Cortical Specimens Research Articles

Longitudinal multi-omics reveals pathogenic TSC2 variants disrupt developmental trajectories of human cortical organoids derived from Tuberous Sclerosis Complex.

Tuberous Sclerosis Complex (TSC), an autosomal dominant condition, is caused by heterozygous mutations in either the TSC1 or TSC2 genes, manifesting in systemic growth of benign tumors. In addition to brain lesions, neurologic sequelae represent the greatest morbidity in TSC patients. Investigations utilizing TSC1/2 -knockout animal or human stem cell models suggest that TSC deficiency-causing hyper-activation of mTOR signaling might precipitate anomalous neurodevelopmental processes. However, how the pathogenic variants of TSC1/2 genes affect the longitudinal trajectory of human brain development remains largely unexplored. Here, we employed 3-dimensional cortical organoids derived from induced pluripotent stem cells (iPSCs) from TSC patients harboring TSC2 variants, alongside organoids from age- and sex-matched healthy individuals as controls. Through comprehensively longitudinal molecular and cellular analyses of TSC organoids, we found that TSC2 pathogenic variants dysregulate neurogenesis, synaptogenesis, and gliogenesis, particularly for reactive astrogliosis. The altered developmental trajectory of TSC organoids significantly resembles the molecular signatures of neuropsychiatric disorders, including autism spectrum disorders, epilepsy, and intellectual disability. Intriguingly, single cell transcriptomic analyses on TSC organoids revealed that TSC2 pathogenic variants disrupt the neuron/reactive astrocyte crosstalk within the NLGN-NRXN signaling network. Furthermore, cellular and electrophysiological assessments of TSC cortical organoids, along with proteomic analyses of synaptosomes, demonstrated that the TSC2 variants precipitate perturbations in synaptic transmission, neuronal network activity, mitochondrial translational integrity, and neurofilament formation. Notably, similar perturbations were observed in surgically resected cortical specimens from TSC patients. Collectively, our study illustrates that disease-associated TSC2 variants disrupt the neurodevelopmental trajectories through perturbations of gene regulatory networks during early cortical development, leading to mitochondrial dysfunction, aberrant neurofilament formation, impaired synaptic formation and neuronal network activity.

Dose-dependent effects of gamma radiation sterilization on the collagen matrix of human cortical bone allograft and its influence on fatigue crack propagation resistance

Fatigue crack propagation resistance and high-cycle S–N fatigue life of cortical bone allograft tissue are both negatively impacted in a radiation dose-dependent manner from 0 to 25 kGy. The standard radiation sterilization dose of 25–35 kGy has been shown to induce cleavage of collagen molecules into smaller peptides and accumulation of stable crosslinks within the collagen matrix, suggesting that these mechanisms may influence radiation-induced losses in cyclic fracture resistance. The objective of this study was to determine the radiation dose-dependency of collagen chain fragmentation and crosslink accumulation within the dose range of 0–25 kGy. Previously, cortical bone compact tension specimens from two donor femoral pairs were divided into four treatment groups (0 kGy, 10 kGy, 17.5 kGy, and 25 kGy) and underwent cyclic loading fatigue crack propagation testing. Following fatigue testing, collagen was isolated from one compact tension specimen in each treatment group from both donors. Radiation-induced collagen chain fragmentation was assessed using SDS-PAGE (n = 5), and accumulation of pentosidine, pyridinoline, and non-specific advanced glycation end products were assessed using a fluorometric assay (n = 4). Collagen chain fragmentation increased progressively in a dose-dependent manner (p < 0.001). Crosslink accumulation at all radiation dose levels increased relative to the 0 kGy control but did not demonstrate dose-dependency (p < 0.001). Taken together with our previous findings on fatigue crack propagation behavior, these data suggest that while collagen crosslink accumulation may contribute to reduced notched fatigue behavior with irradiation, dose-dependent losses in fatigue crack propagation resistance are mainly influenced by radiation-induced chain fragmentation.

Cortical and trabecular mechanical properties in the femoral neck vary differently with changes in bone mineral density.

Graphical Abstract.

133 BMAL1 is Upregulated in Pediatric High-Grade Gliomas

INTRODUCTION: Pediatric high-grade gliomas (pHGG) are highly invasive brain tumors with poor survival outcomes, and novel therapeutic approaches are urgently needed. Recently, a retrospective study found timing of Temozolomide administration affects survival in adult patients with glioblastoma (Damato et al. 2021). There has, however, been limited exploration into circadian gene expression in pediatric gliomas. METHODS: We analyzed expression of BMAL1, a key circadian gene, in pediatric gliomas using bioinformatic approaches, Western blotting, and immunocytochemistry (ICC). We first compared BMAL1 expression in pLGGs and pHGGs using published publicly available data from cBioPortal for Cancer Genomics. Immunoblotting was utilized to determine BMAL1 protein expression levels in patient specimens of pHGG, pLGG and cortex from epilepsy surgery. Immunocytochemistry on synchronized primary tumor lines was used to show localization of BMAL1 protein. RESULTS: Using data from cBioPortal, we found significantly higher expression of BMAL1 in pHGGs compared to pLGGs (0.349 + 1.05 vs 0.0545 + 0.666, p &lt; 0.001). Western blotting showed increased expression of BMAL1 in HGG tumor specimens compared to LGG and cortical specimens. Immunostaining demonstrated cellular localization of BMAL1. CONCLUSIONS: In this study, we found that the circadian gene BMAL1 is expressed in pHGGs, and that expression is higher in pHGGs compared to pLGGs and cortex. Next steps will be to understand how timing of Temozolomide in relationship to the circadian cycle affects tumor cell survival.

Comprehensively characterizing heterogeneous and transversely isotropic properties of femur cortical bones

Comprehensive characterization of the transversely isotropic mechanical properties of long bones along both the longitudinal and circumferential gradients is crucial for developing accurate mathematical models and studying bone biomechanics. In addition, mechanical testing to derive elastic, plastic, and failure properties of bones is essential for modeling plastic deformation and failure of bones. To achieve these, we machined a total of 336 cortical specimens, including 168 transverse and 168 longitudinal specimens, from four different quadrants of seven different sections of 3 bovine femurs. We conducted three-point bending tests of these specimens at a loading rate of 0.02 mm/s. Young’s modulus, yield stress, tangential modulus, and effective plastic strain for each specimen were derived from correction equations based on classical beam theory. Our statistical analysis reveals that the longitudinal gradient has a significant effect on the Young’s modulus, yield stress, and tangential modulus of both longitudinal and transverse specimens, whereas the circumferential gradient significantly influences the Young’s modulus, yield stress, and tangential modulus of transverse specimens only. The differences in Young’s modulus and yield stress between longitudinal specimens from different sections are greater than 40%, whereas those between transverse specimens are approximately 30%. The Young’s modulus and yield stress of transverse specimens in the anterior quadrant were 18.81%/15.46% and 18.34%/14.88% higher than those in the posterior and lateral quadrants, respectively. There is no significant interaction between the longitudinal gradient and the circumferential gradient. Considering the transverse isotropy, it is crucial to consider loading direction when investigating the impact of circumferential gradients in the anterior, lateral, medial, and posterior directions. Our findings indicate that the conventional assumption of homogeneity in simulating the cortical bone of long bones may have limitations, and researchers should consider the anatomical position and loading direction of femur specimens for precise prediction of mechanical responses.

HDAC6-dependent deacetylation of NGF dictates its ubiquitination and maintains primordial follicle dormancy.

Rationale: Primordial follicles are limited in number and cannot be regenerated, dormant primordial follicles cannot be reversed once they enter a growth state. Therefore, the length of the female reproductive lifespan depends on the orderly progression and selective activation of primordial follicles, the mechanism of which remains unclear. Methods: We used human ovarian cortical biopsy specimens, granulosa cells from diminished ovarian reserve (DOR) patients, Hdac6-overexpressing transgenic mouse model, and RNA sequencing to analyze the crucial roles of histone deacetylase 6 (HDAC6) in fertility preservation and primordial follicle activation. Results: In the present study, we found that HDAC6 was highly expressed in most dormant primordial follicles. The HDAC6 expression was reduced accompanying reproductive senescence in human and mouse ovaries. Overexpression of Hdac6 delayed the rate of primordial follicle activation, thereby prolonging the mouse reproductive lifespan. Short-term inhibition of HDAC6 promoted primordial follicle activation and follicular development in humans and mice. Mechanism studies revealed that HDAC6 directly interacted with NGF, reducing acetylation modification of NGF and thereby accelerating its ubiquitination degradation. Consequently, the reduced NGF protein level maintained the dormancy of primordial follicles. Conclusions: The physiological significance of the high expression of HDAC6 in most primordial follicles is to reduce NGF expression and prevent primordial follicle activation to maintain female fertility. Reduced HDAC6 expression increases NGF expression in primordial follicles, activating their development and contributing to reproduction. Our study provides a clinical reference value for fertility preservation.

Efficacy and Safety of CT-Guided Kidney Biopsy for the Diagnosis of Glomerular Diseases in Complicated Patients

Introduction: Kidney biopsy is the cornerstone for the diagnosis of glomerular diseases and to guide treatment. Percutaneous ultrasound-guided kidney biopsy is currently the gold standard to obtain cortical specimens. However, in cases where ultrasound-guided kidney biopsy is not deemed safe (obese patients, deep kidneys, or kidneys with a complicated anatomy), CT-guided kidney biopsy could be a convenient alternative to obtain renal tissue samples. The aim of this study was to describe the diagnostic yield and complications of CT-guided kidney biopsies in patients with glomerular diseases that were previously discarded for ultrasound-guided kidney biopsy. Material and Methods: We performed a retrospective, single-center, observational study including patients who underwent CT-guided native kidney biopsies in our center after being contraindicated for ultrasound-guided biopsy. Patients’ records were reviewed retrieving baseline characteristics and pre-biopsy clinical, laboratory parameters and concomitant medication. The biopsy needle gauge, site of puncture, and number of needle passes were recorded. The diagnostic yield was evaluated by the number of glomeruli obtained, the rate of specimens that were adequate to reach diagnosis, and the number of biopsies that had to be repeated. Complications were defined as minor (hypotension, hematoma) and major (arteriovenous fistulae, major bleeding requiring embolization, or nephrectomy). The diagnostic yield and complications were compared to ultrasound-guided native kidney biopsies performed during the same period. Results: 56 CT-guided native kidney biopsies were performed during the study period. The number of glomeruli obtained per patient was 11.5 ± 6.3, which was inferior to that obtained from ultrasound-guided biopsies (14.08 ± 8.47, p < 0.05). However, the rate of specimens that were adequate to reach a diagnosis was similar (92.9% vs. 90.8%, p = 0.437). The number of needle passes was higher in CT-guided kidney biopsies (2.0 ± 0.7 vs. 1.7 ± 0.5, p < 0.05), as well as the incidence of post-biopsy perirenal asymptomatic hematomas (66.1% vs. 24.5%, p < 0.01). There were no significant differences in other post-biopsy minor complications (1.8% vs. 2.5%, p = 0.621). There were no major complications after CT-guided kidney biopsies. Conclusions: CT-guided percutaneous kidney biopsy is a valid alternative for the diagnosis of glomerular diseases in patients with special characteristics such as obesity or deep kidneys that contraindicate ultrasound-guided biopsy. In this population, CT-guided kidney biopsies are safe and provide a high diagnostic yield, reaching a diagnosis in >90% of patients that had been previously discarded for ultrasound-guided biopsy.

Aberrant adenosine signaling in patients with focal cortical dysplasia.

Focal cortical dysplasia (FCD), a common malformation of cortical development, is frequently associated with pharmacoresistant epilepsy in both children and adults. Adenosine is an inhibitory modulator of brain activity and a prospective anti-seizure agent with potential for clinical translation. Our previous results demonstrated that the major adenosine-metabolizing enzyme adenosine kinase (ADK) was upregulated in balloon cells (BCs) within FCD type IIB lesions, suggesting that dysfunction of the adenosine system is implicated in the pathophysiology of FCD. In our current study, we therefore performed a comprehensive analysis of adenosine signaling in surgically resected cortical specimens from patients with FCD type I and type II via immunohistochemistry and immunoblot analysis. Adenosine enzyme signaling was assessed by quantifying the levels of the key enzymes of adenosine metabolism, i.e., ADK, adenosine deaminase (ADA), and ecto-5'-nucleotidase (CD73). Adenosine receptor signaling was assessed by quantifying the levels of adenosine A2A receptor (A2AR) and putative downstream mediators of adenosine, namely, glutamate transporter-1 (GLT-1) and mammalian target of rapamycin (mTOR). Within lesions in FCD specimens, we found that the adenosine-metabolizing enzymes ADK and ADA, as well as the adenosine-producing enzyme CD73, were upregulated. We also observed an increase in A2AR density, as well as a decrease in GLT-1 levels and an increase in mTOR levels, in FCD specimens compared with control tissue. These results suggest that dysregulation of the adenosine system is a common pathologic feature of both FCD type I and type II. The adenosine system might therefore be a therapeutic target for the treatment of epilepsy associated with FCD.

Accumulation of fluorescent advanced glycation end products and carboxymethyl-lysine in human cortical and trabecular bone

Chemical crosslinks known as advanced glycation end-products (AGEs) are associated with increased bone fracture risk and deteriorated bone mechanical properties. However, measurement of bone AGEs via ex vivo and in vitro methods has been limited to quantification of bulk fluorescent AGEs (fAGEs) and pentosidine only, which is a crosslinking fluorescent AGE. However, a non-crosslinking and non-fluorescent AGE such as carboxymethyl-lysine (CML) is found to be 40–100 times higher in quantity than pentosidine, but only one previous study has reported it in cortical bone, and one study reported it in trabecular bone. In our study, we wanted to investigate if accumulation of CML differs in cortical and trabecular compartments and if they are more strongly associated with bone mechanical properties than with fAGEs. We hypothesized that CML and fAGEs level would be higher in the trabecular compartment and show negative correlations to mechanical properties in cortical and trabecular bone. We obtained human cadaveric cortical and trabecular bone specimens, induced the formation of AGEs via the established in vitro ribosylation method, imaged specimens by microcomputed tomography to assess specimen geometry and microarchitecture, and mechanically tested cortical specimens by cyclic reference point indentation and fracture toughness tests and trabecular specimens by compression tests, followed by measurement of fAGEs and CML. fAGEs were 22 % higher in cortical bone (687 ± 44.8 ng Q/mg collagen) compared to trabecular bone (859 ± 317.1 ng Q/mg collagen), whereas CML levels were found to be 148 % higher in trabecular bone (6189.9 ± 866 ng/mg of protein) compared to cortical bone (924.6 ± 576.3 ng/mg of protein). Pooling the specimens from both the control and ribose groups, Spearman correlation analysis indicated that CML levels, but not fAGEs, are moderately associated with cortical porosity (r = +0.505, p ≤ 0.05) and mechanical properties such indentation depth (r = +0.460, p ≤ 0.05), total indentation depth (r = +0.440, p ≤ 0.05), and average energy dissipated (r = +0.465, p ≤ 0.05) in cortical bone. fAGEs showed a trend towards negative association with crack propagation toughness in cortical bone (r = −0.365, p = 0.055). No significant correlations were observed between CML and microarchitecture or mechanical properties in trabecular bone. CML levels were also associated with fAGEs in cortical bone (r = +0.596, p ≤ 0.05) but not in trabecular bone. Our preliminary findings indicate that CML, a non-crosslinking AGE, may affect bone material and mechanical properties differently than bulk fluorescent AGEs, given the higher accumulation of CML in each bone compartment. This study provides direction to future studies to quantify crosslinking and non-crosslinking AGEs separately as their effect on material and mechanical properties may be different and it would help identify better biomarkers for bone strength prediction.

Comparison Between Cancellous Trabecular and Cortical Specimens from Human Lumbar Spine Samples as an Alternative Source of Mesenchymal Stromal Cells.

Due to their immunosuppressive potential and ability to differentiate into multiple musculoskeletal cell lineages, mesenchymal stromal cells (MSCs) became popular in clinical trials for the treatment of musculoskeletal disorders. The aim of this study was to isolate and characterize native populations of MSCs from human cortical and cancellous bone from the posterior elements of the lumbar spine and determine what source of MSCs yields better quality and quantity of cells to be potentially used for spinal fusion repair. We were able to show that MSCs from trabecular and cortical spine had the typical MSC morphology and expression markers; the ability to differentiate in adipocyte, chondrocyte, or osteoblast but they did not have a consistent pattern in the expression of the specific differentiation lineage genes. Moreover, MSCs from both sites demonstrated an immune suppression profile suggesting that these cells may have a more promising success in applications related to immunomodulation more than exploring their ability to drive osteogenesis to prevent nonunion in spine fusion procedures.

Mapping astrogliosis in the individual human brain using multidimensional MRI.

There are currently no non-invasive imaging methods available for astrogliosis assessment or mapping in the central nervous system despite its essential role in the response to many disease states, such as infarcts, neurodegenerative conditions, traumatic brain injury and infection. Multidimensional MRI is an increasingly employed imaging modality that maximizes the amount of encoded chemical and microstructural information by probing relaxation (T1 and T2) and diffusion mechanisms simultaneously. Here, we harness the exquisite sensitivity of this imagining modality to derive a signature of astrogliosis and disentangle it from normative brain at the individual level using machine learning. We investigated ex vivo cerebral cortical tissue specimens derived from seven subjects who sustained blast-induced injuries, which resulted in scar-border forming astrogliosis without being accompanied by other types of neuropathological abnormality, and from seven control brain donors. By performing a combined post-mortem radiology and histopathology correlation study we found that astrogliosis induces microstructural and chemical changes that are robustly detected with multidimensional MRI, and which can be attributed to astrogliosis because no axonal damage, demyelination or tauopathy were histologically observed in any of the cases in the study. Importantly, we showed that no one-dimensional T1, T2 or diffusion MRI measurement can disentangle the microscopic alterations caused by this neuropathology. Based on these findings, we developed a within-subject anomaly detection procedure that generates MRI-based astrogliosis biomarker maps ex vivo, which were significantly and strongly correlated with co-registered histological images of increased glial fibrillary acidic protein deposition (r = 0.856, P < 0.0001; r = 0.789, P < 0.0001; r = 0.793, P < 0.0001, for diffusion-T2, diffusion-T1 and T1-T2 multidimensional data sets, respectively). Our findings elucidate the underpinning of MRI signal response from astrogliosis, and the demonstrated high spatial sensitivity and specificity in detecting reactive astrocytes at the individual level, and if reproduced in vivo, will significantly impact neuroimaging studies of injury, disease, repair and aging, in which astrogliosis has so far been an invisible process radiologically.

Experimental evaluation of fracture properties of bovine hip cortical bone using elastic-plastic fracture mechanics.

Understanding the fracture mechanics of bone is very important in both the medical and bioengineering field. Bone is a hierarchical natural composite material of nanoscale collagen fibers and inorganic material. This study investigates and presents the fracture toughness of bovine cortical bone by using elastic plastic fracture mechanics. The J-integral was used as a parameter to calculate the energies utilized in both elastic deformation (Jel) and plastic deformation (Jpl) of the hipbone fracture. Twenty four different types of specimens, i.e. longitudinal compact tension (CT) specimens, transverse CT specimens, and also rectangular unnotched specimens for tension in longitudinal and transverse orientation, were cut from the bovine hip bone of the middle diaphysis. All CT specimens were prepared according to the American Society for Testing and Materials (ASTM) E1820 standard and were tested at room temperature. The results showed that the average total J-integral in transverse CT fracture specimens is 26% greater than that of longitudinal CT fracture specimens. For longitudinal-fractured and transverse-fractured cortical specimens, the energy used in the elastic deformation was found to be 2.8-3 times less than the energy used in the plastic deformation. The findings indicate that the overall fracture toughness measured using the J-integral is significantly higher than the toughness calculated by the stress intensity factor. Therefore, J-integral should be employ to compute the fracture toughness of cortical bone.

Increased inflammasome-activated pyroptosis mediated by caspase-1 in Rasmussen’s encephalitis

BackgroundRasmussen’s encephalitis (RE) is a rare, progressive disease characterized by unilateral cerebral hemisphere atrophy. Studies showed that inflammatory response and overexpressed chemokines were present in RE patients. The present study aims to determine whether caspase-1- mediated neuronal pyroptosis occurred in RE. MethodsImmunohistochemistry and Western blotting analysis were used to examine the expression of Gasdermin D (GSDMD), NOD-like receptor protein 1 (NLRP1), NOD-like receptor protein 3 (NLRP3), caspase-1, and pro-caspase-1 in RE and control cortical specimens (n = 14). Perilesional tissue specimens from six focal cortical dysplasia (FCD) cases were used as controls. Double staining showed the colocalization of GSDMD, NLRP1, NLRP3 and caspase-1. Enzyme-linked immunosorbent assay (ELISA) was used to quantify the amount of interleukin (IL)−1β and IL-18 in RE cortical specimens. ResultsCompared with the control cortex, we found higher GSDMD expression in the cytoplasm of neurons in RE cortex but no detectable expression in astrocytes and microglia. Further analysis revealed that NLRP1, NLRP3, caspase-1 and its precursor pro-caspase-1 were also upregulated in the RE, and predominantly localized in the cytoplasm of the neurons. In addition, significantly higher levels of IL-1β and IL-18 were present in the RE group compared with the control group. ConclusionOur results suggest that pyroptosis represents an important pathway for neuronal loss in the pathological processes associated with RE, and that targeting the canonical inflammasome pathway of pyroptosis may provide potential therapeutic value for RE.

Isolation of Adult Human Astrocyte Populations from Fresh-frozen Cortex using Fluorescence-Activated Nuclei Sorting.

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handlingfresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozenpostmortem human temporal neocortex tissue. This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

Is the 0.2%-Strain-Offset Approach Appropriate for Calculating the Yield Stress of Cortical Bone?

The 0.2% strain offset approach is mostly used to calculate the yield stress and serves as an efficient method for cross-lab comparisons of measured material properties. However, it is difficult to accurately determine the yield of the bone. Especially when computational models require accurate material parameters, clarification of the yield point is needed. We tested 24 cortical specimens harvested from six bovine femora in three-point bending mode, and 11 bovine femoral cortical specimens in the tensile mode. The Young's modulus and yield stress for each specimen derived from the specimen-specific finite element (FE) optimization method was regarded as the most ideal constitutive parameter. Then, the strain offset optimization method was used to find the strain offset closest to the ideal yield stress for the 24 specimens. The results showed that the 0 strain offsets underestimated (- 25%) the yield stress in bending and tensile tests, while the 0.2% strain offsets overestimated the yield stress (+ 65%) in three-point bending tests. Instead, the yield stress determined by 0.007 and 0.05% strain offset for bending and tensile loading respectively, can effectively characterize the biomechanical responses of the bone, thereby helping to build an accurate FE model.

Acoustic emissions in vertebral cortical shell failure

Understanding the initiation of bony failure is critical in assessing the progression of bone fracture and in developing injury criteria. Detection of acoustic emissions in bone can be used to identify fractures more sensitively and at an earlier inception time compared to traditional methods. However, high rate loading conditions, complex specimen-device interaction or geometry may cause other acoustic signals. Therefore, characterization of the isolated local acoustic emission response from cortical bone fracture is essential to distinguish its characteristics from other potential acoustic sources. This work develops a technique to use acoustic emission signals to determine when cortical bone failure occurs by characterization using both a Welch power spectral density estimate and a continuous wavelet transform. Isolated cortical shell specimens from thoracic vertebral bodies with attached acoustic sensors were subjected to quasistatic loading until failure. The resulting acoustic emissions had a wideband frequency response with peaks from 20 to 900 kHz, with the spectral peaks clustered in three bands of frequencies (166 ± 52.6 kHz, 379 ± 37.2 kHz, and 668 ± 63.4 kHz). Using these frequency bands, acoustic emissions can be used as a monitoring tool in biomechanical spine testing, distinguishing bone failure from structural response. This work presents a necessary set of techniques for effectively utilizing acoustic emissions to determine the onset of cortical bone fracture in biological material testing. Acoustic signatures can be developed for other cortical bone regions of interest using the presented methods.

Adjustable Cortical Fixation Device for Quadriceps Tendon Repair: A Cadaveric Biomechanical Study.

Background:Adjustable cortical fixation devices have demonstrated utility in orthopaedic applications, such as ankle syndesmosis repair.Purpose:To assess the cyclic gap formation of a quadriceps tendon repair technique using an adjustable cortical fixation device compared with repair with knotless suture anchors and suture tape, a modification of conventional suture anchor repair.Study Design:Controlled laboratory study.Methods:Eight fresh-frozen matched pairs of cadaveric knees were used. Specimens in each pair were randomized to undergo either modified suture anchor repair (control) or adjustable cortical fixation repair. The control repair was performed as previously described. The experimental repair was performed using 2 No. 2 FiberWire sutures placed into the quadriceps tendon in a running locked Krackow configuration and 2 adjustable loop devices passed through transosseous tunnels. The lagging strands of the devices were tensioned to seat the cortical fixation buttons at the inferior patellar pole and then tied to the free Krackow strands at the superior pole to complete the repair. The mean plastic gap (permanent tendon displacement that did not recover with cyclic extension) and mean maximum gap (peak displacement that occurred with cyclic knee flexion and partially recovered with extension) were evaluated during cyclic loading for 500 cycles of full knee extension to 90° of flexion.Results:At all testing intervals, the mean plastic gap was significantly smaller for the cortical fixation group versus the suture anchor group (P < .02). Similarly, the mean maximum gap was significantly smaller for the cortical fixation specimens at all testing intervals (P < .01). After cyclic loading, the mean maximum gap was significantly smaller in the cortical fixation group (4.80 ± 1.56 mm) versus the suture anchor group (8.47 ± 1.47 mm; P = < .001). The mean plastic gap was also significantly smaller in the cortical fixation versus the suture anchor group (3.25 ± 1.10 mm vs 6.57 ± 1.62 mm, respectively; P = < .001).Conclusion:Quadriceps tendon repair using an adjustable cortical fixation device demonstrated superior biomechanical properties in cyclic displacement testing compared with repair using the suture anchor technique.Clinical Relevance:These results suggest that an adjustable cortical fixation device is a biomechanically viable alternative for quadriceps tendon repair.

Irreversible Neuronal Damage Begins Just After Aneurysm Rupture in Poor-Grade Subarachnoid Hemorrhage Patients.

Pathophysiological findings of early brain injury in humans have not permitted conclusive determinations. We explored the essence of this phenomenon by taking intraoperative cortical specimens of Hunt-Kosnik grades IV~V (poor-grade) subarachnoid hemorrhages (SAH). From 2013 to 2017, we treated 39 consecutive poor-grade patients in 226 cases of aneurysmal SAH. Fourteen of the 39 patients agreed to this study following written informed consent. We took specimens from untouched areas prior to surgical intervention: cortex near the ruptured aneurysm for clipping, convexity cortex for cerebral ventricular drainage. Cortical specimens were stained with hematoxylin-eosin, anti-cleaved caspase-3, and anti-DNA/RNA damage staining. Positive signals were calculated in six random, high-power fields for quantitative assessment. Double immunofluorescence was done to evaluate neural damage. Chi-square analyses were carried out to assess the correlation between the Glasgow Outcome Scale at 90days after the ictus and the number of positive cells. Cortical specimens were taken at 12.7±7.00h after the first ictus. All 14 cases showed dense nuclei, with the appearance of acidic and shrunken cytoplasms. Diffuse positivity of anti-cleaved caspase-3 and anti-DNA/RNA damage was detected. Cleaved caspase-3 was detected in 68% of neurons, and DNA/RNA damage was detected in 64% of neurons. Positive reactions of both antibodies indicated poor outcome. With poor-grade cases, irreversible ischemic, apoptotic, and oxidative changes were detected in the cerebral cortex within several hours after the ictus. Those changes occurred far from the aneurysm. Our findings suggest that a revolution is needed in the treatment strategy for poor-grade SAH.

Macroclimate drives growth of hair lichens in boreal forest canopies

Abstract Epiphytic lichens are important biodiversity components of forest canopies world‐wide, significantly contributing to ecosystem function. The RGR, a measure of fitness, drives population dynamics and shapes lichens’ large‐scale distributions. In a climate change scenario, we need to know how external (macro‐ and microclimate, and nitrogen deposition) and internal factors (cortical pigments, chlorophyll and specimen size) affect RGR in these ecologically important canopy organisms. We used dominant pendulous hair lichens widely distributed across the boreal biome to test the hypothesis that precipitation drives RGR of pale (Alectoria sarmentosa, Usnea dasopoga) and dark species Bryoria fuscescens differently across a large‐scale gradient from continental to oceanic climates (precipitation: 450–2,600 mm) in Scandinavia (60–64°N, 5–19°E). After transplanting lichens to lower branches of Picea abies in nine boreal forest sites for 1 year, we used linear mixed effects models to analyse how total precipitation, rainfall, number of days with rain, temperature sum, nitrogen deposition, light, chlorophyll a (an indicator of photosynthetic capacity) and size influenced their RGR. RGR was highest in the pale species (Alectoria and Usnea) and increased with amount and frequency of precipitation, with &gt;3 times higher RGR in the wettest compared to the driest site. The number of days with rain was a better predictor of RGR than total precipitation or rain. By contrast, RGR of the dark Bryoria weakly increased with precipitation. RGR in all species increased with light and decreased with size. Chlorophyll a concentration, boosted by moderate nitrogen deposition, increased RGR of all species. In conclusion, rainfall likely drives the distribution of the pale species due to their higher RGR and abundance in wet climates but cannot explain why Bryoria dominates drier inland forests. Our results highlight that the functional links between rainfall and RGR depends on both colour of the lichens (pale vs. dark pigments) and water storage traits. Synthesis. Our findings may explain the global, regional and local distribution patterns of hair lichens and help us to predict how environmental hazards such as climate change and forestry influence these important boreal canopy components.

Accurate Detection of Hot-Spot MTOR Somatic Mutations in Archival Surgical Specimens of Focal Cortical Dysplasia by Molecular Inversion Probes.

Formalin-fixed, paraffin-embedded brain specimens are a potentially rich resource to identify somatic variants, but their DNA is characterised by low yield and extensive degradation, and matched peripheral samples are usually unavailable for analysis. We designed single-molecule molecular inversion probes to target 18 MTOR somatic mutational hot-spots in unmatched, histologically proven focal cortical dysplasias from formalin-fixed, paraffin-embeddedtissues of50 patients. We achieved adequate DNA and sequencing quality in 28 focal cortical dysplasias, mostly extracted within 2years from fixation, showing a statistically significant effect of time from fixation as a major determinant for successful genetic analysis. We identified and validated seven encompassing hot-spot residues (foundin14% of all patients and in25% ofthose sequenced andanalysed). The allele fraction had a range of 2-5% and variants were absent in available neighbouring non-focal cortical dysplasia specimens. We computed an alternate allele threshold for calling true variants, based on an experiment-wise mismatch count distribution, well predicting call reliability. Single-molecule molecular inversion probes are experimentally simple, cost effective and scalable, accurately detecting clinically relevant somatic variants in challenging brain formalin-fixed, paraffin-embedded tissues.