Live Tissue Research Articles

Expanding super-resolution imaging versatility in organisms with multi-confocal image scanning microscopy

Abstract Resolving complex three-dimensional subcellular dynamics noninvasively in live tissues demands imaging tools that balance spatiotemporal resolution, field-of-view and phototoxicity. Image scanning microscopy (ISM), as an advancement of confocal laser scanning microscopy, provides a two-fold 3D resolution enhancement. Nevertheless, the relatively low imaging speed has been the major obstacle for ISM to be further employed in in vivo imaging of biological tissues. Our proposed solution, multi-confocal image scanning microscopy (MC-ISM), aims to overcome the limitations of existing techniques in terms of spatiotemporal resolution balancing by optimizing pinhole diameter and pitch, eliminating out-of-focus signals, and introducing a frame reduction reconstruction algorithm. The imaging speed is increased by 16 times compared with multifocal structured illumination microscopy. We further propose single-galvo scan, akin to the Archimedes spiral in spinning disk confocal system, to ensure high speed ang high accuracy scan without galvanometer's inertial motion. Benefitting from its high photon efficiency, MC-ISM allows continuous imaging of mitochondria dynamics in live cell for 1000 frames without apparent phototoxicity, reaching an imaging depth of 175 μm. Noteworthy, MC-ISM enables the observation of the inner membrane structure of living mitochondria in Arabidopsis hypocotyl for the first time, demonstrating its outstanding performance.

Massive fabrication of functional hepatic cancer spheroids by micropatterned GelMA hydrogel chip for drug screening

Since hepatic cancer incidence and mortality continue to grow worldwide, it is necessary to develop the biomimetic tumor models for drug development and tumor therapeutics. Cellular spheroids as an excellent simple 3D model can bridge the gap between 2D cell culture and live tissue. In this study, we proposed a biological methacrylated gelatin (GelMA) hydrogel-based microplatform for the massive generation of hepatocellular spheroids and downstream investigation of drug resistance. Micropatterned GelMA hydrogel microwell chip (GHM-chip) with tunable array was easily achieved in standard 24-culture well plates through the micro-molding fabrication strategy. The fabricated GHM-chip induced multicellular self-assembly behavior within the defined topography and further formed spheroidal structure. By regulating cell seeding density and designing microwell size, uniform hepatic cancer spheroids with tunable diameters were obtained in a simplicity, stability and controllable manner. In addition, the screening chemotherapy study of anti-cancer drug was completed through non-destructive recovery of spheroids from the GHM-chip. Beyond that, the recovered functional spheroids have potential application value in various biomedical fields such as tumor biology, pharmacology, and tissue microengineering. Finally, the proposed GHM-chip incorporated into standard cell culture plates with easy to manufacture and operate properties, may be an efficient culture microplatform for cancer research applications.

Terminal ballistics performance of 9 × 19mm cartridges in 10% ballistic gelatin: FBI Protocol based analysis of Brazilian-made law enforcement ammunition.

Understanding that a projectile entering the human body can cause damage or destruction to live tissues through a variety of wounding mechanisms - permanent cavity, temporary cavity, and fragmentation - is crucial for researching terminal ballistics and understand the patterns of gunshot wound configuration. The present work tested four different types of ammunition in caliber 9 × 19mm (Full Metal Jacketed, Gold Hex, Copper Bullet Tactical and Bonded), using ballistic gelatin at 10% as soft tissue surrogate. The tests were based on the Federal Bureau of Investigation Protocol and included shots through bare gelatin, heavy clothing, plywood, steel sheets and auto glass. As a comparison parameter, the American-made Federal™ HST, used by several law enforcement agencies in the USA, was also tested in the same conditions. The Full Metal Jacketed cartridge had a uniform performance throughout the experiment, showing high penetration levels and no expansion, as expected. Gold Hex demonstrated a strong tendency to fragment with low levels of penetration and weight retention. Copper Bullet Tactical did not achieve the 12" minimum penetration in the soft barrier phases but expanded aggressively. Finally, Bonded only failed to achieve the 12" mark of penetration in phase 5 (auto glass), the hardest barrier in the whole Protocol. Tested for comparison purposes, Federal HST showed aggressive expansion in the initial phases (over 100%), after surpassing the 12" threshold. The study concluded that heavier projectiles (CBC Bonded and Federal HST) performed better than lighter and faster bullets in terms of terminal ballistics.

Impact of Live Tissue Training on Provider Confidence for Operative Trauma Management.

Readiness of military health care providers is essential to maintain a prepared fighting force and remains a central issue for today's military. Maintaining skills regarding operative management of trauma is becoming more difficult if not located at an urban trauma center. Live tissue training may be used to practice skills in a facility that does not have exposure to high acuity trauma. The goal of our live tissue skills course is to train health care providers and ancillary staff in the management of various injury complexes and improve confidence and familiarity with management using a porcine model. A live tissue skills course using a porcine model was offered to surgical residents, attendings, and operating room technicians. Before completing the course, each participant completed a survey assessing comfort level, previous training experiences, and deployments. Operative management was simulated with a standardized sequence of abdominal and thoracic injuries. After the course, each participant then completed a survey assessing comfort level, benefit of the course, and comparison to inanimate simulation training. Survey information was obtained via participant completion of a multiple-choice confidential questionnaire. Responses were collected from 34 participants at our institution. Average abdominal trauma comfort increased from 2.79 to 3.73, with no participants reporting feeling not comfortable at all post-lab. Average thoracic trauma comfort increased from 2.47 to 3.48, with one participant reporting feeling not comfortable at all post-lab. Overall, 85% of participants reported the course to be very beneficial in improving their knowledge, 83% reported the course to be very beneficial compared to inanimate simulators, and 92% reported high fidelity of the live tissue training course. Perceived as highly beneficial, live tissue training increased comfort with operative trauma management by surgical residents, attendings, and technicians supporting continued utilization. Further evaluation of live tissue courses would be advantageous in assessing surgical skill improvement, including comparison with simulation training.

Spleen Swabs for Sensitive and High-Throughput Detection of African Swine Fever Virus by Real-Time PCR.

African swine fever (ASF) continues to spread in Africa, Europe, Asia and the island of Hispaniola, increasing the need to develop more streamlined and highly efficient surveillance and diagnostic capabilities. One way to achieve this is by further optimization of already established standard operating procedures to remove bottlenecks for high-throughput screening. Real-time polymerase chain reaction (real-time PCR) is the most sensitive and specific assay available for the early detection of the ASF virus (ASFV) genome, but it requires high-quality nucleic acid extracted from the samples. Whole blood from live pigs and spleen tissue from dead pigs are the preferred samples for real-time PCR. Whole blood can be used as is in nucleic acid extractions, but spleen tissues require an additional homogenization step. In this study, we compared the homogenates and swabs prepared from 52 spleen samples collected from pigs experimentally inoculated with highly and moderately virulent ASF virus strains. The results show that not only are the spleen swabs more sensitive when executed with a low-cell-count nucleic acid extraction procedure followed by real-time PCR assays but they also increase the ability to isolate ASFV from positive spleen samples. Swabbing is a convenient, simpler and less time-consuming alternative to tissue homogenization. Hence, we recommend spleen swabs over tissue homogenates for high-throughput detection of ASFV by real-time PCR.

Single Molecule Imaging of Sp3 Functionalized Ultrashort Carbon Nanotubes for Deep Brain Tissue Investigation

Near-infrared (NIR) fluorescence microscopy has attracted significant attention for in vivo deep tissue imaging due to the combination of low light scattering and absorption by the tissues, resulting in good light penetration depth, especially in the NIR-II window (~1000–1350 nm). In this context, NIR photoluminescence (PL) (λem ~ 900–1400 nm) of single-walled carbon nanotubes overlapping with the so-called “tissue transparency window” laid the foundation for their use in bioimaging and sensing applications. Interestingly, although ultrashort carbon nanotubes (usCNTs) had long been sought for their advantages of mimicking bio-molecular dimensions, it is only when sp3 defects introduced bright fluorescent usCNTs could be generated [1]. In this work, we demonstrate the efficient preparation of bright fluorescent usCNTs specifically designed for biological applications, and we show how to create bright usCNTs wrapped by a biocompatible encapsulating agent (phospholipid-polyethylene glycol, PL-PEG). More precisely, we adapted a synthesis route where the sp3 functionalization is followed by chemical cutting [2]. We will present their full characterization and demonstrate their performance for deep tissue imaging in neuroscience. More precisely, our results performed on live brain tissue allow us to analyze their length specific behaviors in different compartments of mouse brains in comparison to the long sp3 functionalized CNTs [3].

Single-nanoparticle electrophoretic mobility determination and trapping using active-feedback 3D tracking.

Nanoparticles (NP) are versatile materials with widespread applications across medicine and engineering. Despite rapid incorporation into drug delivery, therapeutics, and many more areas of research and development, there is a lack of robust characterization methods. Light scattering techniques such as dynamic light scattering (DLS) and electrophoretic light scattering (ELS) use an ensemble-averaged approach to the characterization of nanoparticle size and electrophoretic mobility (EM), leading to inaccuracies when applied to polydisperse or heterogeneous populations. To address this lack of single-nanoparticle characterization, this work applies 3D Single-Molecule Active Real-time Tracking (3D-SMART) to simultaneously determine NP size and EM on a per-particle basis. Single-nanoparticle EM is determined by using active feedback to "lock on" to a single particle and apply an oscillating electric field along one axis. A maximum likelihood approach is applied to extract the single-particle EM from the oscillating nanoparticle position along the field-actuated axis, while mean squared displacement is used along the non-actuated axes to determine size. Unfunctionalized and carboxyl-functionalized polystyrene NPs are found to have unique EM based on their individual size and surface characteristics, and it is demonstrated that single-nanoparticle EM is a more precise tool for distinguishing unique NP preparations than diffusion alone, able to determine the charge number of individual NPs to an uncertainty of less than 30. This method also explored individual nanoparticle EM in various ionic strengths (0.25-5 mM) and found decreased EM as a function of increasing ionic strength, in agreement with results determined via bulk characterization methods. Finally, it is demonstrated that the electric field can be manipulated in real time in response to particle position, resulting in one-dimensional electrokinetic trapping. Critically, this new single-nanoparticle EM determination and trapping method does not require microfluidics, opening the possibility for the exploration of single-nanoparticle EM in live tissue and more comprehensive characterization of nanoparticles in biologically relevant environments.

Ex vivo model of breast cancer cell invasion in live lymph node tissue.

Lymph nodes (LNs) are common sites of metastatic invasion in breast cancer, often preceding spread to distant organs and serving as key indicators of clinical disease progression. However, the mechanisms of cancer cell invasion into LNs are not well understood. Existing in vivo models struggle to isolate the specific impacts of the tumor-draining lymph node (TDLN) milieu on cancer cell invasion due to the co-evolving relationship between TDLNs and the upstream tumor. To address these limitations, we used live ex vivo LN tissue slices with intact chemotactic function to model cancer cell spread within a spatially organized microenvironment. After showing that BRPKp110 breast cancer cells were chemoattracted to factors secreted by naïve LN tissue in a 3D migration assay, we demonstrated that ex vivo LN slices could support cancer cell seeding, invasion, and spread. This novel approach revealed dynamic, preferential cancer cell invasion within specific anatomical regions of LNs, particularly the subcapsular sinus (SCS) and cortex, as well as chemokine-rich domains of immobilized CXCL13 and CCL1. While CXCR5 was necessary for a portion of BRPKp110 invasion into naïve LNs, disruption of CXCR5/CXCL13 signaling alone was insufficient to prevent invasion towards CXCL13-rich domains. Finally, we extended this system to pre-metastatic TDLNs, where the ex vivo model predicted a lower invasion of cancer cells. The reduced invasion was not due to diminished chemokine secretion, but it correlated with elevated intranodal IL-21. In summary, this innovative ex vivo model of cancer cell spread in live LN slices provides a platform to investigate cancer invasion within the intricate tissue microenvironment, supporting time-course analysis and parallel read-outs. We anticipate that this system will enable further research into cancer-immune interactions and allow isolation of specific factors that make TDLNs resistant to cancer cell invasion, which are challenging to dissect in vivo.

Investigation of Toxoplasma gondii Seroprevalence in Pregnant Women in Çankırı

Toxoplasma gondii can be transmitted to humans through consumption of raw or undercooked meat containing live tissue cysts, consumption of water or food contaminated with oocysts shed from cat feces, and vertical transmission during pregnancy or through tissue, organ, and blood transfusion. The aim of this study was to investigate the seroprevalence of T. gondii in patients admitted to the Obstetrics and Gynecology Department of Çankırı State Hospital (ÇSH). In this study, anti-T. gondii IgG and IgM antibody levels of 62 pregnant women between the ages of 17 and 47 who applied to ÇSH gynecology and obstetrics outpatient clinic for normal pregnancy follow-up between June 2022 and January 2023 were investigated. According to the results of the studies, anti-T gondii IgG positivity is 12.9% and negative 87.1% and anti-T. gondii IgM positivity is 0% while it was found to be 100% negative. According to these results, it is reported that the majority of pregnant women do not encounter T. gondii and; therefore, they should be more careful in terms of congenital toxoplasmosis. T. gondii can be transmitted to humans through consumption of water or food contaminated with oocysts excreted in cat feces and through vertical transmission or tissue, organ, and blood transfusion during pregnancy. Therefore, pregnant women should be carefully monitored for T. gondii.

Scaling-law mechanical marker for liver fibrosis diagnosis and drug screening through machine learning.

Studies of cell and tissue mechanics have shown that significant changes in cell and tissue mechanics during lesions and cancers are observed, which provides new mechanical markers for disease diagnosis based on machine learning. However, due to the lack of effective mechanic markers, only elastic modulus and iconographic features are currently used as markers, which greatly limits the application of cell and tissue mechanics in disease diagnosis. Here, we develop a liver pathological state classifier through a support vector machine method, based on high dimensional viscoelastic mechanical data. Accurate diagnosis and grading of hepatic fibrosis facilitates early detection and treatment and may provide an assessment tool for drug development. To this end, we used the viscoelastic parameters obtained from the analysis of creep responses of liver tissues by a self-similar hierarchical model and built a liver state classifier based on machine learning. Using this classifier, we implemented a fast classification of healthy, diseased, and mesenchymal stem cells (MSCs)-treated fibrotic live tissues, and our results showed that the classification accuracy of healthy and diseased livers can reach 0.99, and the classification accuracy of the three liver tissues mixed also reached 0.82. Finally, we provide screening methods for markers in the context of massive data as well as high-dimensional viscoelastic variables based on feature ablation for drug development and accurate grading of liver fibrosis. We propose a novel classifier that uses the dynamical mechanical variables as input markers, which can identify healthy, diseased, and post-treatment liver tissues.

Modelling and simulation of growth driven by mechanical and non-mechanical stimuli

Living tissues can remarkably adapt to their mechanical and biochemical environments through growth and remodelling mechanisms. Over the years, extensive research has been dedicated to understanding and modelling the complexities of growth. However, the majority of growth laws are based on phenomenological, ad hoc, proposed evolution equations. This work aims to describe a general bulk growth model that developed in the framework of generalised continuum mechanics. This new model of growth is based on a continuum description of the growth process and is an extension of the work of DiCarlo and Quiligotti of the early 2000s. This model builds on the virtual power principle, and the constitutive theory is thermodynamically consistent. The proposed framework allows the inclusion of different constitutive theories linking the elastic strain and stresses, together with accommodating different non-mechanical mechanisms. Moreover, the framework supports anisotropy of both the material and growth, allowing the exploration of complex growth processes further. The descriptive capabilities of the model are demonstrated through numerical benchmarks and simulations describing real-life scenarios, such as the growth of the spine and an artery. The simulation results indicate that the developed thermodynamic consistent growth model is versatile and holds the potential to capture the complexities of living tissue growth, offering valuable insights into biological phenomena and pathologies.

Live cell imaging of cellular dynamics in poplar wood using computational cannula microscopy

This study presents significant advancements in computational cannula microscopy for live imaging of cellular dynamics in poplar wood tissues. Leveraging machine-learning models such as pix2pix for image reconstruction, we achieved high-resolution imaging with a field of view of 55µm using a 50µm-core diameter probe. Our method allows for real-time image reconstruction at 0.29 s per frame with a mean absolute error of 0.07. We successfully captured cellular-level dynamics in vivo, demonstrating morphological changes at resolutions as small as 3µm. We implemented two types of probabilistic neural network models to quantify confidence levels in the reconstructed images. This approach facilitates context-aware, human-in-the-loop analysis, which is crucial for in vivo imaging where ground-truth data is unavailable. Using this approach we demonstrated deep in vivo computational imaging of living plant tissue with high confidence (disagreement score ⪅0.2). This work addresses the challenges of imaging live plant tissues, offering a practical and minimally invasive tool for plant biologists.

Laparoscopic training workshop to assess medical students’ skill acquisition and interest in surgical careers

BackgroundWith its minimally invasive approach, laparoscopic surgery has transformed the medical landscape. As the demand for these procedures escalates, there is a pressing need for adept surgeons trained in laparoscopic techniques. However, current training often falls short of catering to medical school education. This study evaluates the impact of a custom-designed laparoscopic training workshop on medical students’ surgical skills and career aspirations.MethodsThis prospective experimental study was conducted at the E-Da hospital in Kaohsiung City, Taiwan. Medical students from Taiwanese medical schools undergoing Clerk 5, Clerk 6, and Postgraduate Year 1 and 2 were invited to participate. Medical students (n = 44) underwent an endoscopic skill training workshop consisting of lectures, box training, and live tissue training. The trainees performed multiple tasks before and after training using our objective evaluation system. The primary outcome was assessed before and after training through a questionnaire assessing the influence of training on students’ interest in surgery as a career. The secondary outcome measured improvement in skill acquisition, comparing the task completion time pre- and post-workshop. For the primary outcome, descriptive statistics were used to summarize the questionnaire responses, and paired t-tests were performed to determine significant changes in interest levels post-workshop. For the secondary outcome, paired t-tests were used to compare the time recorded pre- and post-training.ResultsPost-training, participants exhibited significant proficiency gains, with task completion times reducing notably: 97 s (p = 0.0015) for Precision Beads Placement, 88.5 s (p < 0.0001) for Beads Transfer Exercise, 95 s (p < 0.0001) for Precision Balloon Cutting, and 137.8 s (p < 0.0001) for Intracorporeal Suture. The primary outcome showcased an increased mean score from 8.15 pre-workshop to 9.3 post-workshop, indicating a bolstered interest in surgery as a career. Additionally, post-training sentiment analysis underscored a predominant inclination toward surgery among 88% of participants.ConclusionThe custom-designed laparoscopic workshop significantly improved technical skills and positively influenced students’ career aspirations toward surgery. Such hands-on training workshops can play a crucial role in medical education, bridging the gap between theoretical knowledge and practical skills and potentially shaping the future of budding medical professionals.

Isophorone-based crystallization-induced-emission sensors detect proteome aggregation in live cells and tissues with breast cancer

BackgroundProtein misfolding and aggregation can lead to various diseases. Recent studies have shed light on the aggregated protein in breast cancer pathology, which suggests that it is crucial to design chemical sensors that visualize protein aggregates in breast cancer, especially in clinical patient-derived samples. However, most reported sensors are constrained in cultured cell lines. ResultsIn this work, we present the development of two isophorone-based crystallization-induced-emission fluorophores for detecting proteome aggregation in breast cancer cell line and tissues biopsied from diseased patients, designated as A1 and A2. These probes exhibited viscosity sensitivity and recovered their fluorescence strongly at crystalline state. Moreover, A1 and A2 exhibit selective binding capacity and strong fluorescence for various aggregated proteins. Utilizing these probes, we detect protein aggregation in stressed breast cancer cells, xenograft mouse model of human breast cancer and clinical patient-derived samples. Notably, the fluorescence intensity of both probes light up in tumor tissues. SignificanceThe synthesized isophorone-based crystallization-induced-emission fluorophores, A1 and A2, enable sensitive detection of protein aggregation in breast cancer cells and tissues. In the future, aggregated proteins are expected to become indicators for early diagnosis and clinical disease monitoring of breast cancer.

Neutrophil NLRP3 promotes cardiac injury following acute myocardial infarction through IL-1β production, VWF release and NET deposition in the myocardium

NLRP3 inflammasome has been implicated in neutrophil polarization and extrusion of neutrophil extracellular traps (NETs) in vitro and facilitates secretion of Il1-beta (IL-1β). Permanent ligation of the left anterior descending artery was used to induce MI in WT and NLRP3−/− mice as well as in NLRP3−/− recipient mice transfused with either WT or NLRP3−/− neutrophils. NLRP3 deficiency reduced infarct size to roughly a third of WT heart injury and preserved left ventricular (LV) function at 12 h after MI as assessed by echocardiography and triphenyltetrazolium chloride staining of live tissue. Transfusion of WT but not NLRP3−/− neutrophils after MI increased infarct size in NLRP3−/− mice and significantly reduced LV function. The key features of myocardial tissue in WT neutrophil transfused recipients were increased H3Cit-positive deposits with NET-like morphology and increased tissue levels of IL-1β and plasma levels of von Willebrand Factor (VWF). Flow cytometry analysis also revealed that neutrophil NLRP3 increased the number of labeled and transfused neutrophils in the bone marrow of recipient mice following MI. Our data suggest a key role for neutrophil NLRP3 in the production of IL-1β and deposition of NETs in cardiac tissue exacerbating injury following MI. We provide evidence for a link between neutrophil NLRP3 and VWF release likely enhancing thromboinflammation in the heart. Neutrophil NLRP3 deficiency conferred similar cardioprotective effects to general NLRP3 deletion in MI rendering anti-neutrophil NLRP3 therapy a promising target for early cardioprotective treatment.

A comprehensive preanalytical protocol for fresh solid tumor biospecimens

Nearly seventy percent of diagnostic lab test errors occur due to variability in preanalytical factors. These are the parameters involved with all aspects of tissue processing, starting from the time tissue is collected from the patient in the operating room, until it is received and tested in the laboratory. While there are several protocols for transporting fixed tissue, organs, and liquid biopsies, such protocols are lacking for transport and handling of live solid tumor tissue specimens. There is a critical need to establish preanalytical protocols to reduce variability in biospecimen integrity and improve diagnostics for personalized medicine. Here, we provide a comprehensive protocol for the standard collection, handling, packaging, cold-chain logistics, and receipt of solid tumor tissue biospecimens to preserve tissue viability.

Deep Red Light Driven Hydrogen Evolution by Heterojunction Polymer Dots for Diabetic Wound Healing.

We describe small heterojunction polymer dots (Pdots) with deep-red light catalyzed H2 generation for diabetic skin wound healing. The Pdots with donor/acceptor heterojunctions showed remarkably enhanced photocatalytic activity as compared to the donor or acceptor nanoparticles alone. We encapsulate the Pdots and ascorbic acid into liposomes to form Lipo-Pdots nanoreactors, which selectively scavenge ⋅OH radicals in live cells and tissues under 650 nm light illumination. The antioxidant capacity of the heterojunction Pdots is ~10 times higher than that of the single-component Pdots described previously. Under a total light dose of 360 J/cm2, the Lipo-Pdots nanoreactors effectively scavenged ⋅OH radicals and suppressed the expression of pro-inflammatory cytokines in skin tissues, thereby accelerating the healing of skin wounds in diabetic mice. This study provides a feasible solution for safe and effective treatment of diabetic foot ulcers.

OpenSTED: open-source dynamic intensity minimum system for stimulated emission depletion microscopy.

Stimulated emission depletion (STED) is a powerful super-resolution microscopy technique that can be used for imaging live cells. However, the high STED laser powers can cause significant photobleaching and sample damage in sensitive biological samples. The dynamic intensity minimum (DyMIN) technique turns on the STED laser only in regions of the sample where there is fluorescence signal, thus saving significant sample photobleaching. The reduction in photobleaching allows higher resolution images to be obtained and longer time-lapse imaging of live samples. A stand-alone module to perform DyMIN is not available commercially. In this work, we developed an open-source design to implement three-step DyMIN on a STED microscope and demonstrated reduced photobleaching for timelapse imaging of beads, cells, and tissue. The DyMIN system uses a fast multiplexer circuit and inexpensive field-programmable gate array controlled by Labview software that operates as a stand-alone module for a STED microscope. All software and circuit diagrams are freely available. We compared time-lapse images of bead samples using our custom DyMIN system to conventional STED and recorded a higher signal when using DyMIN after a 50-image sequence. We further demonstrated the DyMIN system for time-lapse STED imaging of live cells and brain tissue slices. Our open-source DyMIN system is an inexpensive add-on to a conventional STED microscope that can reduce photobleaching. The system can significantly improve signal to noise for dynamic time-lapse STED imaging of live samples.

The muscle and neural architecture of Taenia crassiceps cysticerci revisited; implications on head-tail polarization of the larvae.

Taenia crassiceps has been used for decades as an experimental model for the study of human and porcine cysticercosis. Even though, its life cycle, tissue organization, ultrastructure and immune response elicited in the host, have been extensively described, there are many other biological questions remaining to be addressed. In the present study we revisited the muscle and neural architecture of cysticerci in two of the most frequently used strains (WFU and ORF), using conventional staining and confocal microscopy imaging, aiming to assemble an updated anatomy. Differences between both strains, including polarization processes during development of the young budding larvae, are emphasized. We also performed a search for genes that have been related to peptidergic neural processes in other related flatworms. These findings can help to understand the anatomical and molecular consequences of the scolex presence or absence in both strains.

Knockout of the longevity gene Klotho perturbs aging and Alzheimer’s disease-linked brain microRNAs and tRNA fragments

Overexpression of the longevity gene Klotho prolongs lifespan, while its knockout shortens lifespan and impairs cognition via perturbation of myelination and synapse formation. However, comprehensive analysis of Klotho knockout effects on mammalian brain transcriptomics is lacking. Here, we report that Klotho knockout alters the levels of aging- and cognition related mRNAs, long non-coding RNAs, microRNAs and tRNA fragments. These include altered neuronal and glial regulators in murine models of aging and Alzheimer’s disease and in human Alzheimer’s disease post-mortem brains. We further demonstrate interaction of the knockout-elevated tRNA fragments with the spliceosome, possibly affecting RNA processing. Last, we present cell type-specific short RNA-seq datasets from FACS-sorted neurons and microglia of live human brain tissue demonstrating in-depth cell-type association of Klotho knockout-perturbed microRNAs. Together, our findings reveal multiple RNA transcripts in both neurons and glia from murine and human brain that are perturbed in Klotho deficiency and are aging- and neurodegeneration-related.