Human-specific epigenomic states in spermatogenesis.

Infertility is becoming increasingly common, affecting one in six people globally. Half of these cases can be attributed to male factors, many driven by abnormalities in the process of sperm development. Emerging evidence from genome-wide association studies, genetic screening of patient cohorts, and animal models highlights an important genetic contribution to spermatogenic defects, but comprehensive identification and characterization of the genes critical for male fertility remain lacking. High divergence of gene regulation in spermatogenic cells across species poses challenges for delineating the genetic pathways required for human spermatogenesis using common model organisms. In this study, we leveraged post-translational histone modification and gene transcription data for 15,491 genes in four mammalian species (human, rhesus macaque, mouse, and opossum), to identify human-specific patterns of gene regulation during spermatogenesis. We combined H3K27me3 ChIP-seq, H3K4me3 ChIP-seq, and RNA-seq data to define epigenetic states for each gene at two stages of spermatogenesis, pachytene spermatocytes and round spermatids, in each species. We identified 239 genes that are uniquely active, poised, or dynamically regulated in human spermatogenic cells distinct from the other three species. While some of these genes have been implicated in reproductive functions, many more have not yet been associated with human infertility and may be candidates for further molecular and epidemiologic studies. Our analysis offers an example of the opportunities provided by evolutionary and epigenomic data for broadly screening candidate genes implicated in reproduction, which might lead to discoveries of novel genetic targets for diagnosis and management of male infertility and male contraception.

Sporadic pituitary adenoma with somatic double-hit loss of MEN1.

PURPOSE: Pituitary adenomas commonly arise in patients with MEN1 syndrome, an autosomal dominant condition predisposing to neuroendocrine tumor formation, and typically diagnosed in patients with a relevant family cancer history. In these patients with existing germline loss of MEN1 on one allele, somatic loss of the second MEN1 allele leads to complete loss of the MEN1 protein, menin, and subsequent tumor formation. METHODS: Whole exome sequencing was performed on the tumor and matching blood under an institutional board approved protocol. DNA extraction and analysis was conducted according to previously described methods. RESULTS: We describe a 23 year-old patient with no significant past medical history or relevant family history who underwent surgical resection of a symptomatic and medically resistant prolactinoma. Whole exome sequencing of tumor and blood samples revealed somatic loss of MEN1 at both alleles, suggesting a double hit mechanism, with no underlying germline MEN1 mutation. CONCLUSION: To our knowledge, this is the first case of pituitary adenoma to arise from somatic loss of MEN1 and in the absence of an underlying germline MEN1 mutation.

Single-cell analysis reveals inflammatory interactions driving macular degeneration.

Due to commonalities in pathophysiology, age-related macular degeneration (AMD) represents a uniquely accessible model to investigate therapies for neurodegenerative diseases, leading us to examine whether pathways of disease progression are shared across neurodegenerative conditions. Here we use single-nucleus RNA sequencing to profile lesions from 11 postmortem human retinas with age-related macular degeneration and 6 control retinas with no history of retinal disease. We create a machine-learning pipeline based on recent advances in data geometry and topology and identify activated glial populations enriched in the early phase of disease. Examining single-cell data from Alzheimer's disease and progressive multiple sclerosis with our pipeline, we find a similar glial activation profile enriched in the early phase of these neurodegenerative diseases. In late-stage age-related macular degeneration, we identify a microglia-to-astrocyte signaling axis mediated by interleukin-1ß which drives angiogenesis characteristic of disease pathogenesis. We validated this mechanism using in vitro and in vivo assays in mouse, identifying a possible new therapeutic target for AMD and possibly other neurodegenerative conditions. Thus, due to shared glial states, the retina provides a potential system for investigating therapeutic approaches in neurodegenerative diseases.

Neuropathology of New-Onset Refractory Status Epilepticus (NORSE).

New-Onset Refractory Status Epilepticus (NORSE), including its subtype with a preceding febrile illness known as FIRES (Febrile Infection-Related Epilepsy Syndrome), is one of the most severe forms of status epilepticus. Despite an extensive workup (clinical evaluation, EEG, imaging, biological tests), the majority of NORSE cases remain unexplained (i.e., "cryptogenic NORSE"). Understanding the pathophysiological mechanisms underlying cryptogenic NORSE and the related long-term consequences is crucial to improve patient management and preventing secondary neuronal injury and drug-resistant post-NORSE epilepsy. Previously, neuropathological evaluations conducted on biopsies or autopsies have been found helpful for identifying the etiologies of some cases that were previously of unknown cause. Here, we summarize the findings of studies reporting neuropathology findings in patients with NORSE, including FIRES. We identified 64 cryptogenic cases and 66 neuropathology tissue samples, including 37 biopsies, 18 autopsies, and seven epilepsy surgeries (the type of tissue sample was not detailed for 4 cases). We describe the main neuropathology findings and place a particular emphasis on cases for which neuropathology findings helped establish a diagnosis or elucidate the pathophysiology of cryptogenic NORSE, or on described cases in which neuropathology findings supported the selection of specific treatments for patients with NORSE.

High-plex protein and whole transcriptome co-mapping at cellular resolution with spatial CITE-seq.

In this study, we extended co-indexing of transcriptomes and epitopes (CITE) to the spatial dimension and demonstrated high-plex protein and whole transcriptome co-mapping. We profiled 189 proteins and whole transcriptome in multiple mouse tissue types with spatial CITE sequencing and then further applied the method to measure 273 proteins and transcriptome in human tissues, revealing spatially distinct germinal center reactions in tonsil and early immune activation in skin at the Coronavirus Disease 2019 mRNA vaccine injection site.

Spatial-CITE-seq: spatially resolved high-plex protein and whole transcriptome co-mapping.

We present spatial-CITE-seq for high-plex protein and whole transcriptome co-mapping, which was firstly demonstrated for profiling 198 proteins and transcriptome in multiple mouse tissue types. It was then applied to human tissues to measure 283 proteins and transcriptome that revealed spatially distinct germinal center reaction in tonsil and early immune activation in skin at the COVID-19 mRNA vaccine injection site. Spatial-CITE-seq may find a range of applications in biomedical research.

Atypical thymomas with squamoid and spindle cell features: clinicopathologic, immunohistochemical and molecular genetic study of 120 cases with long-term follow-up.

Thymomas are rare tumors characterized by a broad range of morphologic appearances that can sometimes give rise to difficulties for classification. We have studied a series of 120 thymoma patients in whom the tumors were characterized by sheets of atypical epithelial cells with squamoid and/or spindle cell features. They occurred in 63 men and 57 women and presented as a discrete mass in the anterior mediastinum measuring 2-23 cm (mean: 8.2 cm). Patients' ages ranged from 14 to 86 years (mean: 57.8) and most had symptoms referable to a mass lesion. 20 patients had myasthenia gravis or other autoimmune disorder. 76 cases were characterized by a predominant population of round to polygonal tumor cells while 32 cases were characterized by atypical oval or spindle cells. 12 cases showed mixed features and 16 cases showed the development of thymic carcinoma arising from thymoma. All cases were positive for p40/p63 and cytokeratin AE1/AE3. 23 cases were positive for CD5 (25%), and 13 for CD117 (14%). MIB1 showed a significant increase in proliferative activity (mean = 11.6%). Next generation sequencing in 47 cases did not disclose any variants amenable to current targeted therapies. Clinical follow up ranging from 2 to 29 years showed a progressive increase in aggressive behavior and fatality rate with advancing stage. Overall survival was 87% at 5 years, 67% at 10 years, and 23% at 20 years. Completeness of resection and staging were the most significant parameters for survival. The more aggressive tumors followed a protracted clinical course with multiple recurrences and metastases over a long period of time (mean = 19.8 years from time of initial relapse to death). Atypical thymomas are a distinct category of thymic epithelial neoplasm characterized by a slowly progressive clinical course with increased potential for metastases, transformation to a higher-grade malignancy, and fatal outcome in some cases.

Case Report: Frontoparietal Metastasis From a Primary Fallopian Tube Carcinoma.

Background: Metastatic brain tumors typically arise from primary malignancies of the lung, kidney, breast, skin, and colorectum. Brain metastases originating from malignancies of the female genital tract are extremely rare. We present a case of fallopian tube brain metastasis and in so doing review the pertinent literature. Case Description: We describe a 59-year-old patient with a history of fallopian tube carcinoma who presented with an incidentally identified left frontal brain mass. MRI demonstrated an enhancing lesion in the left centrum semiovale with a second enhancing lesion noted in the cerebellar vermis. She underwent a left parietal craniotomy for resection of the dominant and clinically symptomatic lesion. Immunohistochemical stains were positive for PAX8 and p53, confirming fallopian tube origin. Conclusions: Fallopian tube cancer brain metastasis is extremely uncommon. We highlight the treatment and surgical resection of this patient's BRCA1 metastatic fallopian lesion and systematically review the literature regarding the pathogenesis, diagnosis, treatment, and histologic characteristics of the previously identified fallopian tube metastases to the central nervous system. The optimal course of treatment for brain metastasis of fallopian tube carcinoma has not been clearly defined due in part to the rarity of this condition. Consistent with BRCA1 neoplasms involving the breast and ovaries, the BRCA1 status of the patient's primary tumor likely increased the risk of central nervous system dissemination. This highlights a potential benefit of early screening of individuals with metastatic gynecologic malignancies associated with BRCA1 in the absence of any neurological symptoms.

T lymphocytes and cytotoxic astrocyte blebs correlate across autism brains.

OBJECTIVE: Autism spectrum disorder (ASD) affects 1 in 59 children, yet except for rare genetic causes, the etiology in most ASD remains unknown. In the ASD brain, inflammatory cytokine and transcript profiling shows increased expression of genes encoding mediators of the innate immune response. We evaluated postmortem brain tissue for adaptive immune cells and immune cell-mediated cytotoxic damage that could drive this innate immune response in the ASD brain. METHODS: Standard neuropathology diagnostic methods including histology and immunohistochemistry were extended with automated image segmentation to quantify identified pathologic features in the postmortem brains. RESULTS: We report multifocal perivascular lymphocytic cuffs contain increased numbers of lymphocytes in ~65% of ASD compared to control brains in males and females, across all ages, in most brain regions, and in white and gray matter, and leptomeninges. CD3+ T lymphocytes predominate over CD20+ B lymphocytes and CD8+ over CD4+ T lymphocytes in ASD brains. Importantly, the perivascular cuff lymphocyte numbers correlate to the quantity of astrocyte-derived round membranous blebs. Membranous blebs form as a cytotoxic reaction to lymphocyte attack. Consistent with multifocal immune cell-mediated injury at perivascular cerebrospinal fluid (CSF)-brain barriers, a subset of white matter vessels have increased perivascular space (with jagged contours) and collagen in ASD compared to control brains. CSF-brain barrier pathology is also evident at cerebral cortex pial and ventricular ependymal surfaces in ASD. INTERPRETATION: The findings suggest dysregulated cellular immunity damages astrocytes at foci along the CSF-brain barrier in ASD. ANN NEUROL 2019;86:885-898.

Odontoid cervical gout causing atlantoaxial instability: case report.

The authors present a case report of a patient discovered to have a rotatory subluxation of the C1-2 joint and a large retroodontoid pannus with an enhancing lesion in the odontoid process eventually proving to be caused by gout. This patient represented a diagnostic conundrum as she had known prior diagnoses of not only gout but also sarcoidosis and possible rheumatoid arthritis, and was in the demographic range where concern for an oncological process cannot fully be ruled out. Because she presented with signs and symptoms of atlantoaxial instability, she required posterior stabilization to reduce the rotatory subluxation and to stabilize the C1-2 instability. However, despite the presence of a large retroodontoid pannus, she had no evidence of spinal cord compression on physical examination or imaging and did not require an anterior procedure to decompress the pannus. To confirm the diagnosis but avoid additional procedures and morbidity, the authors proceeded with the fusion as well as a posterior biopsy to the retroodontoid pannus and confirmed a diagnosis of gout.

Histopathological Demonstration of Subacute Endothelialization Following Aneurysm Retreatment with the Pipeline Embolization Device.

BACKGROUND: Endothelial cell coverage along the Pipeline embolization device (PED) is 1 of 2 primary proposed mechanisms of action of the device, along with induction of intra-aneurysmal thrombosis. The temporal course of endothelialization following device deployment is poorly understood in human patients. CASE DESCRIPTION: A 63-year-old female with a persistent aneurysm in the communicating segment of the internal carotid artery was treated with a second PED 14 months after the deployment of a first PED and subsequently developed a fatal intraparenchymal hemorrhage at 3 weeks postimplantation. Histopathological analysis at autopsy showed evidence of endothelialization along the second PED at this time, as well as neointimal growth between both devices. Patency of the vessel lumen with no intraluminal thrombus but thrombus showing early organization (endothelial cell ingrowth) was observed within the aneurysm dome. To our knowledge, this case represents the earliest demonstration of intimal cell growth along the PED. CONCLUSIONS: Aneurysm healing via endothelialization following flow diverter treatment may occur subacutely and not chronically as previously stipulated.

Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.

Expansion microscopy (ExM), a method for improving the resolution of light microscopy by physically expanding a specimen, has not been applied to clinical tissue samples. Here we report a clinically optimized form of ExM that supports nanoscale imaging of human tissue specimens that have been fixed with formalin, embedded in paraffin, stained with hematoxylin and eosin, and/or fresh frozen. The method, which we call expansion pathology (ExPath), converts clinical samples into an ExM-compatible state, then applies an ExM protocol with protein anchoring and mechanical homogenization steps optimized for clinical samples. ExPath enables ∼70-nm-resolution imaging of diverse biomolecules in intact tissues using conventional diffraction-limited microscopes and standard antibody and fluorescent DNA in situ hybridization reagents. We use ExPath for optical diagnosis of kidney minimal-change disease, a process that previously required electron microscopy, and we demonstrate high-fidelity computational discrimination between early breast neoplastic lesions for which pathologists often disagree in classification. ExPath may enable the routine use of nanoscale imaging in pathology and clinical research.

Molecular Testing Turnaround Time for Non-Small Cell Lung Cancer in Routine Clinical Practice Confirms Feasibility of CAP/IASLC/AMP Guideline Recommendations: A Single-center Analysis.

INTRODUCTION: Molecular testing to identify targetable driver mutations is the standard of care for patients with advanced-stage non-small cell lung cancer. Recent guideline recommendations by the College of American Pathologists (CAP), International Association for the Study of Lung Cancer (IASLC), and Association for Molecular Pathology (AMP) established a benchmark turnaround time (TAT) target of 10 working days for results to be available to the treating oncologist and ≤ 3 days for specimens to arrive at a commercial testing laboratory if testing is not performed in-house. METHODS AND MATERIALS: To provide insights regarding the pre-testing, post-testing, and testing intervals that constitute the overall TAT target, we performed a detailed workflow analysis. A total of 157 lung cancer specimens were sent out for molecular testing at a commercial vendor from a single academic medical center during the calendar year 2015. RESULTS: Overall, 128 specimens (81.5%) met the recommended 10-working day TAT, with a median total TAT of 9 weekdays (mean ± standard deviation, 9.17 ± 4.15 days). The pre-testing interval was ≤ 3 days for 146 specimens (93.0%), and the post-testing reporting interval was < 1 day for 116 cases (73.9%). The TAT variance was not related to intrinsic specimen characteristics. CONCLUSION: Overall, the findings indicated that the CAP/IASLC/AMP TAT guideline recommendations are feasible for most lung cancer specimens when a streamlined system is in place.

Short-term dynamics of causal information transfer in thalamocortical networks during natural inputs and microstimulation for somatosensory neuroprosthesis.

Recording the activity of large populations of neurons requires new methods to analyze and use the large volumes of time series data thus created. Fast and clear methods for finding functional connectivity are an important step toward the goal of understanding neural processing. This problem presents itself readily in somatosensory neuroprosthesis (SSNP) research, which uses microstimulation (MiSt) to activate neural tissue to mimic natural stimuli, and has the capacity to potentiate, depotentiate, or even destroy functional connections. As the aim of SSNP engineering is artificially creating neural responses that resemble those observed during natural inputs, a central goal is describing the influence of MiSt on activity structure among groups of neurons, and how this structure may be altered to affect perception or behavior. In this paper, we demonstrate the concept of Granger causality, combined with maximum likelihood methods, applied to neural signals recorded before, during, and after natural and electrical stimulation. We show how these analyses can be used to evaluate the changing interactions in the thalamocortical somatosensory system in response to repeated perturbation. Using LFPs recorded from the ventral posterolateral thalamus (VPL) and somatosensory cortex (S1) in anesthetized rats, we estimated pair-wise functional interactions between functional microdomains. The preliminary results demonstrate input-dependent modulations in the direction and strength of information flow during and after application of MiSt. Cortico-cortical interactions during cortical MiSt and baseline conditions showed the largest causal influence differences, while there was no statistically significant difference between pre- and post-stimulation baseline causal activities. These functional connectivity changes agree with physiologically accepted communication patterns through the network, and their particular parameters have implications for both rehabilitation and brain-machine interface SSNP applications.

Use of frontal lobe hemodynamics as reinforcement signals to an adaptive controller.

Decision-making ability in the frontal lobe (among other brain structures) relies on the assignment of value to states of the animal and its environment. Then higher valued states can be pursued and lower (or negative) valued states avoided. The same principle forms the basis for computational reinforcement learning controllers, which have been fruitfully applied both as models of value estimation in the brain, and as artificial controllers in their own right. This work shows how state desirability signals decoded from frontal lobe hemodynamics, as measured with near-infrared spectroscopy (NIRS), can be applied as reinforcers to an adaptable artificial learning agent in order to guide its acquisition of skills. A set of experiments carried out on an alert macaque demonstrate that both oxy- and deoxyhemoglobin concentrations in the frontal lobe show differences in response to both primarily and secondarily desirable (versus undesirable) stimuli. This difference allows a NIRS signal classifier to serve successfully as a reinforcer for an adaptive controller performing a virtual tool-retrieval task. The agent's adaptability allows its performance to exceed the limits of the NIRS classifier decoding accuracy. We also show that decoding state desirabilities is more accurate when using relative concentrations of both oxyhemoglobin and deoxyhemoglobin, rather than either species alone.

An electric field model for prediction of somatosensory (S1) cortical field potentials induced by ventral posterior lateral (VPL) thalamic microstimulation.

Microstimulation (MiSt) is used experimentally and clinically to activate localized populations of neural elements. However, it is difficult to predict-and subsequently control-neural responses to simultaneous current injection through multiple electrodes in an array. This is due to the unknown locations of neuronal elements in the extracellular medium that are excited by the superposition of multiple parallel current sources. We, therefore, propose a model that maps the computed electric field in the 3-D space surrounding the stimulating electrodes in one brain region to the local field potential (LFP) fluctuations evoked in a downstream region. Our model is trained with the recorded LFP waveforms in the primary somatosensory cortex (S1) resulting from MiSt applied in multiple electrode configurations in the ventral posterolateral nucleus (VPL) of the quiet awake rat. We then predict the cortical responses to MiSt in "novel" electrode configurations, a result that suggests that this technique could aid in the design of spatially optimized MiSt patterns through a multielectrode array.

Comparison of training methods to improve walking in persons with chronic spinal cord injury: a randomized clinical trial.

OBJECTIVE: To compare two forms of device-specific training - body-weight-supported (BWS) ambulation on a fixed track (TRK) and BWS ambulation on a treadmill (TM) - to comprehensive physical therapy (PT) for improving walking speed in persons with chronic, motor-incomplete spinal cord injury (SCI). METHODS: Thirty-five adult subjects with a history of chronic SCI (>1 year; AIS 'C' or 'D') participated in a 13-week (1 hour/day; 3 days per week) training program. Subjects were randomized into one of the three training groups. Subjects in the two BWS groups trained without the benefit of additional input from a physical therapist or gait expert. For each training session, performance values and heart rate were monitored. Pre- and post-training maximal 10-m walking speed, balance, muscle strength, fitness, and quality of life were assessed in each subject. RESULTS: All three training groups showed significant improvement in maximal walking speed, muscle strength, and psychological well-being. A significant improvement in balance was seen for PT and TRK groups but not for subjects in the TM group. In all groups, post-training measures of fitness, functional independence, and perceived health and vitality were unchanged. CONCLUSIONS: Our results demonstrate that persons with chronic, motor-incomplete SCI can improve walking ability and psychological well-being following a concentrated period of ambulation therapy, regardless of training method. Improvement in walking speed was associated with improved balance and muscle strength. In spite of the fact that we withheld any formal input of a physical therapist or gait expert from subjects in the device-specific training groups, these subjects did just as well as subjects receiving comprehensive PT for improving walking speed and strength. It is likely that further modest benefits would accrue to those subjects receiving a combination of device-specific training with input from a physical therapist or gait expert to guide that training.

Optimizing microstimulation using a reinforcement learning framework.

The ability to provide sensory feedback is desired to enhance the functionality of neuroprosthetics. Somatosensory feedback provides closed-loop control to the motor system, which is lacking in feedforward neuroprosthetics. In the case of existing somatosensory function, a template of the natural response can be used as a template of desired response elicited by electrical microstimulation. In the case of no initial training data, microstimulation parameters that produce responses close to the template must be selected in an online manner. We propose using reinforcement learning as a framework to balance the exploration of the parameter space and the continued selection of promising parameters for further stimulation. This approach avoids an explicit model of the neural response from stimulation. We explore a preliminary architecture--treating the task as a k-armed bandit--using offline data recorded for natural touch and thalamic microstimulation, and we examine the methods efficiency in exploring the parameter space while concentrating on promising parameter forms. The best matching stimulation parameters, from k = 68 different forms, are selected by the reinforcement learning algorithm consistently after 334 realizations.