and 200 °C) and three pressures (0.1 MPa, 6.89 MPa and 13.78 MPa). The CO2 concentration was estimated and used in the Sanchez-Lacombe Equation of State (SLEOS) to determine the polymer swelling, as it affects the dimensions of specimens tested in high-pressure rheometers. The correction factors provided a consistent trend in the viscosity with respect to temperature and a decrease of up to 50% in the standard deviation. The results of this study are crucial for an accurate measurement of viscoelastic properties by parallel-plate rheometry.

Chlorophyll-a Pigment Measurement of Spirulina in Algal Growth Monitoring Using Portable Pulsed LED Fluorescence Lidar System.

Chlorophyll-a measurement is important in algal growth and water quality monitoring in natural waters. A portable pulsed LED fluorescence lidar system based on the preliminary algal organic matter and pigments excitation-emission matrix (EEM) of commercialized AZTEC Spirulina powder at varying concentrations was developed. Fluorescence peaks from EEMs showed increasing intensity as the Spirulina concentration increases. Using this information, an LED fluorescence lidar with a wavelength of 385 nm, pulse width of 10 ns, and repetition frequency of 500 kHz was constructed for chlorophyll detection at 680 nm. Turbidity measurements were also conducted at 700 nm emission wavelength at the same excitation wavelength. Range-resolved fluorescence lidar signals from the portable pulsed LED fluorescence lidar system are highly correlated with the standard methods such as optical density at 680 nm (R2 = 0.87), EEM fluorescence chlorophyll-a pigment at 680 nm (R2 = 0.89), and corrected chlorophyll-a concentration (R2 =0.92). The F680/F700 lidar ratio was measured to provide a linear relationship of chlorophyll-a and turbidity in waters. The F680/F700 measurement showed strong correlations with Spirulina concentration (R2 = 0.94), absorbance at 680 nm (R2 = 0.84), EEM chlorophyll-a pigment at 680 nm (R2 = 0.83), and corrected chlorophyll-a concentration (R2 = 0.86). Results revealed that this new technique of chlorophyll-a measurement can be used as an alternative to other standard methods in algal growth monitoring.

In Vivo Photocontrol of Microtubule Dynamics and Integrity, Migration and Mitosis, by the Potent GFP-Imaging-Compatible Photoswitchable Reagents SBTubA4P and SBTub2M.

Photoswitchable reagents are powerful tools for high-precision studies in cell biology. When these reagents are globally administered yet locally photoactivated in two-dimensional (2D) cell cultures, they can exert micron- and millisecond-scale biological control. This gives them great potential for use in biologically more relevant three-dimensional (3D) models and in vivo, particularly for studying systems with inherent spatiotemporal complexity, such as the cytoskeleton. However, due to a combination of photoswitch isomerization under typical imaging conditions, metabolic liabilities, and insufficient water solubility at effective concentrations, the in vivo potential of photoswitchable reagents addressing cytosolic protein targets remains largely unrealized. Here, we optimized the potency and solubility of metabolically stable, druglike colchicinoid microtubule inhibitors based on the styrylbenzothiazole (SBT) scaffold that are nonresponsive to typical fluorescent protein imaging wavelengths and so enable multichannel imaging studies. We applied these reagents both to 3D organoids and tissue explants and to classic model organisms (zebrafish, clawed frog) in one- and two-protein imaging experiments, in which spatiotemporally localized illuminations allowed them to photocontrol microtubule dynamics, network architecture, and microtubule-dependent processes in vivo with cellular precision and second-level resolution. These nanomolar, in vivo capable photoswitchable reagents should open up new dimensions for high-precision cytoskeleton research in cargo transport, cell motility, cell division, and development. More broadly, their design can also inspire similarly capable optical reagents for a range of cytosolic protein targets, thus bringing in vivo photopharmacology one step closer to general realization.

Cytologic analysis of a glomus tumor in the left second toe: Case report.

We present a 66-year-old female with a glomus tumor diagnosed by fine-needle aspiration (FNA) at the subungual region of her left second toe. Cytologic findings include cohesive clusters of round, uniform cells with scant cytoplasm. Nuclei were monotonous with fine chromatin. No cellular atypia, nuclear inclusions, mitotic figures, nor nucleoli were identified. Cells were surrounded by thick wisps of magenta colored myxoid material reminiscent of a pleomorphic adenoma. Few spindle shaped cells could be seen near the border of the tumor clusters. Staining was positive for alpha-smooth muscle actin. This case report presents one of few FNA diagnosed glomus tumors.

Effective Stiffness of Fused Deposition Modeling Infill Lattice Patterns Made of PLA-Wood Material.

Fused deposition modeling (FDM) uses lattice arrangements, known as infill, within the fabricated part. The mechanical properties of parts fabricated via FDM are dependent on these infill patterns, which make their study of great relevance. One of the advantages of FDM is the wide range of materials that can be employed using this technology. Among these, polylactic acid (PLA)-wood has been recently gaining attention as it has become commercially available. In this work, the stiffness of two different lattice structures fabricated from PLA-wood material using FDM are studied: hexagonal and star. Rectangular samples with four different infill densities made of PLA-wood material were fabricated via FDM. Samples were subjected to 3-point bending to characterize the effective stiffness and their sensitivity to shear deformation. Lattice beams proved to be more sensitive to shear deformations, as including the contribution of shear in the apparent stiffness of these arrangements leads to more accurate results. This was evaluated by comparing the effective Young's modulus characterized from 3-point bending using equations with and without shear inclusion. A longer separation between supports yielded closer results between both models (~41% for the longest separation tested). The effective stiffness as a function of the infill density of both topologies showed similar trends. However, the maximum difference obtained at low densities was the hexagonal topology that was ~60% stiffer, while the lowest difference was obtained at higher densities (star topology being stiffer by ~20%). Results for stiffness of PLA-wood samples were scattered. This was attributed to the defects at the lattice element level inherent to the material employed in this study, confirmed via micro-characterization.

Arf-like Protein 2 (ARL2) Controls Microtubule Neogenesis during Early Postnatal Photoreceptor Development.

Arf-like protein 2 (ARL2) is a ubiquitously expressed small GTPase with multiple functions. In a cell culture, ARL2 participates with tubulin cofactor D (TBCD) in the neogenesis of tubulin αß-heterodimers, the building blocks of microtubules. To evaluate this function in the retina, we conditionally deleted ARL2 in mouse retina at two distinct stages, either during the embryonic development (retArl2-/-) or after ciliogenesis specifically in rods (rodArl2-/-). retArl2-/- retina sections displayed distorted nuclear layers and a disrupted microtubule cytoskeleton (MTC) as early as postnatal day 6 (P6). Rod and cone outer segments (OS) did not form. By contrast, the rod ARL2 knockouts were stable at postnatal day 35 and revealed normal ERG responses. Cytoplasmic dynein is reduced in retArl2-/- inner segments (IS), suggesting that dynein may be unstable in the absence of a normal MTC. We investigated the microtubular stability in the absence of either ARL2 (retARL2-/-) or DYNC1H1 (retDync1h1-/-), the dynein heavy chain, and found that both the retArl2-/- and retDync1h1-/- retinas exhibited reduced microtubules and nuclear layer distortion. The results suggest that ARL2 and dynein depend on each other to generate a functional MTC during the early photoreceptor development.

Conditional Deletion of Cytoplasmic Dynein Heavy Chain in Postnatal Photoreceptors.

Purpose: Cytoplasmic dynein-1 (henceforth dynein) moves cargo in conjunction with dynactin toward the minus end of microtubules. The dynein heavy chain, DYNC1H1, comprises the backbone of dynein, a retrograde motor. Deletion of Dync1h1 abrogates dynein function. The purpose of this communication is to demonstrate effects of photoreceptor dynein inactivation during late postnatal development and in adult retina. Methods: We mated Dync1h1F/F mice with iCre75 and Prom1-CreERT2 mice to generate conditional rod and tamoxifen-induced knockout in rods and cones, respectively. We documented retina degeneration with confocal microscopy at postnatal day (P) 10 to P30 for the iCre75 line and 1 to 4 weeks post tamoxifen induction (wPTI) for the Prom1-CreERT2 line. We performed scotopic and photopic electroretinography (ERG) at P16 to P30 in the iCre75 line and at 1-week increments in the Prom1-CreERT2 line. Results were evaluated statistically using Student's t-test, two-factor ANOVA, and Welch's ANOVA. Results: Cre-induced homologous recombination of Dync1h1F/F mice truncated DYNC1H1 after exon 23. rodDync1h1-/- photoreceptors degenerated after P14, reducing outer nuclear layer (ONL) thickness and combined inner segment/outer segment (IS/OS) length significantly by P18. Scotopic ERG a-wave amplitudes decreased by P16 and were extinguished at P30. Cones were stable under rod-knockout conditions until P21 but inactive at P30. In tamDync1h1-/- photoreceptors, the IS/OS began shortening by 3wPTI and were nearly eliminated by 4wPTI. The ONL shrank significantly over this interval, indicating rapid photoreceptor degeneration following the loss of dynein. Conclusions: Our results demonstrate dynein is essential for the secretory pathway, formation of outer segments, and photoreceptor maintenance.